A Versatile Sample Processing Workflow for

A Versatile Sample Processing Workflow for Metagenomic Pathogen Detection Claudia Wylezich1*, Anna Papa2, Martin Beer1, Dirk Höper1*

1

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI), 17493 Greifswald-Insel Riems, Germany.

2

Department of Microbiology, Medical School, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece .

Supplementary file 1: Metagenomics sample processing protocols consisting of reagents, equipment, reagent setup, and the procedure (steps 1-120, with options for RNA extractions from ticks or mosquitoes) with timing specifications. In addition, a troubleshooting table is given for selected steps as well as some examples for anticipated results with Figures A1, A2, and A3.

Protocols for Metagenomics Sample Processing CAUTION Successful RNA extraction demands an RNase-free environment, therefore only RNase-free tubes, aerosol-free pipette tips, and only DEPC-treated (nuclease-free) water must be used. Gloves have to be worn throughout the procedure and changed regularly in order to avoid RNase contamination. In general, only use reagents that do not exceed the expiration date. When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from the manufacturer.

Reagents •Lysis buffer AL (Qiagen; cat. no. 19075) •PBS (Gibco, cat. no. 18912-014): •Disinfectant for dissection instruments (e.g., gigasept®, Schülke) •TRIzol® LS Reagent (Life Technologies; cat. no. 10296-028) ! CAUTION Trizol contains high amounts of chaotropic salts and phenol. Consult the appropriate material safety data sheets. •RNAlater RNA Stabilization Reagent (Qiagen, cat. no. 1017980) •QIAamp® RNeasy Mini Kit (Qiagen, cat. no. 74106) •RNase-Free DNase Set (Qiagen cat. no. 79254) •Ethanol (96 – 100%, undenatured) •Ethanol (75%, undenatured, diluted in DEPC-treated water) •Chloroform •RNA 6000 Pico Kit (Agilent, cat. no. 5067-1513) •High Sensitivity DNA Kit (Agilent, cat. no. 5067-4626) •Roche cDNA-Synthesis System (cat. no. 11 117 831 001) •Roche 400 µM Random-Hexamer-Primer (cat. no. 11277081001) •GeneRead DNA Library L Core Kit (Qiagen, cat. no. 180432) •Barcode Adapters (e.g., Life Technologies, Ion Xpress Barcode Adapters 1-16, cat. no. 4471250) •Barcode Adapters (25 µM, e.g., Bioo Scientific NEXTFlex 96 DNA Adapters, cat. no. 514106) •Cartridges, tubes and tips: provided with SPRIworks System II for Roche GS FLX DNA Sequencer (Beckman Coulter, cat. no A84806) •Agencourt® RNAClean XP beads (Beckman Coulter, cat. no. A63987) •Agencourt® AMPure XP beads (Beckman Coulter, cat. no. A63881) •KAPA Library Quantification Kit Ion Torrent (e.g., Roche, cat. no. 07960212001) •KAPA Library Quantification Kit Illumina® Platforms (e.g., Roche, cat. no. 07960204001) •0.2 M EDTA pH 8.0 •AFA-grade water (Covaris, cat. no. 520101) or highly purified water (at least ASTM Type III or ISO grade 3) for the M220 Focused-ultrasonicator •RNase-free water (DEPC-treated)

Equipment •Covaris cryoPREP CP02 •TissueTUBE TT1 Extra Thick & Plug (e.g., cat no. 520044) and special handle (PN 500231) (both Covaris) •Thermo Savant FastPrep Bio 101 Fp120 Cell Disrupter Homogenizer •NanoDrop 1000 Spectrophotometer connected with a computer running the NanoDrop control software •Bioanalyzer 2100 (incl. Chip Priming Station, IKA Vortexer Model MS3 with chip adapter, Agilent Technologies) •SPRI-TE Nucleic Acid Extractor (Beckman Coulter) (optional, needed for automated library preparation for Illumina platforms) •SPRIworks Method Card for Instrument Library II (REF A85410) •Liquid nitrogen •Container suitable for safe transport of liquid nitrogen •Ice pans suitable for liquid nitrogen (e.g., Magic Touch Ice pans; Thomas Scientific, cat no. 1224J40) •Laminar flow hood •Chemical fume hood •two PCR workstations (one master mix box, one template box) •Thermal block for 1.5-ml tubes •Dissection instruments (e.g., forceps, scissors, petri dishes) •Refrigerated centrifuge (for 1.5 - and 2-ml tubes) •Microcentrifuge (for 1.5- and 2-ml tubes) •Centrifuge for microtiter plates (e.g., Hettich, Rotina 380R) •M220 Focused-ultrasonicator with M220 microTUBE holder (Covaris. cat. no. 500301) and fragmentation tubes (MicroTube AFA Fiber Screw Cap 6 x 16 mm, 130 µl; Covaris, cat. no. 520096) •Thermal shaker (e.g., Eppendorf, Thermomixer Comfort) •Vortex mixer (e.g., Scientific Industries, Vortex Genie 2) •Thermal Cycler •Real-time cycler (e.g., Biorad, CFX96 Real-Time System) •Magnetic particle concentrator (MPC; e.g., DynaMag™-2, Life Technologies) •Pipettors: 2.5, 10, 20, 100, 200, 1,000 µl •Plastic lab ware (0.2-, 1.5- and 2-ml tubes and RNase-free pipette filter tips) •1.5-ml low-bind tubes (sterile, Nuclease-free) •96-well Microtiter plates, white, suitable for the used real-time PCR cycler, with suitable sealing film •Disposable gloves •Cotton gloves to handle liquid nitrogen •Personal protective clothing (protective goggles) •Soft laboratory wipes for cleaning

Reagent setup Calibration of AMPure beads (adapted from Refs 1, 2) Let the bead suspension equilibrate to room temperature; resuspend thoroughly via vortexing. Prepare a 900 µl aliquot of vigorously mixed Agencourt AMPure XP beads in a 1.5 µl tube. Using this aliquot, pipet AMPure XP beads into 1.5 ml tubes for the Bead:DNA ratios as given in the Table below (vortex the bead aliquot and change pipet tips between each sample; pipet and dispense slowly without aspirating any air and without having beads on the outside of the tip): Bead:DNA ratio (by volume)

AMPure XP Beads (µl)

0.40:1

40

0.45:1

45

0.50:1

50

0.55:1

55

0.60:1

60

0.65:1

65

0.70:1

70

0.75:1

75

0.80:1

80

0.85:1

85

0.90:1

90

Prepare sample and control DNA Ladder: Sample DNA Ladder: Pipet 48 µl of DNA Ladder to a new tube and dilute with 1152 µl Nuclease-free water; thoroughly mix by vortexing and spin down briefly. Control DNA Ladder: Dilute 4 µl DNA Ladder with 6 µl Nuclease-free water; thoroughly mix and spin down briefly. Pipet 100 µl of diluted sample DNA Ladder to each bead aliquot from step above; mix by pipetting 15 times and incubate 5 min at room temperature. Place the reaction tube onto the MPC for 5 min to separate the beads from supernatant (until the beads have concentrated on the tube wall). Slowly aspirate the cleared solution from the reaction tube without disturbing the beads and discard the solution; keep the reaction tube in the MPC while aspirating the solution. Dispense 500 µl 70% ethanol to each reaction tube and incubate 30 s at room temperature. Slowly aspirate the ethanol from the reaction tube and discard. Repeat the ethanol step. Air-dry the pellet for 10 min with the cap open (keeping on MPC). Take reaction tube from MPC, elute DNA with 10 μl Tris-HCl by careful pipette mixing and incubate for 2 min. Place the reaction tube onto the MPC, incubate for 2 min (until the beads have concentrated on the tube wall) and transfer the supernatant containing DNA Ladder into the respectively labelled clean reaction tube. Use 1 µl of supernatant of each sizeselected sample DNA Ladder and 1 µl of control DNA Ladder on a single Agilent DNA 7500 chip. When the Bioanalyzer analysis is finished, choose the “All Samples” view from the current chip; in the right-hand global settings tab, open “Integrator” in “Sample Setpoints” and set “Height Threshold [FU]” to 5. Copy the DNA concentrations (in ng/µl) for the 200, 300, 400, 500, and 900 bp peaks from each sample’s peak table to an Excel table and calculate the concentration ratios of the individual peaks and the 900 bp peak that serves for normalization. Make sure that the ratios for the control sample fit those given in the table below, if not repeat the experiment. Select the AMPure XP volume per sample volume that best fits the optimal values as per the Table below.

Peak (bp)

Optimal Ratio (Peak/900 bp)

Control Ratio (Peak/900 bp)

200

< 0.1

0.7

300

0.6

1.1

400

1.0

1.4

500

3.0

3.4

Apply the selected Bead:Sample ratio for all future size selections using the calibrated lot. Aliquot the AMPure XP beads in order to prevent repeated warming and cooling as this will degrade the buffer and render the beads non-functional. DNaseI working solution (Qiagen, prepare on ice, RNase-Free DNase Set) Add 10 μl DNase I stock solution to 70 μl Buffer RDD; mix by gently inverting the tube and centrifuge briefly (do not vortex as DNase I is sensitive to physical denaturation); Buffer RPE (Qiagen, before first use) From the QIAamp RNeasy Mini Kit: add the appropriate amount of ethanol (96 – 100%) to the buffer concentrate as indicated on the bottle; stable for 1 year when stored closed at RT; shake before use PBS Each 5 g Gibco® PBS tablet should be dissolved in 500 ml of distilled water. The pH will be 7.45 and requires no adjustment. DNA Dilution buffer 10 mM Tris-HCl, pH 8.0 + 0.05% Tween 20 (needed for qPCR)

Procedure CAUTION During both Illumina and Ion Torrent sequencing sample preparation, a clonal amplification step is performed. The amplified DNA is released into buffer before starting the sequencing run. To avoid carry-over of the amplified library DNA to original sample material or sequencing ready libraries, use a one-way facility setup with separate areas for sample processing/library construction and run preparation and never carry back materials from the sequencing to the library preparation area. Disintegration of sample material • TIMING 30 min per 2 samples CAUTION Before handling liquid nitrogen, read the appropriate material safety data sheet and operating instructions! Only use the Covaris cryoPREP impactor after having been instructed by trained personnel. 1| Laminar flow (step 1-4): Prepare approximately 20 mg solid or 200 µl liquid sample, place the sample to the centre of the tissueTUBE (TT1) through the top opening using forceps or tweezers. 2| After the sample is loaded, seal the tissueTUBE by screwing the lid on the top of the tissueTUBE but do not screw tightly. 3| While holding the tissueTUBE with the special handle, snap-freeze the sample by immersing the flexible pouch into liquid nitrogen within an ice pan; avoid dipping the cap or transfer tube.

4| Verify that the pouch is not swelled (a sign of trapped air) and that the sample remains centred in the pouch. 5 | Open the cryoPREP lid and use the special handle to insert the frozen tissueTUBE, close the cover, select IMPACT LEVEL 6, and press green ACTIVATE button. 6|

Remove tissueTUBE from the cryoPREP impactor using the special handle.

7|

Repeat steps 3-6 once.

8| Laminar flow (steps 8-10): Immediately re-chill the tissueTUBE by immersing it in liquid nitrogen. 9| Unscrew the tissueTUBE and resuspend the sample in 1-2 ml lysis buffer AL preheated at 56°C. Do not introduce air to avoid foaming of the sample. 10| Transfer the sample solution into two fresh 2-ml tubes. > PAUSE POINT The disintegrated sample in AL buffer can be stored at 4°C over night. We observed integrity of RNA after the disintegrated sample was stored about 3 months at 4°C. Extraction of RNA • TIMING approximately 2 h per 2 samples This protocol (QIAamp RNeasy Mini Kit) is suitable for the purification of RNA from 250 μl sample volume. For higher volumes, the sample can be divided in several subsamples of 250 µl to be process in parallel (steps 11-17) and to load successively on one column (step 18). Sample material can be liquid samples (serum/plasma, cell culture supernatant, EDTA-blood diluted 1:2 in PBS, supernatant from swabs, liquor) or samples homogenized and lysed (tissue and stool homogenates). Alternatively, for ticks and mosquitoes option A and B, respectively, can also be applied. > CRITICAL STEP Successful RNA extraction demands an RNase-free environment, therefore only RNase-free tubes, aerosol-free pipette tips, and Nuclease-free water must be used. Gloves have to be worn throughout the procedure and changed regularly in order to avoid RNase contamination. 11| Pre-cool the refrigerated centrifuge at 4°C. 12| Chemical fume hood: Pipette 750 μl Trizol LS Reagent into a 2-ml tube and add 250 μl sample and thoroughly shake manually for 15 s. 13| Briefly centrifuge for 5 s at 8,000 r.p.m. and Incubate 5 min at room temperature. 14| Chemical fume hood: Add 200 μl chloroform. 15| Thoroughly shake manually for 15 s and incubate 10 min at room temperature. Centrifuge 10 min at 13,000 r.p.m. at 4°C. Note All subsequent centrifugations are performed at room temperature. 16| Chemical fume hood: Carefully transfer the upper aqueous phase without disturbing the DNAcontaining interphase into a new sterile 2-ml tube. 17| Add 600 µl ethanol (75%), mix by pulse-vortexing and briefly centrifuge for 5 s at 8,000 r.p.m. 18| Apply 600 µl of the sample from step 17 to the column, and centrifuge at 10,000 r.p.m. for 20 s.

19| Discard the collection tube and place the column into a clean 2-ml collection tube, apply residual sample from step 17 to the column and centrifuge at 10,000 r.p.m. for 20 s. 20| Discard the collection tube and place the column into a clean 2-ml collection tube, add 350 µl buffer RW1 to the column and centrifuge at 10,000 r.p.m. for 20 s. 21| Discard the collection tube and place the column into a clean 2-ml collection tube, apply 80 µl DNase working solution (10 µl DNase plus 70 µl RD buffer) to the center of the membrane without touching the membrane. 22| Incubate for 15 min at room temperature, add 350 µl buffer RW1 to the column and centrifuge at 10,000 r.p.m. for 20 s. 23| Discard the collection tube and place the column into a clean 2-ml collection tube, add 500 µl buffer RPE and centrifuge at 10,000 r.p.m. for 20 s. 24| Discard the collection tube and place the column into a clean 2-ml collection tube, add 500 µl buffer RPE and centrifuge at 13,000 r.p.m. for 1 min. 25| Discard the collection tube and place the column into a clean 2-ml collection tube and centrifuge 2 min at 13,000 r.p.m. (to let the column dry). 26| Discard the collection tube and place the column into a clean 1.5-ml tube, add 50 µl RNase-free water to the center of the membrane, incubate 1 min at room temperature and centrifuge 1 min at 10,000 r.p.m. CAUTION Apply the water provided with the kit to avoid inefficient RNA elution due to inappropriate pH value. 27| Repeat step 26 twice and elute into the same tube. > PAUSE POINT RNA is stable for up to one year when stored at -80°C avoiding freeze-thaw cycles. When the RNA will be used for metagenomics, we recommend to subsequently continue with quantification of RNA and ds cDNA synthesis. Alternatively, steps 11-17 can be exchanged by option A (for ticks) or option B (for mosquitoes) depending on sampling material. (A) RNA extraction of ticks. (i) Take out of the -80°C freezer one tick at the time and proceed with it fast, avoiding thawing. Cut the tick in half vertically. (ii) Transfer one half of the tick immediately in a 1.7 ml tube containing RNAlater (10 μl RNAlater per 1 mg of tissue) for stabilization and storage. (iii) Store initially at 4°C overnight, then transfer and store at -20°C. (iv) Add 180 μl PBS in a 2-ml micro tube and transfer the other half of the tick and one 3-mm glass bead. (v) Homogenize the tissue by centrifuging 3-4 times at full speed for 20 s each at a FP120 FastPrep Cell Disrupter. Wait 1 min between the centrifuge steps to avoid overheating of the samples. (vi) Centrifuge homogenized tissue for 3 min at 14,000 r.p.m. (vii) Transfer 100 μl of supernatant in 250 μl RLT buffer (and store the rest of supernatant in tube

at -80°C) (viii) Incubate at RT for 10 min and centrifuge for 3 min at 14,000 r.p.m. (ix) Transfer the supernatant (about 350 μl) to a new microcentrifuge tube by pipetting and add 350 μl of 70% ethanol to the cleared lysate. Mix immediately by pipetting. Do not centrifuge. (x) Proceed with step 18 of the RNA extraction protocol above. (B) RNA extraction of mosquitoes. Number of mosquitoes

PBS

≤40

400 µl

40-70

500 µl

≥70 - 100

600 µl

Volume of PBS used for homogenization according to the number of mosquitoes. (i) Add the appropriate volume of PBS to the homogenization tubes according to the Table above. (ii) Add to the homogenization tube glass beads. (iii) Rinse the mosquitoes with nuclease-free water. Discard carefully the water. (iv) Add mosquitoes in the homogenization tube containing the culture medium and one 3-mm glass bead. (v) Homogenize the tissue by centrifuging 3-4 times at full speed for 20 s each at FastPrep centrifuge. Wait 1 min between the centrifuge steps to avoid overheating of the samples. (vi) Centrifuge homogenized tissue for 5 min at 14,000 r.p.m. and then transfer 100 μl of homogenized tissue in 250 μl RLT. (vii) Transfer the remaining 300 μl or 400 μl or 500 μl, depending on the sample volume (see also Table above) of the supernatant to a new microcentrifuge tube for stock storage at -80°C. (viii) Incubate at RT for 10 min and centrifuge for 3 min at 14,000 r.p.m. (ix) Transfer the supernatant (about 350 μl) to a new microcentrifuge tube by pipetting and add 350 μl of 70% ethanol to the cleared lysate. Mix immediately by pipetting. Do not centrifuge. (x) Proceed with step 18 of the RNA extraction protocol above. Quantification and integrity check of RNA • TIMING approximately 1 h per 2 samples CAUTION Since the NanoDrop quantification can be unreliable [3], quality-checked RNA can be accurately quantified using the Quant-iT RiboGreen RNA reagent. 28| Quantify the purified RNA on a NanoDrop 1000 Spectrophotometer. 29| Confirm RNA integrity using an Agilent Bioanalyzer RNA assay (with the Eukaryote Total RNA Series II.xsy assay). If necessary, adjust the RNA concentration to fit the requirements of the selected assay. From the generated electropherogram, the software calculates an RNA Integrity Number (RIN) that can help in assessing RNA quality. cDNA synthesis • TIMING approximately 4 h per 2 samples

30| Approximately 500 ng high quality RNA in a maximum volume of 17 µl is needed for cDNA synthesis. In case the concentration is too low, RNA needs to be concentrated with Agencourt RNAClean XP beads (steps 31-38). CAUTION To avoid contaminations: Tubes containing purified template RNA/DNA must exclusively be opened and handled in a template PCR workstation. Buffers and reagents for preparing mastermixes must exclusively be opened and handled in a clean mastermix box. Change gloves after handling template RNA before working in the mastermix box. Buffers and reagents once opened in the template box must never be used and opened in the mastermix box. Concentrating RNA before cDNA synthesis (optional) • TIMING approximately 45 min per 2 samples 31| Template box: (steps 30-38): Pipette 1.8 volumes of well-mixed Agencourt RNAClean XP beads to RNA (within low-bind tube) and mix the DNA-bead-solution thoroughly by pipette mixing (15x). Incubate 10 min at room temperature on a shaker at 500 r.p.m. 32| Place the reaction tube onto the MPC for 5 min to separate the beads from supernatant (until the beads have concentrated on the tube wall). 33| Leaving the tube in the MPC, slowly aspirate the supernatant from the tube without disturbing the beads and discard the solution. 34| Leaving the tube in the MPC, dispense 1 ml 75% ethanol to reaction tube and incubate 30 sec at room temperature and slowly aspirate the ethanol from the reaction tube and discard. 35| Repeat step 34 while the reaction tube keeps on MPC. 36| Air-dry the pellet for 10 min with the cap open (keeping on MPC); do not over-dry! 37| Take reaction tube from MPC, elute DNA with 20 μl Nuclease-free water by careful pipette mixing and incubate for 2 min. 38| Place the reaction tube onto the MPC, incubate for 2 min, and pipette 17 µl of the concentrated DNA solution into a clean reaction tube. cDNA synthesis • TIMING approximately 4 h per 2 samples 39| Mastermix box: Prepare a mastermix for the first strand synthesis using the Roche cDNASynthesis System: Components

1x

Vial 1 (5× RT-buffer AMV)

8.0

µl

µl

Vial 3 (DTT, 0.1 M)

4.0

µl

µl

Vial 7 (dNTPs, 10 mM)

4.0

µl

µl

Vial 4 (Protector RNase Inhibitor, 25 U/μl)

1.0

µl

µl

Vial 2 (AMV RT, 25 U/μl)

2.0

µl

µl

19.0

µl

µl

Total volume

x

40| Mastermix box: Pipette 4 µl of the 400 µM Random-Hexamer-Primer in a 0.2-ml PCR tube.

41| Template box: Add a maximum of 500 ng template RNA in a total volume of 17 µl (from step 27 or optionally from step 38) to the Random Hexamer-Primers, briefly vortex and centrifuge. 42| Incubate 2 min at 95°C in a thermal cycler with hot-lid on and subsequently cool down the reaction for 2 min on ice. Add 19 µl of the first-strand mastermix. 43| Incubate 10 min at 25°C, 60 min at 42°C with hot-lid on (110°C). After the incubation is finished, keep on ice. Note Briefly before the incubation is finished, start preparing the mastermix for the second-strand synthesis (see below). Before proceeding to the mastermix box, change gloves. 44| Mastermix box: Prepare mastermix for the second strand synthesis using the Roche cDNASynthesis System: Components

1x

x

Vial 9 (5× 2nd strand synthesis buffer)

30.0

µl

µl

Vial 12 (Water, PCR Grade)

72.0

µl

µl

Vial 7 (dNTPs, 10 mM)

1.5

µl

µl

Vial 10 (2nd strand enzyme)

6.5

µl

µl

110.0

µl

µl

Total volume

45| Template box: Add 110 µl second strand synthesis mastermix to the first-strand reaction. 46| Briefly mix and centrifuge. Incubate 2 h at 16°C (hot-lid off) in a thermal cycler. 47| Add 20 μl of vial 11 (T4 DNA Polymerase), carefully mix and centrifuge briefly. Incubate 5 min at 16°C. Stop reaction by adding 17 µl 0.2 M EDTA, pH 8.0. > PAUSE POINT Store cDNA at -20°C or continue directly with the DNA fragmentation. Library preparation: fragmentation • TIMING approximately 30 min per 2 samples CAUTION Never run a method without AFA-grade water in the acoustic assembly; this could damage the transducer. Do not leave water in the water bath for an extended time. Empty the water bath and wipe it dry after use with a lint-free cloth. Do not employ isopropyl alcohol, ammonia-based or abrasive cleaners on the acoustic assembly. Store the tube holder in a dry place. 48| Pipette 130 µl sample in the sample tube (if the sample volume is less than 130 µl fill up to 130 µl with water). Place the tube in the microTube holder and fix it with the sample weight. 49| Start protocol via the start button, repeat fragmentation once by pressing the repeat button. Use the pre-installed DNA shearing protocol DNA_0500_bp_130_ul_Snap_Cap_microTUBE. 50| Pipette the fragmented sample into a clean 1.5 ml tube. If the sample volume is greater than 130 µl, repeat steps 48-49 using the same microTube. Purification and concentration of fragmented cDNA synthesis products for samples to be sequenced with the Ion Torrent platform • TIMING approximately 45 min per 2 samples CAUTION When opening a new lot of AMPure XP beads, perform a bead calibration before first use. Aliquot the AMPure XP beads in order to prevent repeated warming and cooling as this will degrade

the buffer and render the beads non-functional. Make sure the Agencourt AMPure XP beads are at room temperature and are thoroughly mixed. The reagent (magnetic particle solution) should appear homogenous and consistent in colour. Do not freeze. 51| Add 1.8 volumes of well-mixed Agencourt AMPure XP beads to each cDNA sample and mix the DNA-bead-solution thoroughly by pipette mixing (15x) and incubate 10 min at room temperature on a shaker at 550 r.p.m. 52| Place the reaction tube onto the MPC for 5 min to separate the beads from supernatant (until the beads have concentrated on the tube wall). 53| Slowly aspirate the cleared solution from the reaction tube without disturbing the beads and discard the solution; keep the reaction tube in the MPC while aspirating the solution. 54| Leaving the tube in the MPC, dispense 1 ml 75% ethanol to the reaction tube and incubate 30 s at room temperature; slowly aspirate the ethanol from the reaction tube and discard. 55| Repeat step 54 while the reaction tube keeps on MPC. 56| Air-dry the pellet for 10 min with the cap open (keeping on MPC); do not over-dry! 57| Take reaction tube from MPC, elute DNA with 27 μl Nuclease-free water by careful pipette mixing and incubate for 2 min. 58| Place the reaction tube onto the MPC, incubate for 2 min. Keeping the tube on the MPC, pipette 25 µl of the concentrated DNA solution into a clean reaction tube. Library preparation There are 2 possibilities for library preparation, one manual preparation to be sequenced with the Ion Torrent PGM (Steps 59-77) and one automated preparation to be sequenced with the Illumina MiSeq (Steps 78-92). CAUTION To avoid contaminations: Tubes containing fragmented and concentrated DNA must exclusively be opened and handled in a template box. Buffers and reagents for preparing mastermixes must exclusively be opened and handled in a clean mastermix box. Do not use the same barcode as in the sequencing run before. Change gloves after handling template DNA before working in the mastermix box. Library preparation for sequencing with Ion Torrent PGM: end-repair • TIMING approximately 45 min per 2 samples 59| Thaw, mix, and shortly centrifuge both end repair and ligation buffer, adapters, and dNTPs and keep them on ice. 60| Mastermix box: Pipette 2.5 µl of End-Repair Buffer in one 0.2 ml PCR tube per DNA sample. Add 2 µl End-Repair Enzyme Mix and keep the PCR tube on ice 61| Template box: Keep DNA samples on MPC some minutes before pipetting to avoid transfer of residual AMPure beads. Add 20.5 µl DNA sample to buffer and enzyme mix for a final total volume of 25 µl. Briefly vortex and centrifuge. Optionally, save 2 μl of fragmented DNA for troubleshooting. 62| Incubate in the thermocycler with following temperature profile:

Temperature

Time

25°C

20 min

70°C

10 min

8°C



63| Briefly before the incubation is finished, start preparing the mastermix for the adapter ligation (see step 64). Before proceeding to the mastermix box, change gloves. Library preparation for sequencing with Ion Torrent PGM: adapter ligation • TIMING approximately 30 min per 2 samples 64| Mastermix box: Prepare mastermix for the adapter ligation: Components

1x

RNase-free water

8.0

µl

µl

Ligation Buffer (2x)

40.0

µl

µl

dNTP Mix (10 mM)

1.0

µl

µl

Universal adapter P1 (to 0.5 µM final conc.)

1.0

µl

µl

Ligation and Nick Repair Mix

4.0

µl

µl

54.0

µl

µl

Total volume

x

65| Briefly vortex and centrifuge the mastermix. 66| Mastermix box: Pipette 54 µl of mastermix in one 0.2 ml PCR tube per DNA sample. 67| Mastermix box: Add 1 µl of the chosen barcode adapter to each mastermix aliquot. CAUTION To avoid contaminations, open only one barcode adapter tube at a time and change gloves between pipetting the different adapters to avoid cross-contamination. 68| Template box: Add 25 µl end-repaired DNA (from part End-repair, above), briefly mix and centrifuge. Incubate in the thermocycler with pre-set temperature profile: Temperature

Time

25°C

10 min

72°C

5 min

8°C



Purification and concentration of IonTorrent Library • TIMING approximately 45 min per 2 samples CAUTION The end-repaired and adapter-ligated library has to be purified to remove excess of polyethylene glycol (PEG), salts, and enzymes. Otherwise, the high concentration of PEG in the ligation mix will interfere with proper size selection. 69| Template box: (steps 69-77): Pipette 144 µl (1.8 volumes) of well-mixed Agencourt AMPure XP beads to 80 µl of end-repaired and adapter-ligated DNA library and mix the DNA-bead-solution thoroughly by pipette mixing (15x). Incubate 10 min at room temperature on a shaker at 500 r.p.m.

70| Place the reaction tube onto the MPC for 5 min to separate the beads from supernatant (until the beads have concentrated on the tube wall). 71| Leaving the tube in the MPC, slowly aspirate the supernatant from the tube without disturbing the beads and discard the solution. 72| Leaving the tube in the MPC, dispense 1 ml 75% ethanol to reaction tube and incubate 30 sec at room temperature and slowly aspirate the ethanol from the reaction tube and discard. 73| Repeat step 72 while the reaction tube keeps on MPC. 74| Air-dry the pellet for 10 min with the cap open (keeping on MPC); do not over-dry! 75| Take reaction tube from MPC, elute DNA with 27 μl Nuclease-free water by careful pipette mixing and incubate for 2 min. 76| Place the reaction tube onto the MPC, incubate for 2 min, and pipette 25 µl of the concentrated DNA solution into a clean reaction tube. 77| Adding 25 µl Nuclease-free water, repeat the elution (steps 75-76) to increase the yield of library, pool both elutions. Optionally, save 2 μl of the library for troubleshooting. Automated library preparation with the SPRIworks Fragment Library System II for sequencing with Illumina MiSeq: • TIMING approximately 4 hours per 2 samples (including pre-arrangements and running time). CAUTION Blunt end adapters are not compatible with SPRIworks Fragment Library System II. Do not change the method card while instrument is powered on. To avoid contaminations, do not use the same barcode as in the sequencing run before. 78|

Completely thaw, mix, and briefly centrifuge all components.

79| Mastermix box: Prepare 3 labelled screw cap tubes provided with the kit for each library: sample tube, adapter tube, library tube. Take off all caps of the tubes; keep the caps of the library tubes. 80| Pipet 2,5 µl of one of the different barcode adapters to the adapter tube and dilute with 2,5 µl Nuclease-free water. CAUTION To avoid contaminations, open only one barcode adapter tube at a time and change gloves between pipetting the different adapters to avoid cross-contamination. 81| Template box: Transfer 500 ng fragmented DNA in a maximum of 400 µl to the sample tube, fill up to 400 µl with Nuclease-free water. 82| SPRI-TE Nucleic Acid Extractor (Steps 82-92): Set up the instrument: Turn off the instrument Press the EJECT button, remove any method card if necessary and carefully insert the SPRIworks method card for Fragment Library System II (REF A85410) - Close the instrument door - Turn the power on. 83| Open the door only when the screen displays the TOP MENU.

84| When cartridges have completely thawed, ensure that all solutions including magnetic beads are at the bottom of the wells by gently tapping on the bench top. 85| Remove tip/tube rack and reagent rack (in this order) from the instrument. 86| Insert one cartridge per library into the grooved channel of the reagent rack by grasping the labelled lip and sliding it to the back of the channel. 87| Arrange the tubes and tips provided with the kit as displayed in the Table below: Position in the tip tube rack

Consumables

1

Sheath with Piercing Tip

2

Sheath with 1 ml Tip

3

Sheath with 200 µl Tip

4

Uncapped 2 ml Adapter Tube with Adapters

5

Uncapped 2 ml Library Tube

88| Load reagent rack and tip/tube rack (in this order) into the instrument; the back edge of the tip/tube rack overlaps the reagent rack when loaded correctly. 89| Load the uncapped sample tube containing the fragmented DNA into the circular hole in position 13 of the reagent rack. 90| Start the run: Close the instrument door - Select the green START button - Specify 1 = no size selection and select ENTER to confirm - After a heat block test, the run begins automatically. 91| The run is finished when the instrument displays “Run Complete” - Open the door - Retrieve and cap the library tubes - Discard used sheaths, tips and cartridges. 92| To clean the instrument, select UV-Clean from the TOP MENU. Size selection of library • TIMING approximately 45 min per 2 samples 93| Template box: (steps 93-108): Resuspend an aliquot of the magnetic particle solution via vortexing. For the upper size selection, dilute Agencourt AMPure XP beads per the Table: 1x

x

AMPure XP beads

104.0

µl

µl

Water (PCR grade)

80.0

µl

µl

184.0

µl

µl

Sum

94| Fill the generated library (from step 77 or 91 depending on sequencing platform) up to 100 µl with Nuclease-free water. 95| Add 160 µl diluted Agencourt AMPure XP beads per DNA library to remove large fragments and mix the DNA-bead-solution thoroughly by pipette mixing (15x). Incubate 5 min at room temperature on a shaker at 550 r.p.m.

96| Place the reaction tube onto the MPC for 5 min to separate the beads from supernatant (until the beads have concentrated on the tube wall). 97| Slowly aspirate the cleared solution from the reaction tube without disturbing the beads and pipette the solution into a clean 1.5 ml tube in two steps á 125 µl (exact final volume 250 µl). 98| Add 30 µl undiluted Agencourt AMPure XP beads per 250 µl supernatant from step 97 to remove small fragments and mix the DNA-bead-solution thoroughly by pipette mixing (15x). Incubate 5 min at room temperature on a shaker at 550 r.p.m. 99| Place the reaction tube onto the MPC for 5 min to separate the beads from supernatant (until the beads have concentrated on the tube wall). 100| Leaving the tube in the MPC, slowly aspirate the supernatant in two steps á 138 µl from the tube without disturbing the beads and discard the solution. 101| Leaving the tube in the MPC, dispense 200 µl 75% ethanol to reaction tube and incubate 30 sec at room temperature. 102| Slowly aspirate the ethanol from the reaction tube and discard. 103| Repeat step 101-102 while the reaction tube keeps on MPC. 104| Air-dry the pellet for 10 min with the cap open (keeping on MPC); do not over-dry! 105| Take reaction tube from MPC, add 18 μl Nuclease-free water by careful pipette mixing and incubate for 2 min. 106| Place the reaction tube onto the MPC, incubate for 2 min and pipette 15 µl of the DNA solution into a clean reaction tube. 107| Take reaction tube from MPC, add 15 μl Nuclease-free water by careful pipette mixing and incubate for 2 min. 108| Place the reaction tube onto the MPC, incubate for 2 min and pipette 15 µl of the DNA solution into the same reaction tube. > PAUSE POINT Store the completed library in a safe-lock tube at 4°C or continue directly with quality check. Quality check and quantification of library • TIMING approximately 3 h per 2 samples 109| Ensure the absence of too short or too long fragments, and of primer dimers using an Agilent Bioanalyzer High Sensitivity DNA assay (with the High Sensitivity DNA.xsy assay). 110| Continue directly with quantification of the library using the KAPA Library Quantification Kit for Illumina or Ion Torrent sequencing. CAUTION Make sure to select the appropriate kit for the used sequencing platform and the real-time PCR cycler you use! 111| Completely thaw, mix, and briefly centrifuge all kit components.

112| Mastermix box: Before first use, prepare buffer qPCR/Primer mix from the Kit: Add 1 ml of Primer Premix (10×) to 5 ml bottle of KAPA SYBR FAST qPCR Master Mix (2×) and mix thoroughly. This mix is stable for 1 year when stored closed at -20°C. 113| Mastermix box: Prepare a mastermix using the following Table; calculate 6 reactions per DNA library plus 12 reactions for standards plus additional positive controls, if used, plus 2 no-templatecontrols (NTC) plus 0.5 reactions in excess. Component

1x

Nuclease-free water

x

4.0 µl

µl

KAPA SYBR FAST qPCR Mastermix (including Primer mix)

12.0 µl

µl

Total volume

16.0 µl

µl

114|

Vortex and briefly centrifuge.

115| Mastermix box: Pipette 16 µl mastermix in each used well of a 96-well microtiter plate. 116| Template box: Dilute sample DNA only with dilution buffer: 3 dilutions with duplicates are needed. Keep sample DNA on MPC during pipetting to avoid transferring AMPure XP beads. Based on the Bioanalyzer data (see step 109), select adequate library dilutions (each 3 dilutions); use the following values as guidance: • No detection with Bioanalyzer DNA HS Chip → use dilutions 1:20; 1:40; 1:80 • Weak signal with Bioanalyzer DNA HS Chip → use dilutions 1:200; 1:400; 1:800 • High signal with Bioanalyzer DNA HS Chip → use dilutions 1:2,000; 1:4,000; 1:8,000 • Amplified Libraries → use dilutions 1:20,000; 1:40,000; 1:80,000 117| Template box: Add 4 µl of diluted DNA library or standard to the respective wells, final reaction volume 20 µl. 118| Seal the plate with sealing film, press well on. Briefly centrifuge the plate using a microtiter plates centrifuge. 119| Insert the plate into the cycler and wipe the seal clean before closing the lid. Enter parameter, sample names and dilutions in the cycler software and start the KAPA PCR program. Step

Temperature

Time

1

95°C

5 min

2

95°C

30 sec

3

60°C

45 sec

1x

4

4°C



1x

Melting curve analysis

1x 35x

120| After completion of the run, check the integrity and plausibility of the PCR results, e.g. fluorescence intensity, amplification curves, melting curves, R2 of the standard curve ≥ 0.95, PCR efficiency ≥ 0.95, efficiencies between 0.90 and 0.95 are acceptable if R2 is ≥ 0.99, Cq values of different sample dilutions and the positive control (Illumina).

Calculate DNA library molarities from the qPCR results and optionally pool several libraries with different barcode adapters for one sequencing run.

Timing The time is calculated for 2 samples. Steps 1-10, disintegration of sample material: 30 min Steps 11-27, extraction of RNA: 2 h Option A, RNA extraction of ticks (optional to steps 1-17): 1-2 h Option B, RNA extraction of mosquitoes (optional to steps 1-17): 1-2 h Steps 28-29, quantification and integrity check of RNA: 1 h Steps 30-38, concentrating RNA (optional, if RNA concentration is too low): 45 min Steps 39-47, cDNA synthesis: 4 h Steps 48-50, library preparation: fragmentation: 30 min Steps 51-77, (optional to steps 78-92), manual library preparation for sequencing with Ion Torrent PGM: 3 h Steps 51-58, purification and concentration of fragmented cDNA synthesis products: 45 min Steps 59-63, library preparation for sequencing with Ion Torrent PGM, end-repair: 45 min Steps 64-68, library preparation for sequencing with Ion Torrent PGM, adapter ligation: 30 min Steps 69-77, purification and concentration of Ion Torrent Library: 45 min Steps 78-92 (optional to steps 51-77), automated library preparation for sequencing with Illumina MiSeq: 4 hours Steps 93-108, size selection of library: 45 min Steps 109-120, quality check and quantification of library: 3 h

Troubleshooting Step

Problem

Possible reason

Solution

28

Very low RNA concentration is measured

Depends on the sample

Repeat RNA extraction with sufficient material if possible and eluate with nuclease-free water supplied with the kit, Concentrate RNA (steps 31-38) before cDNA synthesis

29

RNA is fragmented and RIN value is low or not calculated

Depends on the sample and its pre-treatment (e.g., processing, fixation)

Proceed with the protocol

109

Size distribution is out of the suitable range

AMPure beads are not previously calibrated

Carefully repeat the size selection procedure with calibrated beads, Check residual Ion Torrent library from step 61 and 77 using Agilent HS chip to follow the library status

109

No DNA detected

Very low initial input for library preparation

120

No library determined

Improper library dilutions used for qPCR or very low initial input for library preparation

120

Lower amount of library determined than expected

Improper library dilutions used for qPCR or very low initial input for library preparation

Check sample before (step 61) and after library preparation (step 77) using HS chip to find the possible error source Proceed with library quantification by qPCR. Use expected fragment size for KAPA calculation Repeat KAPA qPCR with appropriate dilutions, use DNA dilution buffer

If measurable amount of library is detected, proceed with sequencing

Anticipated results Step 29. Typical results of the Bioanalyzer 2100 RNA pico assay are shown in Figure 3. In lanes marked with C intact total RNA is evident from rRNA peaks. In lanes marked with T (Fig. 3, A+B) and M (Fig. 3, C), rRNA peaks are partly or completely missing because of degradation of RNA. The Bacillus subtilis samples (spore) are also shown as electropherograms (Fig. A1). The cryoPrep sample shows the most intact RNA whereas the TissueLyser sample shows very degraded RNA. From the generated electropherogram, the software calculates an RNA Integrity Number (RIN) that can help in assessing RNA quality. According to our experience, however, there is no correlation between pathogen detection or sequence data output and the RIN value.

Figure A1 RNA quality after RNA extraction on RNA 6000 Pico Assay (Agilent) for the Bacillus subtilis spore sample using different disintegration tools. nt, nucleotides.

In some cases, the extracted RNA had only a low concentration and needed to be concentrated (using steps 31-38 of the protocol) before cDNA synthesis. This was done for the bird feces, tap water and the pizza samples. Compare Fig. A3 for resulting libraries.

Figure A2 RNA quality after RNA extraction on RNA 6000 Pico Assay (Agilent) for different samples disintegrated with the cryoPREP before the RNA concentration step. Compare Fig. A3 for the resulting DNA libraries. nt, nucleotides.

Step 109. The size-selected library is composed of fragments ranging from 300 to 1,000 bp with a peak at about 500-550 bp. Ideally, the library should show a representative or weak peak (Fig. A3, sheep pool ID 1454, containing an astrovirus4, or wild boar with trichina ID 1806, respectively). For the trichina-sample, we obtained 0.02% trichinellid reads (data set of 1,627,077 reads). But also in cases without a peak, virus reads can be detected depending on the viral load. This was for example the case in the meat loaf sample ID 1488 (Fig. A3) that contained 319 reads (0.1%) of a pepper mild mottle virus 6.36 kb genome (99% identity, RNA virus) representing a plant pathogen (total data set 360,148 reads).

Figure A2 Sequencing libraries visualized with a High Sensitivity DNA Assay (Agilent) for sequencing with Illumina (squirrel transudate5) and Ion Torrent PGM (sheep pool4, meat loaf, meat with trichina). Compare Fig. A2 for the quality of RNA. bp, base pairs.

Step 120. After completion of the run, check fluorescence intensity, amplification curves, melting curves for integrity and plausibility of the PCR results. The relative standard deviation of replicates should be < 10% and R2 of the standard curve ≥ 0.95, PCR efficiency ≥ 0.95, efficiencies between 0.90 and 0.95 are acceptable if R2 is ≥ 0.99. Check also the Cq values of different sample dilutions for plausibility.

Additional references 1. GS FLX Titanium General Library Preparation Method Manual, Document USM-

00048.B, Roche Diagnostics, April 2009. 2. GS FLX System Technical Bulletin TCB No. 017-2009, Roche Diagnostics, November 2009. 3. Troubleshooting. In: ND-1000 Spectrophotometer V3.5 User’s Manual. NanoDrop Technologies, Inc. Rev 6/2007 4. Pfaff F, Schlottau K, Scholes S, Courtenay A, Hoffmann B, Höper D, Beer M. A novel astrovirus associated with encephalitis and ganglionitis in domestic sheep. Transbound Emerg 2017; 64:677–682.

Supplementary file 2: Extractions of the RIEMS result protocols graphically summarizing the 100 most abundant families found in the sequencing data sets of samples given in Tables 2 and 3 of the main document, ordered by library IDs. In order to represent all detected superkingdoms, the displayed families are selected from the complete result applying the HighestAverages algorithm from the R package SciencesPo. Families are sorted alphabetically, color-coded according to superkingdoms. Note that the graphs are in logscale. The original sample name created by the sequencing platform is included for each data set that refers to the Library IDs given in Table 2 and 3. For example, the name “lib12611-reg2-RL2-run2011-11-02-resultprotocol.pdf” refers to library ID 126. Known false positive viral families in the graphs are listed in the legends. Causes for the occurrence of false positives may be (i) reagent contamination and taxonomic misclassification (Retroviridae, Arenaviridae) and (ii) missorting of reads during data deconvolution or run-to-run carry-over. The former workflow-specific false positives are labelled WF (workflow), the latter where the taxonomic families are known to emerge from other samples in the same run/previous run are labeled RS (run specific). For details on the possible causes of false positives, please refer to the main text.

Zoogloeaceae Yersiniaceae ae onadace Xanthom idae r tilion Vespe ae e c nella Veillo ae eae Suid c a t yce ae om ace ae ept e occ Str c a toc ae cc ep co tace ae Str ylo a ce ph sm ada Sta n pla e o a iro e ce om Sp g na rida la i So hov p Si

10.0

hin

Sp

7.5

5.0

0.0

Poaceae

ar

ur

M

Mi cr

M

N oc

e ea ae ac il d lacid b p e By isto dae ea ll ani a ac C e C cteri ea ba rac rno bacte a C ulo dae e Ca Cebi cida ithe p o c r e e a C id Cerv eae nadac domo y m la e Ch a ce tia Chroma riaceae Chromobacte Cladosporiaceae Clostridiaceae Clupeidae Comam onadace ae Cordy cipita Cory ceae neba cteria ceae Cric etid ae C Cy ulic ph ello idae De Cy pho rac p ba ea Di ryo rinid e ae ph m Dr yllo yce tac ac bo ea un thr e ii cu lid dae ae

Superkingdom Archaea Bacteria Eukaryote Virus

False positives: - Arenaviridae (WF) - Retroviridae (WF)

ae ce e ia er cea ct a ba occ e ro te roc ida En nte Equ ceae E ica ceae Er ia win dae e Er li cea Fe eria act e vob hagida Fla p y c ae Gly iace Hafn ae teuthid Histio ae Hominid eae Lactobacillac Lamprophiidae

Plasmodiidae Piscicolidae e Pinacea nidae ia s a e Ph atida som colo idae ir Phas v ya e ibun ilacea Per h ae nip e o c t lla ae Pep ce ure ste ctera ae Pa d a i b c ae alo ocer hid p e Ox h e c r t a On as did m i Om Num

di oi da or ce i d ax a ae e om ell ac on Mi cro osp eae o co Mic cc rac rob act acea eae Me er i thy ac e lop Met hila eae hylo cea bac teria e Meth cea anoc e occa ceae Malas sezia ceae Lophiotr ematace ae Lipotidae Leporidae

log.counts

2.5

Sa Rh l od mo n Rh os pir idae od ob i ac llace Rh te a izo bia race e Re ae ce tro a e viri Psy da e cho Pse did udo a e mon ada Pseu cea doco ccida e Psath e yrella ceae Propion ibacteri aceae Procaviidae

Acetobacteraceae Actinomyceta ceae Albugin aceae Alcali genac eae Allig atorid ae Apt eryg idae Are As navir ida pe e r g il Ba cill lacea ac e Br B e ad ov id ae y Bu Bru rhiz ae rk ce obi ac ho lla e c ld er eae ae ia ce ae

Result Report

Family

Figure 3: Graphical representation of read counts for the 100 most abundant families. In order to represent all detected superkingdoms, the displayed families are selected from the complete result applying the HighestAverages algorithm from the R package SciencesPo. Families are sorted alphabetically, color-coded according to superkingdoms. Note that the graph is in log-scale.

lib126-11-reg2-RL2-run2011-11-02-resultprotocol.pdf

6 of 24

ph

No

O

Nu

ae ae e rid to erac s ae t Ca bac ecid h o t l i u e p ae Ca erco rvida dace a C Ce on ae e m ac ydo teri e lam obac a h e c C rom poria Ch dos ceae a d Cla a n amo ceae Com cipita Cordy ae ce la o ri Co riaceae Corynebacte Cricetidae Cylindroleberididae Cyprinidae Delphin idae Diapo r thace Diph ae yllob Dys othrii gon dae amo nad ace En Emyd ae ter ida ob e act eri Eq a cea uid Er w ae e Fa iniac b e Fl a a e c av ivi eae rid ae

Superkingdom Bacteria Eukaryote Virus

False positives: - Retroviridae (WF) - Flaviviridae (RS)

ae ce e lla ea se llac ae ci hid an ne Fr llio onc idae a h t m G u e e ae ae Ha tiot s nid ace Hi mi ter Ho libac iso dae nd i ae mu Janir Im ace spir hno Lac ridae Lepo ae hyphid Lepto ae ematace Lophiotr Lyophyllaceae Macraucheniidae

Polymixiidae Pipridae eae Pinac ceae ia Pich ae ocid llod e Phy a cid Pho nidae e a a i ce e as ila Ph iph acea n ll pto ae ae ure Pe ste urid race y Pa e Ox act b lo xa O

nc ho ce r m ca id cid id rd a Ne ioid ae e ac rei Ne e d oty id a len ae e Ne od iplo chid a sto e m Mu rida idae Mor e axe llac Micr eae omo nosp orac eae Micro hylida e Microco ccaceae Methylophilac eae Malasseziaceae

log.counts

0.0

Zoogloeaceae

la na ce ov ae i ng rida oe e Sa c l m Ru on idae min ida oc e Rh odo occac spi rilla eae Rho cea doc e ycla Rho cea doba ctera e ceae Rhizo biace ae Retroviri dae Psychodidae Pseudomonadaceae Propionibacteriaceae

lpi

2.5

So

Si

Sa

5.0

Yersiniaceae ae onadace Xanthom eae nellac Veillo ceae gina e Ustila dida ae e ona c a hom por Tric nos mo e e rmo ida acea The Su et eae yc c tom cca ae e o ep id ea e Str toc ur c a rep han cca ce St ep oco ada St l on hy ap gom St n hi Sp

7.5

Actinomycetaceae Alcaligenacea e Anaero myxoba cterace ae Anatid ae Aotid a e Apt Asp erygida e e Az rgillac on exa eae Br ad Bovi ceae da yr Bu B hizo e r b C ar rkh ami iace no old da ae ba er e ct iac er ea ia ce e ae

Result Report

Family

Figure 3: Graphical representation of read counts for the 100 most abundant families. In order to represent all detected superkingdoms, the displayed families are selected from the complete result applying the HighestAverages algorithm from the R package SciencesPo. Families are sorted alphabetically, color-coded according to superkingdoms. Note that the graph is in log-scale. lib127-11-reg2-RL3-run2011-11-02-resultprotocol.pdf

6 of 20

So rra Se

9

Sc iu rid ae os o Sa m a lm t i da on Rh ida e izo e bia ce Rh ae ino lop hid Pte ae rop odid Pseu ae dono card iace Propio ae nibac teriac eae Prevotella ceae

Sc

his t

6

0

ea

ac

bi izo

h

yr

ad Br

Polystomatidae

e

e da

eli

m Ca

ae nid Ca ae bid ae Ce cid ithe p o rc e Ce vida Cer e leida iroga Che e c a d ea mona Coma ae ce acteria Coryneb Cyprinidae Delphinidae

Poaceae

Diphyllobothr

Plasmodiidae ridae Physete ae oenid Phoc e cida Pho ae nid e sia ea Pha rac e ct e a a ib id lag thy Pe ch i l e ae ra ea r id Pa yu ac x er O t c ba lo xa O

Malvaceae

Ny

O nc

Dracun culidae Dros ophil idae Eim eriid a e Ele pha En ntid ter ae ob act eri Eq ace uid ae Fa ae ba ce Fe ae lid ae

Malasseziaceae

My

iidae


False positives: - Flaviviridae (RS)

e da ae ir i ce iv ia r av te Fl e ac a ob ae ffid av tid ra Fl Gi ma ne ae ylo hid ng onc Go em ae Ha erid d i pos Hip e inida Hom idae Iguan e spiracea Lachno Lottiidae

ho ce m rc ph id co ae al pla id a My sm e co ata ba ce cte ae r ia Mu ce ste a e lida e Mu rida e Micr oba cter iace ae Meth anop yrace ae Manida e

log.counts

3

Adrianichth yidae Aptery gidae Asca ridid ae Bab esii d a e Ba cilla Ba cea c e Ba teroid lae ac e n ae o Ba thy pteri da e Bo ergi da vid e ae

Vitaceae

go

hin

Sp

Ursidae

dae Tupaii ae Suid idae loid ae ngy Stro ace occ e loc ea c phy ata ae Sta sm ce da pla iro na Sp mo ae e ac e lan da ni

12

Actinomycetaceae

Result Report

Family


6 of 11

6

3

ha ro

Ro sac

Rhin

olop

ea

ter ac

As

ae

on ida

e

e

idae eryg

Apt

arid ida

ae

daceae

Aotid

r id

lm

be Ba sii la da en e op te rid ae

Sa cc

iu

Ba

Sc Sa

Asc

9

Aeromo na

ceae

ae

l So

mo

go

hin

Sp

Vespertilionidae

lla Veillone

Ursid

ae Suid ae idid ylo e ea ong ac Str e cc co ea ylo ac ph ad n Sta e ea ac an

12

Actinomycetaceae

Result Report

rt Ba

e

my c

ce ae

hida e Rhab ditida e

e

ida

Bo

eta

eae

ae ce

la

el on

ae vid Bo e lida me Ca idae Can ae

Cebid

log.counts

Pteropo didae

0

ecidae

Cercopith

Propionibacteriaceae

Cervidae

Polystomatidae

Cheirogaleidae

Plasmod Plano

Chinchill

iidae

idae

Colum

e rbida

Cric

e

ida eter

s Phy

Ei

e

Indriidae

Manid ae

cea

ae

cca

Lipotida e

ae

idae

Mic

roco

rid

nid

ae

lid ae

Mo ro

ae

dae

Mu

ae

ste

ae

e

al

ae

da

tri

False positives: none

ce

lid Fe

affi Gir

Mu

Eukaryote

rid

ini d

da e

Bacteria

atid

potam

nem

eridae

Hominidae

Hipposid

gylo

Gon

ba Fa

ba

ce

Eq

er ii

ae

ida e

yu

lph

m

Hippo

Na

Da s

De

ui

e ida ari ae Ot cid er c o ae ch id al On h p m Ny

prin

Archaea

bidae

etid

Cy

dae

ae

o Ph

ni coe

Superkingdom

Family


6 of 10

Se

Xantusiidae e Vesper tilionida dae Toxocari iridae Thyas ae evid Ther ae ontid aod Tetr dae e Sui a cid e nis he ida Sp ios ae e mn e ac So da lan leci ae o So id isc in e h m a or yli ncid i Sc

15

Sc

Sc

10

log.counts

5

0

hi

Sa

st

rc

oc

os

om

Sa ys atid lm a tid o ae e n Sa lica idae Rh ce ab dov ae irid Ra ae nid ae Pyx icep hali dae Pyth onid ae Pseud omon adace ae Propion ibacteri aceae Pomacanthida e Polypteridae

Acipenseridae Adrianichthyid ae Alligato ridae Amby stoma tidae Anati dae Ang Ant uillidae enn arii Ap dae Ar teryg thr ida o e lep As ti c Bl arid dae en id Bo niid ae Br vid ae am ae id ae

Result Report

ae ce e ria e ida d ol ch e kh rhin a r lid Bu allo me e C Ca nida ae Ca hinid ar rch ridae a C sto Ca idae Ceb ae ecid opith Cerc e a Cervid ae Channid e Clostridiacea

Polynemidae Poaceae

M ur or ax ida el e l a o Mo don cea lpa tid e a Mi cro diida e e hy Ma lid cro a e po did Lep orid ae ae Lep ism atid ae Leio gnath idae Kur tid ae Iguanid ae Hylidae Hyaenidae M

on

M

Bacteria Eukaryote Virus

FaOse positives: none

ae ce ia er ct e e ba ida u da ro te Eq teri En ae op m ce Et aba ae F lid Fe lidae via dae Ga ydi e oem Ge cida ieso Gob ae ingiid Golf ae matid ylone Gong idae Hexanch Holothuriidae Hominidae

Pipridae e Pipida idae trach a b o n a Phry id e sian ae Pha erid ae g lan tid n o Pha yz e om da dae r t ati i Pe lob nch e e P rhy nida o h o e nit ph ida Or m d Ny umi N

Coelacanthidae Comamonadaceae Corynebacte riaceae Cricetid ae Croco dylida Cylin e drole berid Cyn idae oce pha lida Cy e De prinid ae nd rob Dic atid rog ae Di Did loss elp ida ph e yll hid ae Em obo yd thri ida id ae e

Superkingdom

Family


lib00300-S4-L001-R1-001-resultprotocol.pdf

7 of 33

Vespertilionidae Ursidae dae Therevi ae dontid Tetrao idae nath Syng ae Suid ae ntid ae ma ace abo S tr occ e a loc nid phy rae ae e Sta y e h ac Sp da lan leci o So e isc ida m ur dae i ci in Sc

Se

Sc

Sc

10

ino

log.counts

0

st

os

om at id i d ce ae ae Rh rot ab ida do vir e Ra i nid dae a e Ra jida Pyx e icep hali dae Pyth onid ae Ptero podid ae Pseudo monada ceae Procaviidae Pomacanthidae

Rh

5

hi

Sa

lm

on

Acipenseridae Adrianichthyid ae Alligato ridae Anatid ae Angu illida e Apt er Are ygidae nav irid As ae car Ba idida e gri d ae Br Bovi da ac C Br hion e al a lo mi idae rh d in ae ch id ae

Result Report

ae id el e m idae ea a C an iac C cter e a ob torida rn Ca Cas idae b ae Ce cid the opi ae c r id ale Ce irog e a Che id ll de Cica e iacea id tr s Clo ceae a d a n o Comam Cordylidae

Corvidae Cricetidae Crocodylidae Cynoce phalida e Cypri nidae Deb aryo myc etac Delp eae De rma hinidae b act Dic e rac rog eae Ele Did lossid ae uth elph ida ero En e Em da te ro ydi ctyli da ba da e e ct er ia ce ae

Poaceae Pipridae

Lipotidae

Virus

False positives: - Arenaviridae (WF)

ae

ae

N ot ho br er a t od nch M er yc i m ida t at e Mu oph ste ida ida e Mu lida e e rid ae Mo rid Mo ae rax ella cea Mon e odo ntid ae Micr ohyli dae Micro cocca ceae Manida e Macropodidae m

Eukaryote

Iguanid

Lepidosirenid Leporidae

Ne

Bacteria

e da dae ui ri Eq te e op cea m Et aba ae F ae lid Fe ractid ae ph lliid mi He miscy e ida He nch a Hex inidae Hom e nida Hyae e Hylida

Pipidae e terida Physe e a id Phoc idae sian ae Pha erid ae g lan tid n o Pha e yz om obida r t Pe tol iidae c e ed idae Or rst n Oe pho dae i m id y N m Nu

Superkingdom

Family



7 of 23

in cid a so Sa m e lm Rh on atid od a i ob ac dae e Rh ter ino ac l op ea Rh hid e ino ae cer otid Rha ae bdo virid ae Ran idae Rajida e Pteropo didae Pomacanthida e his

to

ae id idae am nch r B hi e r da llo eli Ca am idae e C n cea Ca teria c a b e rno rida Ca Casto ae id b Ce ae ecid opith Cerc e a Cervid aleidae Cheirog e Clostridiacea

Polystomatidae

Cordylidae Cricetidae Crocodylidae Cynoce phalida e Cypri nidae Dasy po Delp didae hinid Dic ae rog los D Dip idelp sidae hid hy ae Dr llobo os t h En Em ophi riidae te ro ydi lidae ba da e ct er ia ce ae

Poaceae

Mu api da r Mu idae e llid ae Mo rid Mo ae rga nel lac Mon ea odo ntid e ae Micr ohyli dae Manid ae Lipotida e Leporidae

id

ac

el

Kurtidae

M

us

cic

ob

us t

M

M yc


False positives: - Arenaviridae:)

e da e ui a Eq ace e b a Fa elid e F alida e vi ida yd Ga m ae oe liid Ge iscyl e m rida He ide pos ae Hip inid Hom e nida Hyae e Hylida ae Hylobatid Hynobiidae Iguanidae

e Plasmodiida Pipridae e Pipida ae terid e s y Ph idae c o Ph e ae nid acea sia c Pha ococ t e ep ida str en dae e pto i ob a Pe n d O ho hiid mp anc e y a r N ob inid th c No Nar

te ria c ae eae

log.counts

e

0

Sc

Yersiniaceae

Vesper tilionida

5

Sc

Ursidae dae Tupaii tidae odon ae Tetra thid gna Syn dae ae Sui id oid ceae gyl a on cc Str co ae ylo scid ph ni e Sta he da e a Sp pari ce S na dae la i So ciur S

10

Acipenseridae Adrianichthyid ae Alligato ridae Anatid ae Angu illida e Aot idae Api a cea Ap e t Ar erygi en da e av A i s rid Ba c ae la ari Ba eno dida e pt th e Bo yerg rida i e vi da dae e

Result Report

Family



7 of 25

Vitaceae

e Vesper tilionida Ursidae tidae erma astod Thaum tidae odon Tetra dae Teii e lida Syl e ida idae Su d e loi a d e gy nti cea on ma a Str o e cc rab oco ae cea St l lid da hy ua na ap Sq mo o ng hi St

Sp

So m

10

So

yli

0

ni os

e ae da bi ricid e Ce loth ida a ec e ph pith da e C rco galei e o e C eir ida e Ch ricet ida C hal e c p e o a n id ss Cy oglo e Cyn a id in Cypr inidae Delph ssidae lo g Dicro Didelphidae

ac

Rajidae

Mustelidae

Ph

Pe t



ae e a id id yl ae H at b nid lo re Hy dosi dae e i ea pi or ac Le Lep er i ha sp lidae pto u Le Lim ceae lva Ma idae Man idae oveli ae Mes inace nosarc Metha idae Microhyl e Monodontida Muridae

Diphyllobothriidae Drosophilidae Emydidae Equidae Fabac eae Felid ae Gek kon Ge idae o He emyd ida mis e cyl He liid Hi xan ae c pp os hida Ho e ide Hy min rida e ae ida e ni da e

Pyxicephalidae e Pycnophyida didae Pteropo ae adace omon ae Pseud id tr acen ae Pom nthid aca m e a Po e c e Poa ida ect ron iidae u e Pl od sm ae Pla iprid idae P er e et ys cida h P o Ph

Ph as ia al an nid ro ge ae m rid Pa y ral zon ae ep t idi idae Ox d yu Or rid ae ect ae olo On bid cho ae cer cid Odo ae ben id Noto ae nem ourid ae Noca rdiace ae Nephilid ae Narcinidae

log.counts

5

lan

id ae orh eae ini Sc d iur Sc ida ae his tos e om Sar atid coc ae y Salm stidae onid a Rhin e olop hida e Rhino ceroti dae Rhabdo viridae Ranidae

Sc

Acipenseridae Adrianichthyid ae Apteryg idae Ascari didae Bovid ae Bra ssic Ca ace llor ae hin Ca Cam chida e mp e tob lidae Ca asidi a Ca C ni ce ae rn apr dae o o C bac idae as te r to rid iace ae ae

Result Report

Family



7 of 51

Yersiniaceae

Vitaceae ilionidae Vespert ae Ursid iidae Tupa e iida Tars ae pid Tal e ida idae Su e d loi ea gy ac eae on cc c o ae a Str ce toc cc rep loco teria St e hy ac ida ob c ng ala Sp

hi Sp

ap

St

So

10

Sc

his

Sa

0

na

tos

Poaceae

M

M

M

Cucurbitaceae Cynocephalidae Cyprinidae Dasypo didae Delph inidae Dide lphid Dip ae hyllo both Dra riida cun e cul Dro ida sop e E h En lep ilid a h te En rob antid e ac ae te ter ro c Eq occ iace a a ui da cea e e e


False positives: - Retroviridae:) - Paramyxoviridae 56

ae ce e ia in cea w Er aba dae ae ce F li ria Fe ae cte ce ba teria vo c e Fla oba ida s g Fu Gala e ida Glir idae avir adn idae Hep romy Hete dae sideri Hippo ae Hominid Hyaenidae Hyalellidae

Plasmodiidae Pipidae ridae Physete ae id c o Ph e nida ia s a Ph idae m o t ta ae Pen ace e rell a u e t irid Pas xov my idae e a r r Pa yu ida Ox cerc ae o id h nt c ae On todo iace r c e O iss Ne

us te ur lida o id Me e ra a xe tha lla e no Me ce s a tha a no rcina e co cc cea Ma a ce e nid ae a Ma lvac e Lep eae totric hiac ea Lepo ridae e Lemu ridae Lactoba cillacea e Indriidae Hylobatidae

log.counts

5

ae ce ra e ct ba ae lo d py ani idae m C tor e Ca s Ca viida Ca idae b ae Ce cid the opi c r e Ce ervida C e leida iroga Che e a d hilli Chinc iaceae Clostrid Cricetidae

la

iur cea ida e om e ati lm o d Rh ino nidae ae lop Rh h ida ino e cer oti Ret rovir dae id a e Pter opod idae Pseud omon adace ae Propion ibacteri aceae Polystomatida e

Sc

Actinomycetaceae Adrianichthyid ae Alcalige naceae Aotida e Apte rygid ae Asc arid idae B Ba acillac lae eae Ba nopt eri thy da e e Bo rgid ae Bu Bras vid sic ae rk h C old ace am er ae el iac e id ae ae

Result Report

Family


lib00648-S2-L001-R1-001-1M-resultprotocol.pdf

7 of 62

Vitaceae e Vesper tilionida aceae Ustilagin ae Ursid iidae ae Tupa atid som ae ano nid Tryp me y rah ae Tet siid e Tar ida ae Su cid e ea ala ac e Sp ae lan ida tid r a iu m so to

10.0

So

is

Sa

rc oc ys ti Rh lmo nid dae ino lop ae Rh ino hid ce Py rot ae xic ida ep e ha Pte lid rop Pse odi ae dae udo mon ada cea Poxv e irida e Poma canth idae Poacea e Plasmodiida e Sa

ae eae vid iac Bo lder ae lid ho rk me e Bu Ca nida e a C ida r sto e a Ca d i i Cav ae id b e C ae ecid opith Cerc e a id rv Ce aleidae Cheirog Chinchillidae

Pipidae

br an c ac hiid co e ria eae ae sp ce h Mu aere ae lla ste l ida cea Mu e e rid ae Ma nid a e Lipo tida e Lepo ridae Indriid ae Hylobatid ae Hyalellidae Hyaenidae

My

Ne

iss

ot N

No

Cleomaceae Comamonadaceae Cricetidae Culicida e Cyno cepha lidae Cypr inida Cyt e oph ag De Dasyp aceae ba o did ryo a De myce e lph tac D ea ini Di i d e d ph elp ae Dr yllo hida os bo e op thr ii hi lid dae ae


False positives: - Arenaviridae:) - Paramyxoviridae56 - Poxviridae 56

ae id si rd dae wa erii dae Ed im anti E e h ep ida e El Equ ida ce ina eae c Er a b e Fa ida Fel e gida ae Gala race bacte ae Geo matid ylone Gong yidae Heterom ae Hipposiderid Hominidae

Physeteridae Phocidae idae Phasian idae m to ta Pen ae virid o myx idae Para d o e op ida cter nch Or y rhy idae o h t i c cer Or n e cho nida e On be tida o d n O o ae tod onid e t Oc o da h di Oc mi u N

ho

0.0

oc

log.counts

2.5

st

5.0

Sc

h Sc

7.5

Adrianichthyidae Anatidae Aotidae Aplod ontiid ae Apte rygid ae Are nav irida As e Ath caridi dae eri n op Ba si Ba cill l ac dae Ba aen e r to opt ae Ba n er Bo thy ellac idae ea rn erg e av id a iri da e e

Result Report

Family



7 of 15

Sc

hi st os o oc ma Sa ti ys lm tid dae Ru on a mi ida e no co Rh cc e ino lop acea Rh h ida e ino e cer otid Ret ae rovir id a e Pter opod idae Pseud omon adace ae Prevote llaceae Polystomatida e Sa

rc

ae ra c ce Ma nida eae ae e lva c ea Le po e r Lem idae u rida Lac e hno spir ace ae Indrii dae Idiom arinac eae Hylobatid ae Hyalellidae ca

ur

oc

Hyaenidae

Ma

M

os

on om icr

icr

M

M


False positives: - Retroviridae:) - Herpesviridae56 - Paramyxoviridae 56

ae ce e ia in cea w Er aba dae ae ce F li Fe teria c e ba ida vo ae ag Fla Gal erace act ob eae Ge afniac e cea H ada mon e Halo dnavirida a Hep ae svirid Herpe yidae Heterom ae Hipposiderid Hominidae

Cynocephalidae Cyprinidae Dasypodidae Delphin idae Desu lfurom onada ceae Dide lphid Dip ae hyllo both riida Dip e Dro odida e sop Ed h ili wa rds dae E El imer iidae ep iid ae h Eq ant id ui da ae e

Plasmodiidae Pipidae ridae Physete ae id c o Ph e nida ia s a Ph idae m o t ta dae Pen viri ae yxo m hid a ync ae Par h r o d nith donti ae Or to nid ae o Oc t e ho ac Oc ialb dae i rt el Na ust M

id

0

Poaceae

po

log.counts

3

e da vii ae e a C bid cida Ce ithe p e rco vida ae id Ce Cer ale g o dae eir Ch inchilli e cea Ch ia strid ceae a Clo d a n amo Com ae Conid ae tid Crice eae Cyanothecac

oc

6

Vespertilionidae

9

Ursidae e Tupaiida dae Tarsii idae Talp ae Suid ae idid ae ylo e ong tac eae S tr yce c tom nada eae c ep Str mo eria t go hin obac dae i Sp e g c hin pala cea Sp S na dae la i So ciur S

12

Acidilobaceae Adrianichthyid ae Aotidae Aptery gidae Asc Bala arididae eno pter Bar idae to Ba nellac thy eae erg Bu Bo ida e rkh vid ae o l d Ca er iac m e C ea C an lida e as id e a to e rid ae

Result Report

Family



7 of 31

Vombatidae

10.0

Vitaceae ilionidae Vespert ae Ursid iidae Tupa e iida Tars ae pid Tal e ida idae e Su d loi acea e gy et cea e on yc a Str a tom occ ace eae rep yloc at ac St h sm teri ap la p St ac iro ob Sp ing h Sp

12.5

Sp al a lan cida a e Sc c e Sc his iurid ae ae tos om Sa rco a cys tida Sal tida e mo e n ida Rhin e olop h id Rhin ae ocer otida e Retro virida e Pteropo didae Pseudomona daceae Propionibacteriaceae So

5.0

0.0

m

O

Nu

tro

Ni


False positives: - Retroviridae :) - Paramyxoviridae56

ae ce e ba ae ea Fa lid iac ae Fe cter e iac a ob cter e a av a Fl ob gid s ae a Fu Gal avirid dn ae pa yid m He tero idae He ider pos e Hip inida Hom nidae Hyae dae Hyalelli Hylobatidae Indriidae

Cyanothecaceae Cynocephalidae Cyprinidae Dasypo didae Delph inidae Dide lphid Dip ae hyllo both Dra riida cun e c D En roso ulidae ter ph ilid ob ae ac te E Er quid riace i ae Er nac ae wi eid ni ac ae ea e

Polystomatidae Poaceae diidae Plasmo ae id ip P ae eterid e s y h P ida m to llos Phy dae i c Pho nidae ceae a a asi cocc Ph to idae e ep r a t m s to irid pto nta xov idae Pe Pe y c m cer ra ho Pa nc O

ct od on ti My sos idid dae ph a co ae e pla r Mu sma ace tac ae ste l i da eae Mu e rid Mo ae rax ella Met han cea oba cter e iace Man ae idae Malva ceae Leptotric hiaceae Leporidae

log.counts

2.5

e da ni dae Ca tori e s a Ca aviid e C ida ae b Ce thecid pi o e c a r Ce rvid Ce idae gale o ir dae Che li chil Chin ceae ia id tr Clos ae tid Crice ae Cucurbitace

Lactobacillaceae

7.5

Adrianichthyidae Anatidae Aotidae Aptery gidae Asca ridid ae Ba Bal cillace ae aen opt Ba erid thy ae e Bo rgida Br v e i d Bu as a e sic rk h ac C am C old ea e py am eria lo elid ce ae ba a e ct er ac ea e

Result Report

Family



7 of 37

10

0

ha

ae cid ae e ae ith ce op ella dace rc n a dis Se Ce atto mon ae tae r ce Ch ydo e a i id Inc lam str III. Ch Clo ily X m e a a s F Conid iale idae avir trid n s o r o dae Co Cl r o idii tosp p y r C ceae ophora Cyan ae ri cte ace Cycloba Cyprinidae Debaryomycetaceae Desulfovibrionaceae Eggerthellac eae Eimeriid ae Entero bacte riacea Ente e roco c Ent erom caceae ona dida Equ Ery e ida sip e Eu elotr ich ba Ex ace c ob teria ae as ce Fl a i F d e iac av ab ob ac ea e ac ea e te ria ce ae



e ea e ac er cea ct a ae ba or ce eo sp G iga era e G lom llida e y G ida Gr e ch on cea em era Ha bact ae ryid lico He teroph idae He amit Hex e inida Hom nidae Hyae ceae bactera Hymeno eae Hyphochytriac Kofleriaceae

ng ob ac te e Sp rosp ria ath or cea id i d So e lan iida ae Se e ac len e om a e on Sch ad isto som acea Sap a tida e role e gnia Sacc cea haro e myc etac Rumin eae ococc aceae Rikenella ceae Pycnococca ceae Prevotellaceae Porphyromonadaceae e Polyangiacea e a ce a Po e odiida Plasm ae adid n o e om cea a Plan t e yc tom ae c d i n r Pla avi e orn Pic cea hia idae eae c c Pi n ca ia as oc eae Ph ptoc c e a ea tre hil tos onip cac p pt oc Pe oc Pe pt e P Sp

Pa Pa ste en ure ib ll Ox aci ace Os yu llac ae cil rid ea a e On losp ira e ch ce oc erc ae Mu Mo rid idae nob ae lep h Met arid han o sarc aceae Meth inac anom eae assil iicoc Metha cace nobac ae teriac eae Leporid ae Lactobacillac eae Lachnospiraceae

log.counts

5

hi

Veillonellaceae Tubulanidae ceae onasca Trichom is didae Sed mona Tricho r tae ae nce tiace IV. I Tille mily s Fa rale e ida acte ridi erob e The e ana ida acea rmo Su The n rio dae i e vib d a i ini e ce cc ylo g ca cea Su ron e oc a St at ea oc c m pt re as eta St pl a iro ch Sp iro Sp

Sp

Acidaminococcaceae Aerococcacea e Agarica ceae Alicyc lobac illacea Apte e rygid ae Astr ovir id Ba ae Ba cillac eae cte Ba roida lan cea Ca t e m Bo idiida vid py e lob ae C ar a C no an cte ba ida rac ea ct e e er ia ce ae

Result Report

Family

Figure 3: Graphical representation of read counts for the 100 most abundant families. In order to represent all detected superkingdoms, the displayed families are selected from the complete result applying the HighestAverages algorithm from the R package SciencesPo. Families are sorted alphabetically, color-coded according to superkingdoms. Note that the graph is in log-scale. lib00721-S11-L001-R1-001-resultprotocol.pdf

6 of 80

Vitaceae ae Veillonellace e onadida Trichom aceae ssiosir Thala ae Suid idae loid ngy ae Stro ylid eae ong ac Str occ ceae toc ep eta e Str a ha c d iro dii ae ae Sp thi e e a c c e a Sp ana ad tida l a on So m m no oso le st hi Sc

Se

0

Sa

rc

pr

ole

ur

nc

Mo n

M

M

O

ae e id de cida b e ry the ea Ca opi llac e ae rc n e e e C atto diac cea y h da C lam na ae Ch domo sidace y p o i m d i la Ch ococc iaceae Sedis id ro r tae Ch Clostr Ince I. II yX amil e les F riacea tridia oriobacte s lo C C e a d ri vi Corona iidae Cryptosporid Cucurbitaceae Cyprinidae Delphinidae Diphyllo bothriid ae Dracu nculid ae Dros ophil Dro idae so Egg phyllac eae e En r the te ll En robac aceae ter ter oc iac o ea Er e ys Equ ccac ea ida Eu ipel e e o ba tri c ct er hac ia ce eae ae


False positives: - Asfarviridae56

ae ae ce ce ba eria eae Fa act riac e ea ob acte av rac Fl sob ige eae e Fu emin rac e cea G da om a n Gl mo e nio ida Go Gryll ae chid mon eae Hae terac obac e Helic mitida Hexa ae Hominid Hyaenidae Hydridae

ho c

log.counts

5

Sa

oc ys ti ch g nia dae aro Ru ce my mi ae ce no t co Py cc ace cno coc ace ae Pre cac ae vot ea ella Por e phy c eae rom ona dac Poac eae eae Plasm odiida e Planoco ccaceae Planctomyce taceae Picornaviridae Picomonadidae e Picobirnavirida ae d ri te e Phys llidae Phiale e nida e ia cea Phas cca o c o t e p a e r e tost ilac Pep iph ceae ae ton a c ce c Pep da co e na pto Pe mo acea o s ill e hy c p a ra ae ib da Pa en yuri ace r Pa x i O sp illo sc O

Sa c

er id cid uc or ae ae ob Me lep ace a tha ha Me no ri e tha s arc dace no ma i a Me ssi nace e tha nob liicoc ae c a a cte Mas riac ceae tiga ea moe bida e Malv e acea e Lepori dae Lactoba cillacea e Lachnospira ceae Hyphochytriaceae

10

Acidaminococcaceae Aeromonada ceae Apidae Apter ygida e Asfa rvirid ae Bac Bac illacea e Ba teroid ace lae no ae B p te Bif ala ido ntid ridae iid ba Ca ae c te B m py ovi riac da ea lo e C bac e an t id era ae ce ae

Result Report

Family


6 of 111

Sp

Yersiniaceae

10.0

Vitaceae e rnithida Threskio aceae ococc Therm ae Suid ae rnid e Stu ida ion e uth ida S tr oid ceae gyl on eta eae yc Str c e tom cca cea o ep a e Str toc occ cea ae e c rep a St ol dac ylo b h a io ap id on St or om Sp ng hi Sp

12.5

hi ng

e ea ac er ae a h r id i e isp ld liciv ida e Ca Ca har t nida t o Ca pog ae o cid rat the Ce rcopi a d e Ce iconii eae C itac vicip eae Cla c tridia Clos idae b m lu Co ae onadace Comam e ea Cordycipitac

ob ac he nis teria So lan cid ce ae a Sc ac olo ea e Sc pa e his c id tos om ae Sal atid mo Sac n ida ae cha e rom yce Rho tac doba ctera eae ceae Rhizo biace ae Rhabdo viridae Rhabditidae Retroviridae Sp

7.5

5.0

0.0

Op h

Corvidae Corynebacteriaceae Cricetidae Cucurb itaceae Cypri nidae Diph yllob othrii Dra dae cun culi Dro dae sop En te hi En robac lidae ter ter oc iac ea Es occ e a t Fa rildid ceae ae Fa bac lco ea e ni da e


False positives: - Retroviridae:) - Caliciviridae56 - Rhabdoviridae56

ae ce ria e te a id ac ae ob mic av For gillid Fl in iidae e Fr v ida Ga ch on e em ida Ha omin eae H eac ocr eae Hyp spirac hno ae lace Lac bacil Lacto dae Lepori ziaceae Malasse Malvaceae Methanosarcinaceae

Rajidae daceae Pseudomona ae acteriace ib n io p Pro aceae ll te o Prev e atida stom Poly e a ce Poa ae diid mo s a ae Pl d i r e e Pip ea cea ac a e Pin cteri e ea ida ac ba llo sian occ y a Ph oc Ph pt tre s o pt Pe

Pa st eu Pa rell ioc rid ac ea On ordy ae e ch c oc ipit erc ac Nu ea m id No car idida ae e dio e i Ne isse dace ae riac Nec e triac ae e a Mus cicap e idae Murid ae Moraxe llaceae Micrococcac eae Microbacteriaceae

log.counts

2.5

Accipitridae Actinomyceta ceae Alcalige naceae Amary llidace ae Anati dae Apt e rygid Arh ae yn As chob atid pe ae Ba rgillac cill ea e a Br ad Bov ceae id y Bu Bra rhiz ae rk ssi obia ho ca ce c ld er eae ae ia ce ae

Result Report

Family



7 of 65

Yersiniaceae e Vesper tilionida thyidae Trachich ae dontid Tetrao ae Suid ae ace e occ cea ptoc isca eae Stre nod ac pha occ Ste loc dae phy ci ae Sta ala d i s e Sp o a i mn neid e e a So a oli id rc atid nu pe ni som to is

Si

Si

h Sc

Sa

10 Rh

od o

on

ys

tid

ae

Pipidae

N

la

op

M yc

ae rid to ae s ae d Ca ebi ecid C ith p e rco vida e Ce Cer ida n rylo eidae Ce gal e eiro adacea Ch mon o e a o r e ac Ch icipit Clav ae idiace tr s lo C ae Clupeid ceae Comamonada Conidae Cordycipitaceae Corynebacte riaceae Cricetid ae Cypri nidae Desid ae Did elph idae Din i Ec hin dorida os e Em toma En tida te y e En rob dida e a te ro cter co i a ce Eq c a ui cac da ea e e e


False positives: - 5etroviridae:) - Caliciviridae56 - Flaviviridae 56

e ea ac e a en gl ace Eu ab idae F el ae F irid ae viv id Fla ntom e jie lida Fu via eae Ga rac ige e min cea Ge hylla e eop Glo acea erell Glom cidae oscole Gloss idae phthalm Gymno Hexanchidae Hominidae

Philasteridae iridae Phenuiv nidae ia s a Ph idae nger Phala ae id t ba ae Pelo bid o l o ae Ors bid e o l o t da c e rci Or ce ae e ho c i dd da On mi ouri dae i Nu m hi ne anc to r o N ob th No

ec tri a sm cea M at e ur Mo a rax idae ce ae ell Mo a no do ceae Mic nti d roh Mic ylid ae rob ae act eria Mala ce sse ziac ae eae Lepo ridae Lachn ospira ceae Intraspo rangiace ae Iguanidae Hylobatidae

log.counts

0

oc

lm

ida ba c e tro terac vir ida eae Ra e Pse nid ae udo mo Pro n a pion dac ibac e teria ae Prev cea otell e acea e Poma canth idae Poeciliid ae Poaceae Plasmodiidae

Re

5

rc

Sa

Adrianichthyidae Agonidae Alligato ridae Aotida e Apte r Ar th ygidae role ptid Bab ae esi ida Bo e v Br ac idae h Bu Bruc ionid e ae rk ho llac C Cal lder eae i al lo civi iace rh r in idae ae ch id ae

Result Report

Family


6 of 17

Xanthomonadaceae Vitaceae ilionidae Vespert ae Ursid tidae oma anos ae Tryp ontid aod Tetr ae siid Tar e ida ae e Su id ell acea e loc a et Sty yc ace ae tom cc ce rep toco cca e St a p co lo ace hy n ap ola S

St

7.5

5.0

0.0

Si

lu rid ae i d to a Sa som e lm ati Rh on od ida dae ob e act Rh era ino cea lop e hid Rhin ae oce r o tida Rha e coph orida e Rhab dovirid ae Ranidae Pyxicephalid ae Pycnophyidae

Sc

his

Sc

iur

Manidae

ae id or ae t s e d Ca ebi nida e C ole ida r c t n he Ce opit ae rc id ae Ce Cerv eid gal ae o r i e iace r e t Ch c eba n y e r a Co etid Cric eae rbitac Cucu e a h p lida Cynoce Cyprinidae

Dasypodidae Delphinidae Dicroglossida e Didelph idae Diphy lloboth riidae Emy dida Ent e erob acte riac Eq eae Eri uidae na Fa ceida ba e Ha Fe ceae lid l o ae He ar m cul isc ac e yll iid ae ae


False positives: - Arenaviridae :)

ae id ae d ch i an ider e ex H pos inida e p Hi Hom nida ae e Hy lida ae Hy atid lob laceae l Hy aci tob idae iren Lac idos dae Lep mati Lepis dae Lepori e Lipotida ae Malasseziace Malvaceae

Pteropodidae daceae Pseudomona ae cteriace a ib n io Prop ceae a ll te Prevo idae ygon motr ae id Pota h nt aca ae m i o P at d om yst e a e Pol e ac da Po dii mo ae s a id ae Pl Pip terid e ae ys id Ph hoc P

Ph Pe asia l ec nid Or an a ec id e t o Od a l ob obid e en a Nu ida e m Mu idida e e ste l Mu idae rida Mor e axe llac eae Mon odon tidae Mitsu kurinid ae Microhyl idae Micrococcac eae

log.counts

2.5

re

St

10.0

Aclididae Actinomyceta ceae Adrianic hthyidae Amby stoma tidae Aotid ae Apt eryg idae Are nav irid A ae Ba scar id lae no idae p te Ca Bo llo vid ridae Ca rhin ae ch m i C an elid dae id ae ae

Result Report

Family


6 of 26

Xanthomonadaceae Vitaceae e rnithida Threskio pidae Thrau ae Sulid ae Suid ae rnid ae Stu id ion dae i uth d Str loi gy e eae on da c ae Str igi ta r e e St yc ac ae om occ ace pt t c re lo ma St hy s ap pla iro Sp

St

10

log.counts

5

0

Sp h So enis ci lan ac dae Sc ea op Sc i da e olo e Sc pa his c i d tos om ae Sar atid coc ae y Salm stidae onid a e Rivu laria ceae Rhab ditida e Retroviri dae Pteroclidae Psittacidae

Accipitridae Adrianichthyid ae Alcalige naceae Alliga torida e Anati dae Anh imid ae Apo did Ap ae ter Ar ygida e Ba deid a cil lac e B ea Bu Bra ovi e rk ssi dae ho ca ce ld er ae ia ce ae

Result Report

ae ce ra e ct ae ba lgid lo u py rim idae m p m Ca Ca aria tidae C har ae t cid a C ithe op ae c d i r i Ce aradr eae h C gac pha o in e Chit niida Cico ae id n io C iaceae Clostrid Columbidae

Procellariidae Poeciliidae

ba ci lla id cea l Op at ae e o ist r i a ho On co cea ch oc mid e ae er Nu mid cida e i No toth dae eni Mus ida e oph agid Mus cicap ae idae Murid ae Mimivirid ae Methanosarci naceae

ni

ur

xy

Meropidae

Os

cil

Pa e

O

Archaea Bacteria Eukaryote Virus

False positives: - Retroviridae:)

ae id e ld tri cea Es ba idae ae Fa con iace l r Fa acte ae b lid vo gil Fla Frin idae vi eae Ga rac cte oba Ge idae Gru idae esvir Herp idae Homin midae Leptoso Malvaceae Megapodiidae

e Poacea dae ii d o Plasm a id e Pipr ae Pipid ae nid dae sia i Pha corac e cro ntida a l a ae tho Ph ae anid ae Ph c id e l ell Pe e er ss rida a P Pa

Comamonadaceae Corvidae Cracidae Cucurb itaceae Cyno gloss idae Cypr inida Dicr e oglo ssid D ae Dip idelph ida hy e Dr llobot os h r iida Eim ophil e id En Em eriid ae te ae ro ydi ba da e ct er ia ce ae

Superkingdom

Family


lib01003.IonXpress-001.R-2014-05-10-10-58-40-user-SN2-6-lauf006.2015-02-27-resultprotocol.pdf

7 of 40

Vitaceae

ap

St

Tytonidae e rnithida Threskio e Sulida ae Suid ae rnid e Stu ida ion e uth ida Str oid gyl e e on da cea Str igi e eta ea Str yc ac tom occ ae id e rep yloc St sc h ni cea he a Sp lan So

Accipitridae Acholeplasma taceae Adrianic hthyidae Alliga torida e Anati dae Apt eryg idae Ard eid As ae car idid Ba ae b Ba esii da cil e lac B ea Bu Bra ovi e rk ssi dae ho ca ce ld er ae ia ce ae

Result Report

his

log.counts

0

ol

ae ce ra e ct ba ae e lo d py ani lgida m C u ir m idae Ca p Ca ariam dae C r ti tha dae Ca itheci p o e c r iida Ce radr Cha ceae a g a oph Chitin niidae Cico e Cionida e Clostridiacea

op

to so

ac

id ae ida ida ula e e ria Rh ce ab a e diti da Pte e roc lida Psit e tacid Pseu ae dom onad acea Proce e llariid ae Polysto matidae Poeciliidae

Riv

5

Sc

Sc

10

Sa

lm

on

m

at

Poaceae Plasmodiidae Pipridae



Methanobacteriaceae

O O sc xyu ri Op illa to dae is r On thoc iac ch o m ea e o Nu cerc idae mi i did dae No a sto My cac e cop ea las e m ata Mus ce oph agid ae Mus cicap ae idae Murid ae Morgan ellaceae Mimiviridae Methanosarcinaceae

Archaea

ae ae id ce on ia lc ter c ae Fa a d illi ob av ring iidae ae Fl F v ce Ga era t ac e ob da e Ge Grui ida svir rpe ae He inid Hom idae som Lepto lidae Lingu ae Malvace Meropidae

Pipidae e terida nicop Phoe e a id ian ae Phas racid roco e c a la e d i Pha ont cea eth cca o c Pha to ae ep str nid ca lidae pto e l Pe l Pe ere ae ss e a rid da P Pa oni ili p Pa

Columbidae Corvidae Cracidae Cucurb itaceae Cypri nidae Dicro glos Dip sida hyllo e both Dra riida cun e cul Dro ida sop e h E En ilid ae ter myd ida ob Es acte e t r iac Fa rild ea ba ida e e ce ae

Superkingdom

Family



7 of 48

St

Vitaceae Vespidae pfiidae Vahlkam atidae nosom ae Trypa nadid omo e Trich rida oca e Tox ida ridi The ziidae e a ela nid Th me ae hy tra itid r Te e ph ae Te ida did i e Su ylo ida ng gyl ro n ro St

12.5

10.0

St re

Sp

7.5

5.0

0.0

oc

ch

oc c

ac ea ta la Sip nac cea e e e ho Se a e v len om irida e on Scl ad ero ace tini ae Sch a c e isto ae som a tida Salic e acea Sacch e aromy cetac eae Rumino coccace ae Rosaceae Rikenellaceae

So

ae

Pa e

Drosophilidae Echinostomati dae Eggerth ellaceae Eimeri idae Erys ipelo trich Eub acea acte e riac F eae Fib abac e r o a e b Fla vo acter ba ace cte ae F o r G G lom rmic iace ae on ida e gy re e ll lo ne ace ae m at id ae



ae id ae ch llid ae on m one atid ae ae H ligm som lace o iel He m lig rich dae He rpot ini om ae He H d ae dri Hy epidid l e a eno ace Hym spir hno ae lace Lac bacil Lacto ae Lottiid ae Malvace riaceae Methanobacte Methanomassiliicoccaceae

Rhabditidae daceae Pseudomona llaceae te vo re P eae nadac yromo dae Porph ti a stom Poly idae ovir Pod eae ceae c Poa tida cys idae i mo u e od Pn ae e sm ae Pla ipid tace cea a e P yc occ fom toc f a p Ph stre o pt e P

e ea e ac ida c i s ec ae as pith r e B co ac eae r idi c Ce lostr cca ae C toco diid r oi yp ae Cr ptosp e c a Cry curbit e Cu cida Culi ae lionid u c r Cu ae id n ri p Cy ceae omyceta Debary riidae Diphylloboth Dracunculidae

Pa r Pa am e pi lio ciid nib a ni d a ae e ci Ox yu llac Os e ri cill os dae ae p Or chi irace da Op ae c hry osc eae ole Onc cid hoc ercid ae Nym ae phali dae Nectr iacea e Myovirid ae Muridae Methanosarcinaceae

log.counts

2.5

pt

iro

Adrianichthyidae Anisakidae Aphidid ae Apida e Apte rygid ae Asc Asp aridida e e Ato rgillac eae p Ba obiac e c B Bi acte illace ae fid ob roid ae a a Bo cte ceae Br ria v i ce ac da ae on e id ae

Result Report

Family

Figure 3: Graphical representation of read counts for the 100 most abundant families. In order to represent all detected superkingdoms, the displayed families are selected from the complete result applying the HighestAverages algorithm from the R package SciencesPo. Families are sorted alphabetically, color-coded according to superkingdoms. Note that the graph is in log-scale. lib01005.IonXpress-003.R-2014-05-11-14-07-43-user-SN2-14-lauf014.2015-04-23-resultprotocol.pdf

6 of 180

Sp

Zamiaceae Vitaceae llaceae Veillone nidae e Tubula acea nasc e omo dida Trich ona dae hom eni Tric ym rah e e Tet ida a Su did ae loi ce ae gy ca on oc tace e Str a toc ea ae rep lasm tac ce St p ria ae iro ch cte iro ba o ng hi

12.5

5.0

0.0

So

xy

sp

My

Or

ch

O

illo

sc O

ae

ae ce da eae a n c to on is at m era ed Ch ydo glom ae eS e m o r ta c a e e a l i c d Ch aroi strid III. In Cl Clo ily X m ae Fa nid les Co ae idia virid r a t s on ceae Cor Clo a h p mor Cryo iidae d spori Crypto ceae a ri e ct a Cyclob Cyprinidae Debaryomycetaceae

ce

la el

Dermateaceae Desulfovibrio naceae Dinobry aceae Endo gonac eae Ente Ery rococcac sipe eae Eub lotrich a Ex acteria ceae ob cea as e Fla Fab idiace ac ae vo ea ba e c G Form teri ig as ici acea po dae e ra ce ae



e ea ae e ac er ac m lac e lo e G yau llida ida on Gry ch ae G on race em te e ac Ha ida b y r lico ph He etero ae itid H am Hex inidae Hom teraceae ac enob e Hym iacea ochytr Hyph didae homona Hypotric ceae Lachnospira Lactobacillaceae

Plasmodiidae idae Planomonad ccaceae co o n la P ae etace e tomyc Planc acea id s y hr ae uioc g id in ir P av birn e Pico cea hia e c i P ida ae e n a e asi tac cea e Ph a c my cocc ae o f e af pto cac ae Ph e r t c ce tos oco illa p pt bac Pe i Pe en Pa

ur id ae i r ac co i d a e s p c My ha ea ae co pla erel e lac sm e a Mu sci tace ae da ae e Mu ri Muc dae orac Mon eae oble phar Metha idac eae noma ssiliic occac Methan eae obacteri aceae Mastigamoeb idae Leporidae

log.counts

2.5

Sp

at hi d len lana iida om ce e Sc a on his ad e t Sa cch osom ace a aro ati da e my Ru e c min oco etace cca ae Rike ce nell ace ae Pycn ae ococ cace ae Prevo tellac eae Porphyr omonad aceae Polystomatida e Poaceae

Se

7.5

Sp

Sp

10.0

Acaulosporaceae Acidaminoco ccaceae Apiacea e Apter ygida e Bacil lace Bac ae tero id ace Bal ae ant idiid Ba ae ng iac B ea Br e ac ovid ae Ca Bur hys m kho pira ce C pyl lde a er ob r co ac iace e pi th tera ae ec ce id ae ae

Result Report

Family


6 of 103

Veillonellaceae

idae dis Trichomonad rtae Se III. Ince s Family acterale anaerob ridae Thermo Temo ae Suid eae nac ibrio ae ciniv idid Suc ylo e ong ida ae Str gyl e on ac e c Str oc ea toc sac ae e ep ce ea mu Str a t c oro ia ae Sp och cter e ir a da ob idii Sp ng ath Sp hi

Sp

0

len

id

ur

N ec

Me

tha

Mo

M

uc

M

e da ae ni id a ae C bde cid ceae y r he Ca opit nada o rc e Ce ydom acea it edis lam vicip ceae h eS C Cla tridia er ta c n s I III. Clo ily X ae Fam itace les ip c ia y rd rid e t o s C riacea Clo bacte Corio e a d ri vi Corona iidae Cryptosporid Cyanophoraceae Cyclobacteriaceae Cyprinidae Dermog lyphidae Desu lf Desu ovibrionace ae lfuro mon adac D eae Dip ileptida hy e Dro llobo thri sop i d ae En Eim hyllac ea t e e En erob riida a e Er te ys rom cter iac ip el ona ea ot ric dida e ha e ce ae


False positives: - Asfarviridae56 - Orthomyxoviridae56

ae ce ia e e er ea ct ea ba bac riac ae Eu Fa cte ce a a ob cter e av a da Fl eob ae ylli G hid Gr c on em nidae Ha i e c i a l He race acte ae cob riace Heli bacte e Helio mitida Hexa ae Hominid raceae Hymenobacte Hypocreaceae

Poaceae e Plasmodiida e a ce ia Pich nidae Phasia eae tylac e odac cea cca Phae o c o t e p a e r ace tost occ ceae Pep toc illa Pep c iba dae e en ea e uri Pa y ac a Ox pir irid eae s v lo xo igac cil s y t O om as th llim Or a oc Ne

tri ac

log.counts

5

So la n om ac Sa on eae rco a d Sa pro cyst ace Sa ida ae leg cch aro niac e ea my Ru e c min oco etace cca ae Rike ce nell ace ae Pycn ae ococ cace Pseud ae omon adace ae Prolixib acterace ae Prevotellace ae Porphyromonadaceae Se

ea ae e o rax race a e no ma llac e Me ea ss tha i l iico e no ba Ma cte ccac stig r ea ia am oeb ceae e Malv i ace dae ae Mala ssez iace ae Lepori dae Lactoba cillacea e Lachnospira ceae Hypotrichomonadidae

10

Accipitridae Acidaminoco ccaceae Anatida e Apter ygida e Asca ridid ae Asfa rv Bab iridae esi ida Ba e ci Ba ct llace Ba eroid ae ac lan e C Ca Bo tidiid ae am m v a i py pan dae e u lo ba lar iid ct a er ac e ea e

Result Report

Family


6 of 88

Yersiniaceae

Vitaceae e Viperida ae Vespid ceae nella ae Veillo atid e som tida ano ma Tryp ram ae hog rid Tric oca ae d Tox dii eri iidae e Th z da ela eni Th m ae hy id dae r it i tra Te ph din e Te hr nt Te

15

Te n

10

eb rio n ida ida e g ylo e Sta idi ph d y a lin e So id lan ace ae Sim ae uliid ae Silp hida e Scle rotin iace ae Sciari dae Schisto somatid ae Salicaceae Saccharomycopsidaceae

St

ron

log.counts

5

0

Adrianichthyidae Agaonidae Aleyrod idae Amoe bophil aceae Aphid idae Ap Apt idae ery gid Ar ae As acea e ca Bo rid id mb yc ae B ida B ov e Br rac ida e as on id si a ca ce e ae

Result Report

ae eae c tid es tera r p e c Bu loba yiida py dom ae m i d e Ca Cec ephi ida C byc dae m i n ra o e C pog e rato hecida Ce it p o c r e ida Ce obor Cha midae o n o ir Ch melidae Chryso Cimicidae

Su

Clostridiaceae Cucurbitaceae Culicidae Curculio nidae Cypri nidae Deb aryo myc Dip etac hyllo eae both Dra riida cun e cul Dro ida En e ter soph ilid ob ae ac Er ter wi iac n Fl ea av Fab iace e ae ob ac ac ea e te ria ce ae

Saccharomycetaceae Rhabditidae

Muridae

Pe n Pe tato Pa m di cu id ra a m yx lida e Pa pil ovir e ion i da Ox yu idae e r Or chi idae da On cea cho e c Nym ercida e pha lida e Noctu idae Nitidu lidae Mycopla smatace ae Muscidae


False positives: - Paramyxoviridae56 - Poxviridae 56

ae dae id i ic at ae rm em e Fo lon dac ae tid gy ona ma on G alom oso ae m H e nid i a lig d He Hom lepidi no ae me reace Hy c o Hyp oniidae Lim e acea Malv e chilida Mega aceae obacteri Methan naceae Methanosarci Mimiviridae

e Pyralida dae li a m Ptero ae d ho id e Psyc a ace ll e t vo Pre dae e i r i v tida Pox ma sto e y l ea ae Po ac Po odiid sm ae e Pla ipid ida P n ia as Ph

Superkingdom

Family



7 of 247

Vitaceae

Viperidae e Vespida eae nellac Veillo tidae mma ogra ae Trich ricid e Tor t ida ridi ae The ziid ela hidae Th c e ny tra ritida ae Te ph nid e Te redi ida n th io ae br id in ne Te ach T n Te

10

Su ro i ng dae ylo ap hy idid li So a lan nida e Sim acea e e uli Silp idae hid ae Scle rotin iace Schis ae toso mati dae Scato psida e Sarcocy stidae Salicaceae Saccharomycetaceae St

St

0

Cladosporiaceae Clostridiaceae

No ali d c Nit tuida ae idu e cop lid a las ma e t Mu scid acea e ae Mur idae Mim ivirid ae Micro cocca ceae Methan osarcin aceae Methanobacte riaceae Megachilidae

id

rc

xy ur

My

Ny

m

ph

ce

O

ho

nc O

Conidae Cucurb itaceae Culicid ae Curc ulion idae Cyp rinid Dip ae hyl Dra loboth riid cun ae cul En Dros ida o ter ob philid e ac Er ter ae Er win iac iace yt hr e ae ae ae id ae


False positives: - Paramyxoviridae 56

ae eae c ce ba eria Fa act dae ae i tid ob mic r av ma ae Fl Fo ne ce ylo da na ng ae Go lomo aniid ae Ha rrim atid Ha osom ligm inidae He ae Hom irace nosp Lach ceae bacilla Lacto ae Limoniid Limulidae Malvaceae

Rhabditidae Pyralidae alidae Pterom e llacea te Prevo dae ti a stom Poly e cea e Poa ida d o i sm e Pla a id e Pip mida o tat lidae ae n u id Pe dic vir Pe yxo dae m oni ra li Pa api P

ae ce ica idae eae s st as ac Br upre cter ae B oba iid l my py o m d e i Ca Cec phida e ida Ce byc ae ram id e n C go opo t a ae r Ce ecid opith Cerc e a d bori Chao ae id m o n Chiro ae Chrysomelid

id ae ae

log.counts

5

Adrianichthyidae Agaonidae Albugin aceae Aleyro didae Amo ebop hilac eae Aph idid ae Api dae Ap te As rygid ca ae rid Bi i Bo bion dae ida m e B by Br ov cida ac ida e on e id ae

Result Report

Family



7 of 107

St

Zodariidae Xeniidae teriidae Trochan atidae gramm Tricho ae inellid ae Trich hid nyc eno e Tria rida asi ae Thy nid rio eb ae ae Ten llid tace e Sy a e yc atid ae tom m ce rep erne cca ae St o ein loc linid y St y h ap aph St

12.5

Sp

10.0

hi

Sc hiz os

7.5

5.0

0.0

ob

yto

ac

te

di riac aro dae eae m tos om ycet Sc a ara ati da cea ba e e Sal ida e mo nid e ae Rom aleid Rho ae palo nem atida Rhizo e biace ae Rhabditi dae Retroviridae Pseudonocardiaceae

Sc

ac

his

ch

ag

af

Monocelididae

Pa n

Pe l

Ph

ae ce e ia a r te ce e ac eria b i ea d ev ol iac Br rkh ann ae Bu urm barid e B am da C inii vol idae a C p rco ae Ce onid it h ae C oridid mod e c Chro a d ea mona Coma e a lid a d Cory e Crisuliporida

Cucumariidae Cyprididae Cyprinidae Dermab acterace ae Dinob ryace ae Dros op Ent erob hilidae ac Ero teriace ae tyli Fla Faba dae vo c e ae b Fr an acte Ge cise riace l ae l o a G plan cea e ry llid idae ae


False positives: - Retroviridae :)

ae id ar ae d ac al lioti ae H Ha brid ae He rid ste idae e Hi n cea mi eta Ho ha c no e me odida Hy Ix ae ulid Lim iidae taen e Lino tacea trema Lophio ae opediace Merism eae Micrococcac Migidae

Pseudomonadaceae ae Prorhynchid ae acteriace ib n io p Pro idae m o id Prod idae yllod toph e Proc a e ce tida e Poa a rma ode ellace m u r e ae Pne a h p d s ii od ae cto d Ple sm oli e Pla c i c da Pis olci idae h n P ia as Ph

fo m yc et ac i i Pa nari dae eae ac mp e h Or ag ae so lob idae Op eco idae eli Oo nop dae i d ae Num idid ae Noca rdioid acea e Neog oveid ae Neodiplo stomida e Nectriaceae

log.counts

2.5

ng

Sc

Acrididae Alvinellidae Amauro biidae Anap lasma tacea e A Anc natidae ylod isco Ap idid ae Ap helinid hro ae Ap phor Ap seud idae ida ter A rg ygid e A As sco asid ae ae pe ide rg ac illa ea ce e ae

Result Report

Family



6 of 31

bo

gli

ae e id th ida Bu bar ae m niid a e C po da Ca copi r ae Ce tonid i dae Ch doridi mo o e r Ch nida Cio ae dalid y r o C ae liporid Crisu ae id ri a m Cucu Cyprididae

el id ae a e so i d sp ac ha Ne e era ae og Ne ov ce od ae iplo eida e sto Ne mi me siid dae ae Nec triac eae Myc obac teria ceae Murid ae Munidid ae Milleporidae

Pseudomonadaceae lidae Proteocepha chidae Prorhyn ae Poace tidae erma eae d o m c u e ll n P re a hae tosp dae c i l le o P e cic ida Pis sei yto idae e h P ea olc ac Ph ycet ae d om agi ae h aff Ph amp obid l P o rs O

pe

op

O

on

O

ca r

Migidae

Ni tro

Membracidae

No


False positives: - Retroviridae :)

ae e rid yida eb H roph ae ae te erid tace e He Hist ha c e no da me xodi e I Hy ida can ae Le ulid e Lim iida taen Lino ae riace e Liste tacea trema Lophio e Lucinida Mantidae

Cyprinidae Dictynidae Diphylloboth riidae Diplurid ae Droso philid ae Faba ceae For m icid Fra ae nc Glo isellac eae me rac Go ea e H mp Ha alac hida e ar lic ida Ha ho e n pl op driid or ae id ae

dio

0

Sp

io ni da e n ch ida Sc aro his my e tos ce om Sc tac ara ati ea d ba Sar eid ae e coc ae yst ida Salm e onid ae Rom aleid ae Rhop alone matid ae Rhabditi dae Retroviridae Pseudonocardiaceae

ac

Si

co

log.counts

5

Sc hiz os

id

10

Zodariidae Xeniidae ae Uloborid e chida Tubilu ae torid lorap idae Trog mat ram ae hog hid Tric nyc e eno a irid e Tria yas e a d Th ea dii tac ae eri ce Th d i e y at ea tom m c rep erne cca e a St o ein loc inid y St yl h ap aph St St

15

Acanthopidae Acrididae Adrianic hthyidae Agele nidae Ama urob Ana iidae plas mat Aph ace ae e l Ap hro inidae Ap phor ter ida yg e A ida As rga e s c i Br Asp oide dae ev er a ib gill cea ac a e te cea ria e ce ae

Result Report

Family



7 of 94

hin

Sp

Yersiniaceae aceae Xanthomonad ceae Vibriona idae r tilion Vespe ae Ursid ae ontid aod Tetr ae siid Tar e ida idae e Su d ea e loi c gy ca ea on oc riac Str loc e cte hy ap ea ae ba St go nac ace l la el So an ew

15

Sh

0

ae ae id d am nchi r B hi ae or elid l l Ca am dae e i C n cea Ca teria c a b e rno rida Ca Casto ae id b Ce ae ecid opith Cerc e a Cervid aleidae Cheirog ceae Comamonada

Psychodidae



e ea eae c ac ni ada ae af H on erid m d lo osi idae p n Ha i p ae Hi om H nid ae idae t Hy a lob dae Hy luri Icta ae er tid Lac ridae Lepo dae Lipoti ae ematace Lophiotr Malvaceae Manidae

Cricetidae Cynocephalidae Cyprinidae Dasypo didae Delph inidae Emy dida Ent e ero Ent bacter iace ero ae coc cac Eq eae uid Er ae wi Fa niace ba ae Fl av Fe cea e ob lid ac ae te ria ce ae

Pseudomonadaceae Poaceae dae diophori sm Pla o dae ii d o Plasm ae cace e ococ ea c Plan ia tts icke e ir a c lid Pis cico e Pis a id e Pip ida ter se dae y i e Ph oc ae da Ph iani ace i s r a e Ph act b to c Pe

O O nc tari id h Od oce ae No o r tho ben cida bra ida e Ne e od nc ipl h o sto iida Ne e iss m i eri ace dae Mu ae ste lida e Mur idae Morid ae Morax ellace ae Monodo ntidae Migidae

log.counts

5

Sc i Sc urid a i n his cid e tos a om e Sa ati lm o d Rh ino nidae ae l op Rh h ida ino e cer otid Rha ae bdo virid a Rha bditid e ae Rajida e Pyxicep halidae Pteropodida e

Sc

Methanosarcinaceae

10

Adrianichthyidae Aeromonada ceae Alterom onadace ae Amiid ae Anis akid ae Aot Apt idae ery gid Are ae n As avirid ca ae r B i d Ba ac illa idae la Ba eno cea e pt th e Bo yerg rida id e vi ae da e

Result Report

Family



7 of 113

ty ae

lid

Anatidae Aphele nchoid idae Apiac eae Apte rygid ae Are nav As irida tro e pec tini Bo dae vid Br am ae B ida Ca upr e e C llorh stid al ae om inc ys hid a ci da e e

ae

ac od

log.counts

ida

Su

0.0

er

2.5

llid

5.0

ui

le m yid rra nid ae Sc ae iur ida Sa lm e on Rh i d ae izo bia cea Rha e bdo virid ae Retr ovirid ae Ptero malid ae Pseudo monad aceae Pseudokero nopsidae Procellariidae

Se

7.5

So

Sq

ha Sp

10.0

Vespertilionidae

n Sy

ae Vahlkampfiid idae Thyasir e atida derm masto idae Thau ianth lass idae Tha ont aod ae Tetr nid edi ae Ter nid ae ba tid Ta ac all e

12.5

Adrianichthyidae

Result Report

Ca

ba

ae

r id

e da eli ae m d a i r C sto e Ca bida e Ce ida p o e rc cida Ce e h t i cop e a Cer id Cerv ceae cerota to e a Ch ae Chalinid

m

e Cheirogaleida Cicadellidae

Clupeidae Coelacanthid ae Comam onadac eae Cord ycipit acea Cory e neb acte riace Cric ae Cy e tida lind e rol Cy eberid pri i dae did Cy ae pri Di nid c ae Di tyn pl id od isc ae id ae

Pratylenchidae Polynoidae

Herpotrichiellaceae

Eukaryote Virus


Gryllidae Hebridae

N em er to de M yx rm id iid ati M ur ae da i d e Mu ae r ic Mo rax idae ella Ma cra ce a uch eni e Luc ida ern e ariid Lep ae ilem urid ae Lamia ceae Hominid ae Holothuriida e

Bacteria

e da pi io e a e om yid ida til e ip him mat D ea o t Ec ac os eri hin act e Ec rob ea te ac ab ae En F ace lidi Ge idae plan Geo eae erac Glom ae socid Gobie e Gobiida

Pipidae eae Pinac ae iace cter lloba e a Phy cid Pho ae nid eae sia a rac e Ph e ct ida ba o l a erc e e Ox oc h da ida c idi On m om Nu lost ip od Ne

Superkingdom

Family



7 of 12

log.counts

rid

ae

ae

eb

id

Ca

ae

sto

Bo vid

Ba

cilla

cea e

Asilid ae

gidae Aptery

ae

Vahlkampfiidae

hid Tubiluc

ae

Rhodo

C

e

lo ha

bid

p

Ce

e

isto som

3

ida

ida

Sch

Salm

ae id di bi

inc

6

br

ae

nid

m m

Sc

ae sirid Thya

e

ur

ro St Sc o

tid don rao Tet

ida

Su St

9

Aotidae

Result Report

ae

ac

rn Co

atid a

e

ea

ae

rvid

Co

e

onid ae

ne Cory

spirilla c

ceae

eria bact

idae Cyprin

eae

Rhizobiaceae

Didelphidae

Rhabdoviridae

Echinostomatidae

0 Entero


bacteri aceae

iridae Retrov

ae alid

m

eae

e ce a

od

Numididae

Oligac

anthorh ynchid

ae

e

cida

tyla dac

hoce r Onc

aeo

e

eae

Ph

e

cea

e ida

e

da

ian

e

da

as

ae

ryi

Ph

ae

di

as m

da

iid


e

ea ac alv

Pl

lax

ace

ae

idae

ylid roh

ph

go

Me

Mic

Mur

urellac Nakam

Nothobranchiidae

Ph yll o

ini

bac teri

po

ro ac M

ut Pl

Ho

e ida

M

ium on

Ga

ae

om

e

a ce

da

a on

r ia

P

iid

h cep yxi

Faba c

Fus o

ba cte

idae Ran

d eu Ps

Superkingdom

Family



7 of 11

Sa

rc

Planococcaceae taceae Planctomyce aceae si tt e Piscirick idae irnavir Picob ae id r te e Phys ae id n eae sia cac Pha o c c ae o t e trep ilac ae tos iph e Pep ton ccac ae p Pe co llace o t i p c e Pe ae iba da en yuri ace r Pa x i O sp illo sc O

ae ce ria e e ct ida e ba ae no rybd llace dis r Se Ca Ca tone ceae tae r t e a a di nc I. I Ch ostri XII Cl ily eae c m a t a s F ochae e l ae idia Cole navirid str o ceae Cor Clo r te ia ebac n y r iidae Co sporid Crypto e a Cyprinid e da ini lph De

N eo

Desulfovibrionaceae Drosophyllaceae Enterobacter iaceae Enteroco ccaceae Entero mona Ento didae mop htho Ery race sipe ae Eub lotricha cea Ex acteria e ob cea asi e Fla Fab diace ae Fu vob acea a e G soba cter eo iac ba cter ea iac e ct er ac eae ea e



e ea ae ac ace er e m nad cea lo G omo laca i e a au on G ony ryllid idae G G ch e on cea em Ha oraga ceae l a Ha ytri h ae c rpo race Ha acte cob ae Heli mitid Hexa idae Homin ae Hyaenid raceae Hymenobacte Hyphochytriaceae

ca llim a M M ur stig o id Mo a ra xe ae ce no Me ae lla ble tha ce ph no ae ar ma Me i d ss tha iliic acea no o e ba Ma cte ccac stig r ia ea am oeb ceae e ida Lipo e tida e Liag orac eae Lepori dae Lactoba cillacea e Lachnospira ceae Hypotrichomonadidae

log.counts

0

Veillonellaceae idae Trichomonad dae Tiaropsi ridae Temo ae Suid e lida e ngy cea e Stro cca a oco ace ept occ ceae Str loc ta phy sma ae ce Sta a l a t p e iro ae ch diida e Sp ae iro a hi ce Sp ce at na ada Sp a l on m no

id in ge oc rotr ich p y rol s Sa eg tida ida cc ha nia e e ro c Ru min myce eae oco tac e c Rik ene cace ae ae llac Pyc e ae noc occ Pseu ace ae dom onad acea Prevo e tellac eae Porphyr omonad aceae Poaceae Plasmodiidae

Sa

5

le

m

So

Sc h

Se

10

Acidaminococcaceae Aerococcacea e Apteryg idae Apuso mona didae Astro virid ae Bac il lace Bac ae Ba teroid lae ace ae Ba nopte lan rid ae tid B i i da ov Br e id a C am C ssic ae py am ace ae lo elid ba ae ct er ac ea e

Result Report

Family


6 of 61

10.0

So

le

no filo m lem or Sm p int yida hida hu e Sil rid e uri ae da Sc e om bri d Sci urid ae ae Scin cida e Scia enid ae Schis tosom atidae Sarcocy stidae Salpingoecid ae So

7.5

5.0

0.0

Salmonidae

id ian ae ida e nith s orh idae ync Op hid hic hth ae Not ida hob e ranc Neo h iida diplo e stom Neme idae r tode rmatid ae Muridae Monocelidida e Migidae

oc

Or

Pe rc

Ph

as

Ph

op

pi

id

Pi

nn

Pi

e da ni idae a C tor s ae Ca ebid idae b C alo ae ph id Ce rcop dae Ce itheci p o c r Ce ervidae C ae dellid Cica e a Clariid ae id e p lu C Cricetidae Cupressaceae Cylindroleberididae Cynocephali dae Cynoglo ssidae Cypri didae Cypr inida e Delp hinid Did ae Dip elphi dae tilo m Eq iopid ae u id Fa ba ae Fe cea G lida e ad e id ae



ae lid eae ia c av era G ae m rid ae lo G Heb riid hu e lot ida Ho min Ho lidae Hy e rida Lab ae er tid Lac idae gnath Leio dae Lepori ridae Longido taceae Lophiotrema Microhylidae

Sagittidae Rhamnaceae dae Rhabditi ae Ranid lidae h p e a Pyxic idae ae d cho ace Psy nad ceae mo o ria d e u ct Pse iba dae i ion n p e r tu Pro ea Po ac idae o l P o e ic sc rida Pi p i P

da e ae

log.counts

2.5

Vespertilionidae eae Tricholomatac dae Thyasiri aceae ococc Therm e idiida Ther ae revid ae The tid don rao e Tet iida e s da Tar thi na ng dae e i Sy da Su ini l ae hy ap onid dae St i lli Sp de a Sp

12.5

Acipenseridae Adrianichthyid ae Ambysto matidae Amiid ae Apte rygid ae Astr ovir Bag idae Be ridae ry Bo cidae v Ca Bra idae llo mid C Ca rhin ae am m c pa eli hida nu dae e la rii da e

Result Report

Family

Figure 3: Graphical representation of read counts for the 100 most abundant families. In order to represent all detected superkingdoms, the displayed families are selected from the complete result applying the HighestAverages algorithm from the R package SciencesPo. Families are sorted alphabetically, color-coded according to superkingdoms. Note that the graph is in log-scale. lib01454.IonXpress-032.R-2014-05-10-10-51-59-user-SN2-48-lauf047-resultprotocol.pdf

6 of 65

Vespertilionidae

Umbridae dae Thyasiri iidae Therid tidae odon Tetra e iida Tars e lida Syl e e ea ida c Su eta yc ae tom nid ae ep ae id Str id pio S ell rph o ad e Sp om ida l i y of en olem S

l So Sm

10 Si

hu rid cid ae ur ae i d Se rpu ae li Sc om dae bri da Sci e urid ae Scin cida e Scia enid ae Schis tosom atidae Sarcocy stidae Salmonidae Sagittidae

Sil

log.counts

5

0

in t

nip

Acipenseridae Adrianichthyid ae Agamid ae Agari cacea e Amii dae Aot id ae Ap As terygid pe ae r g illa As cea tr e Ba oviri da be e s Ba Bag iida e rid t ae Be hye ry rgid ci da ae e

Result Report

ae vid ae Bo mid idae a h Br hinc r ae lid llo e a m C Ca nidae Ca ridae sto Ca idae Ceb ae opid Cerc ae hecid it p o Cerc e a id rv Ce Cicadellidae

er

Clariidae Clupeidae Cottidae Cricetid ae Cupre ssace Cylin ae drole berid Cyn idae oglo ssid Cyp ae r Cy ididae pri nid De ae lph Di pti ini En Dro lomi dae o s te ro oph pida ba e i ct lida e er ia ce ae

Rhinocerotidae Rhabditidae

as ia n hy ida hic nc e No h h tho bra thida idae Ne od nc e ipl os hiid a to Na rcin mid e ae ida Mu rida e Mon e oce lidid ae Migid ae Micro hylida e Lophiotr ematace ae Longidoridae

Ph

or

Leporidae

Op

ith

rn

O



e da ui ae Eq ace e b a Fa elid ae F did ae Ga lid via ae Ga lanid op eae Ge rac me e Glo rida Heb e inida Hom e Hylida ae Ictalurid Labridae Lacertidae

Ranidae ae rigyrid Quad dae li a eph e Pyxic dida cho Psy ae e c Poa lidae o cic ae Pis rid Pip dae i e Pip ida nn idae Pi c o Ph

Superkingdom

Family


lib01455.IonXpress-017.R-2014-05-10-10-51-59-user-SN2-48-lauf047-resultprotocol.pdf

7 of 61

Sa

cc

0

Ru

ae

ar

N

ph

os

Irpicaceae

M yc

e ea ac dae t le ci Bo mby ae ae o ce vid B Bo obia e z ea i c h ia yr ad ibasid ae r e B ler iac l der Bu e hol a k e r iac Bu nod ae Cap iace id s toba ae id Cera n pogo Cerato dae ci e ith p Cerco Chaoboridae

Chironomidae Chytriomycetaceae Cordycipitac eae Coriolace ae Cor tic iacea e Cor ti naria Cor ceae yne bac teria cea De Cypr e inid ba ae Div ryom y cet En ers ace is te ae En rob pora ce a ae En tero cter iac to coc lo m cac eae at ac eae ea e


False positives: - Orthomyxoviridae56

ae iid en e en ea ae ko ac ace e ea Eu Fab sid p tac a ito ae m erm ce d Fo lla no hy e Ga oeop racea Gl ome dae Gl ntii e Gra cea icia Her e inida Hom aceae Hydn e horacea Hygrop taceae Hymenochae Inocybaceae

Plasmodiidae Planorbidae ae Pichiace ceae a ri c la Physa ae id ian e cea Phas aeta ae h c e ro e c n ta e Pha yce da e ffom oviri a x ce Pha y a t i m cip eae tho y Or d r tac e co e hio alo a ea ph idid llac Op m e m O i Nu wsk ko wa No

na vi r id a M ur rell e Mo ida ac r ti ea e Mo erel e lac rax e Mic ellac ae ea rov e irid Me ae mb rac Mar ida e asm iace ae Malv acea e Lyoph yllace ae Lycoperd aceae Lipomycetace ae

log.counts

4

ha

ro m y ss ula ceta izo ce ce ae Pte biac ae ea Ps rul eu a e ce do a mo na e Psa da thy cea rell Pro ace e pion ae ibac teria Poly cea stom e atida e Polyp orace ae Poacea e Pluteaceae Pleurotaceae

Rh

8

Yersiniaceae Waddliaceae aceae Ustilagin ae scace mona eae Tricho atac olom ae Trich lace aer u sph Tela dae e Sui cea ria e ha e e a op ce ea ea Str rea cac tac Ste oc ce loc omy eae hy ar llac e ap a ch y St h id ac op yst os z hi hiz oc Sc arc Sc S

12

Acanthoecidae Actinomyceta ceae Agarica ceae Aman itacea e Anati dae Ang iost r o ngy Aph lida ro e Ap phorid t ae e ryg As i co ide dae A ac Au the ric liac eae ea Ba ula e Bo rna riac ea lb vir e id iti a ac ea e e

Result Report

Family


lib01469-S14-L001-R1-001-lauf0118-resultprotocol.pdf

7 of 30

Yersiniaceae

re

St

Virgaviridae ae Violace idae r tilion Vespe ae Ursid idae asir e Thy tida don ae rao Tet uid ass ae Tay d lpi e ae Ta ida ace c Su oc ae oc pt ricid eae c So na la So

12.5

10.0

Sc iu r oc idae Sa ys lm tid on ae Sa lica idae Rh ce izo ae bia Rh cea ino e lop hid Rhin ae oba tida Pter e opod idae Pseud omon adace ae Protope ridiniace ae Poaceae Pleosporaceae Sa

rc

7.5

5.0

log.counts

2.5

0.0

Adrianichthyidae Alligatoridae Apteryg idae Aquifo liacea e Arali acea Asc e oide ace Ast ae e Ba racea e be siid Ba ae lae Bo nid Br vida ae B a e C ras mid ab si a om cac e ba ea ce e ae

Result Report

e da hi nc ae i rh lid e llo e e Ca Cam nida ea ac Ca cteri a e b da rno tori Ca Cas dae i b Ce idae cop ae Cer ecid h opit Cerc e a Cervid ceae Cleoma Cricetidae Cucurbitaceae

Piscicolidae Pipridae

id ra ida nc e hi m i ipl ila da Na osto cea e mi rci d e nid Mu ae ae ste lida Mu rida e e Mig idae Meg alon ychid ae Malva ceae Macrosc elididae Macrauchenii dae Lulworthiaceae tro

Ne

od

so

pu

N um

ob

th No

Ni



e e da ffi ea ae ira rac atid G e m m e lo one l G ea c y ng fnia dae Go Ha cari ae la rid Ha ide s po ae Hip minid e cea Ho cilla toba ridae Lac emu Lepil dae Lepori ae ostocace Leucon Lipotidae Lophiotremataceae

Pipidae ae stomid y Ph llo ae id lc Pho idae sian atidae Pha om los ae sco sid i Pha rk n ceae Pe a i ae l da race e Pe lo ida e f i a ir ss Pa r titiv rcid ce Pa o h nc O

Cupressaceae Cyprinidae Dasyuri dae Delph inidae Echim yida e Eim Ent eriid ero ae bac t e Er Equi riacea em da e e ae o Fa ba zetid ae Fe cea G lida e ad e id ae

Superkingdom

Family


6 of 33

10.0

So

le m yid ida ae Sc iur e i Sc iae dae Sc n his tos idae om Sar atid coc ae y Salm stidae onid ae Rhin olop hida e Rhino ceroti dae Ranidae Si

lur

7.5

5.0

ae id el e m da Ca ani dae C tori e s Ca bida dae Ce hylli p o rat dae Ce rcopi e Ce cida h it e cop e a Cer id Cerv idae ogale Cheir e a d ri Cle

Pteropodida

0.0

Clupeidae Cricetidae Cupressaceae Cylindrolebe rididae Cynoce phalida e Cyno gloss idae Cypr inida e Delp hinid Did ae elp Eq hidae uid Fa ba ae c e G Felid ae ae G eop lo m lanid er ac ae ea e

e Psychodidae

Pe r

Ne

Macraucheniidae

th

ob

le

O

No



ae iid e ob da dae G bri ri He ide e os da ini pp Hi om idae H n ae Hy lidae Hy dae luri Icta e rida Lab ae lace bacil Lacto dae rophii Lamp ae Lecanid Leporidae Lophiotremataceae

Pseudomonadaceae aceae Protoperidini e Poacea lidae o ic c Pis idae Pipr ae ip P id e ida n e Pin ida ter s y e idae Ph e olc Ph cida ae ae o d Ph iani ace t s e a c Ph my fo af Ph

ki ns ac ida ea e ra od nc e ipl hi o My stom idae t Mu ilidae idae ste l Mu idae rida Mor e axe llac eae Migid ae Micro tromb idiida e Microhyl idae Manidae

log.counts

2.5

Vespertilionidae eae Tricholomatac ae rammatid Trichog dae emati ssosin Trava ae sirid Thya dae ridii ae The niid ae tigo Tet ntid do ae rao uid Tet ss e ya ida Ta rsi e Ta lida l e Sy ida ae Su nid io Sp

12.5

Adrianichthyidae Alligatoridae Aotidae Aptery gidae Aren avirid ae Ba Bal laenid ae aen opt Ba erid thy ae Be ergid ryc a ida e Bo Br vida e B a C ras mid e al s ica ae lo rh in cea ch e id ae

Result Report

Family



7 of 40

Sc le ro his tini Sc a nd ace en a ra ed ce e e Sa rco sma ae ce c y Sa a sti da e nta e lac Sal ea e mo nid ae Sali cac eae Ruta ceae Rubia ceae Rosace ae Rhizophorac eae Rhinolophidae Sc

e e ea ea ac ct thac ae a C an lace lyc nu e e Ca mpa pida cea Ca erco eridia C a h p eae tos ae harac eae Ch C diac o op n ceae e Ch nada omo d y m e Chla llacea Chlore daceae si p io id cc Chrooco Cleomaceae

ce a oc ce e ae er Ol cid ea Ny ce ae mp ha ae ea Nu ce cle a ari No ida e sto cac e Neo e ae diplo stom Nar th idae ecia ceae Myr ta ceae Morgan ellaceae Micromonosp oraceae ta

rc hi

ys

Mesodiniidae

On

ch

O

oc

O

Comamonadaceae Convolvulaceae Cucurbitace ae Cupress aceae Cyan obacte riacea Cyno e moria Dip c eae oda sca cea Er e Eu icace ae ph orb i F a Ge abac ceae e n Ge tian ae G ran ace lo m iace ae er ac ae ea e



ae e ce ia cea m a i rim nd ae e G rna inid ea He om eac H ng eae c dra Hy amia eae L c ura e La cea Lilia e cea Lina eae etac myc e Lipo tacea trema Lophio aceae Loranth Luteoviridae Malvaceae

Pseudomonadaceae nopsidae Pseudokero e Poacea oridae h p io od Plasm ae d li ico Pisc eae c a Pin ae ace eae ym ac Phr e c t my ae o f f i id a Ph lag ceae e e P lia cea e da a a Pe iflor ace ss nch a P ba ro O

da

log.counts

0

Zygophyllaceae

5

Zosteraceae ae Zamiace e racea Winte eae Vitac e cea Viola ceae a rion e Vib cea ica ae e Ur t ce a e ae ea Th rac ace u hy ad ac on eae ae St m ac ace go hin olan lari Sp u S ph ro Sc

10

Acanthamoebidae Anacardiace ae Apiacea e Aquifo liacea e Arac eae Ara liac eae Are cac As eae c As oide ace pe ae As rgilla t c e ea Ba rac e c B e Bu ras illac ae rm sic ea e an ac e ni ac ae ea e

Result Report

Family



7 of 41

len

Sp

Su

ion

Aotidae Aphrop horidae Aptery gidae Aren avirid ae Asc Asc idiidae oid eac B eae Ba abes lae iida n e Ba op te th Be yerg ridae ida r Bo ycid e Br vid ae am ae id ae

So

Vespertilionidae

Ursidae e Turdida idae Triops tidae mma ogra e Trich llida hine ae Tric d i e r asi cea Thy cca oco ae erm ridiid e Th a e e iid Th a er eil ontid e Th a od id ra ssu ae ya iid Ta ars T t Te

10

Adrianichthyidae

Result Report

ae id ch ae n i id rh llo aen ae Ca am elid C am ae C nid Ca ridae sto Ca ae bid Ce e pida o c r e Ce cida e h it op Cerc e a id Cerv aleidae Cheirog e ell Chlor acea Clupeidae Colpodellidae Cricetidae Cupress aceae Cyno cepha lidae Cypr inida e Delp hinid ae En Eime riid ter ae ob act eri E ace Er quid ae wi ae n iac Fa ea ba e Fe ce lid ae ae

id

ae

i ofi lom dae lem orph id y Sm inth idae ae uri d Silu ae r id Sciu ae rida Schis e toso mati dae Sarco cystid ae Salmon idae Rosaceae Rhinolophidae

So

0

Manidae

No

No



e ea ac er ae m id e lo bi G Go rida ae b rid He de i os idae pp n Hi omi ae H d i l Hy ae irid Jan e rida Lab ridae Lepo dae Lipoti ridae Longido taceae Lophiotrema Macraucheniidae

Rhinocerotidae Ranidae halidae Pyxicep e odida p ro Pte ae id d o h Psyc e cea Poa ae diid o e sm ida Pla l o cic ae Pis d ri Pip dae e i a Pip terid e se ida y Ph hoc P

Ph as to ne iani d th m ob ou ae Ne ra od nc rida ipl h e os Ne to iida od as mid e Na a y ida ssu e e lop sid Mu ae ste lida e Mur idae Mora xella ceae Mono celidid ae Migidae Microhylidae

log.counts

5

Family

Figure 3: Graphical representation of read counts for the 100 most abundant families. In order to represent all detected superkingdoms, the displayed families are selected from the complete result applying the HighestAverages algorithm from the R package SciencesPo. Families are sorted alphabetically, color-coded according to superkingdoms. Note that the graph is in log-scale. lib01806.IT-031-Standard.R-2016-09-01-09-24-56-user-SN2-92-lauf085-resultprotocol.pdf

6 of 49

Sc

10.0

5.0

0.0

Sa

lm

e da idae bi Ce pom idae x ro e nt my da Ce rato heci t e i ae d C cop i r nth Ce laca nidae e Co nagrio e e Co rduliida Co eae c a n Cor ae Corvid Cottidae

Parabathynellidae

Mu

M

ur

al

oc

yc o

yc

M

M

Cricetidae Cucumariidae Cynocephalidae Cynoglossida e Cyprinid ae Darw inellid ae Delp hacid Ech ae inos tom atid Ect ae ob En Eim iidae eri ter ida ob Ep acte e im ria ce Fa eri Fa bac idae ae sc ea io e lid ae



ae ae id lid Fe tom eae n c ra jie ae Fu lome ocid dae G bies mati Go lone ae y d i ng ad e Go Goni ida licin He ae inid Hom e nida Hyae e Hylida ae Ictalurid eae Labyrinthulac Lacer tidae

Panagrolaimidae Oxalidaceae ae Orussid ae ynchid rh o h Ornit e a iurid e Oph tida rma e e d a io h d i p s O op ae alin cid Op cer dae o h i c n ae On ho iid mp nch e y N ida e bra tho ops da No erit phi N to yc M

pl as m

log.counts

2.5

st

os om at o tst roe nida idae e Rh mi a ag Rh ion cea ab e id do ple ae uri Ra da nid e ae Psy cho dida Pom e acen trida e Poace ae Pipidae Phasianidae

Ru

7.5

hi

Vespertilionidae Vesicomyidae ae Unionid vidae There ae rogid Tetra ae ontid aod Tetr dae e Sui da elli ad e Sp da e uri cea e Sil a a cin ace i ba n i Se ae e ot ler ncid ea i c Sc Sc ai a in Sc

12.5

Result Report

Adrianichthyidae Amiidae Apteryg idae Areca ceae Aren avirid ae Bab esii Bov dae id Bra chi ae o Ca Bufo nidae nid llo Ca rhin ae ch m C Ca elid idae ap a n no ida e e di ac ea e

Summarized Results

a ici tac a e ida cea ae e e gil Mi i d cro ae h Me tzg ylida e eri ac Ma lvac eae eae Mac rom iida Luce e rnar iidae Libell ulidae Leporid ae Leiognathida e Lamiaceae

4

Family


lib01949.IonXpress_001.R_2016_10_07_08_47_09_user_SN2-102-lauf095-resultprotocol.pdf

5 of 32

Summarized Results of basic Analysis

Se

Sc h

5.0

0.0

Sc

ina

in

cid

ae

iac ist e o Sa som ae lm ati on da i Sa gitt dae e Ru ida tstr oem e iac Rha eae gion idae Rha bdop leurid ae Ranid ae Psycho didae Pomacentrida e Poaceae

N

ob

Ne

th

No

ae id ch e n i rh lida e llo e Ca Cam nida e ea Ca diac o n e p a Ca ebid ae C cid ithe p o rc e a e C vid Cor e etida Cric didae ri e b role Cylind e lid pha a Cynoce e ida ss glo Cyno Cyprinidae Darwinellidae Delphacidae Diphyllo bothriid ae Eimeri idae Elep h a Ent erob ntidae acte riac Equ eae ida Fa e ba cea Fa sc e io Fu Fel lidae jie ida G nto e al ax mid a iid ae e



ae ce e ra a po ace idae t r as ig me ema G o e l G ylon dida ng nia e o ida Go r G b e He inida lic ae He ace loti ae He inid Hom dae athii Horm nidae Hyae Hylidae Hynobiidae Hypsibiidae

Plectosphaerellaceae Pipidae e Pinnida ae almid th h p o Phil idae n ia ae Phas tace yce ae ffom d a i l h l P e e ser ida Pas olaim e r a g e c na da e Pa ali rida e Ox iu sida e h p a p O no rcid ali e Op hoc nc O

ym ph a ra lid nc ae rit hi op sid ida Mu e a ri Mi dae e Mic ridae roh Me y cys ma lidae uch eni Lep ida orid e ae Leio gnath idae Lamia ceae Lacertid ae Labyrinthulac eae Ictaluridae

log.counts

2.5

Sc

Se

10.0

7.5

Vespertilionidae Vesicomyidae ae Unionid didae mona Tricho ae evid Ther ae ontid aod Tetr dae ceae Sui ta yce e tom llida ep e ae Str ad lid Sp cu ae un Sip urid dae e l i Si ea ul rp nac ci ba

12.5

Result Report

Acipenseridae Adrianichthyid ae Amiidae Aphe lench oidida Apte e rygid Ara ae ch n o idid Are ae Are cacea e nav Ar i r ida en e Ar icoli Ba istei dae d b Br B esi ae id ac ov hy ida ae cl ad e iid ae

4

Family



4 of 26

10.0

Sa

rc op h rc oc agi d Sa ys lm tid ae on ae Rh i d ag ion ae ida Ra e nid Pyx ae ice pha l i d Pyr ae alid ae Psyc hodid ae Pseud omon adace ae Pomace ntridae Poaceae Sa

7.5

5.0

log.counts

2.5

0.0

Vespertilionidae Thyasiridae dae Therevi ae dontid Tetrao ae Syllid ae Suid dae lini phy dae Sta yi lem ae So d e uri da Sil ini orh dae dae yli i i at e Sc inc Sc som cea o sa st hi as Sc arg S

12.5

Acipenseridae Acricoactinid ae Adrianic hthyidae Alliga torida e Amii dae Apt eryg idae Are n Are avirid ae n i Ar thr colid Ba olep ae ti b Be esii dae d r Bo ycid ae Br vid ae am a id e ae

Result Report

e da hi nc ae i rh lid e llo e Ca Cam nida e Ca orida t s Ca bidae ae Ce pid e rco Ce cida e h it cop e a Cer id Cerv e ellida Cicad e a id e Clup Cornaceae

Piscicolidae

Cottidae Cricetidae Cupressace ae Cynoce phalida e Cypri nidae Dicro glos sida D e Ech ictynid ae ino sto ma Eim tida eri e ida En E e ter myd ida ob ac e te E Fa quid riace ae ba ae ce ae

Pipridae Pipidae

Janiridae

Ne Ne o m er das to de yida M ur r ma e ida M t e ida Mo ugili e d no do ae Mig ntida e ida Mic e roh Lop ylid hiot a e rem atac e Lepo ridae ae Leiog nathid ae Lacertid ae Kurtidae



ae lid io e sc da ae Fa Feli mid o e nt jie ida e Fu Gad dida my eae oe c Ge mera ae Glo esocid ae bi atid Go nem gylo Gon ebridae H idae Homin Hylidae Ictaluridae Iguanidae

idae Phasian ae matid o s lo o Phasc idae r e g n e Phala llida sere idae Pas ss o l g e teo ida Os e uss cida Or r e e e c a o a iid ch On rsted chiid ae d n e a omi O r b t ho los t ip No od Ne

Superkingdom

Family



6 of 32

Summarized Results of basic Analysis

St

Vitaceae Vespidae llaceae Veillone ae scace mona eae Tricho atac olom ae Trich ontid aod Tetr dae i hin e Tac ida rph ae Sy ce illa e e Su ida ea Su iac ae e d i ar a ph loid ce a ro y cc ng ro oco pt

12.5

Result Report

St

Sp

7.5

5.0

0.0

ap

hin

hy lo

ath i

m

Ph

Ph

ae ce e ica rida s ae as ho Br llip iace e Ca nod yiida p e Ca cidom cida e Ce opith dae i rc Ce ironom e a Ch ellid d a Cic ae d li h Cic ceae cipita Cordy ce ita ae Cucurb Cyprinidae

Debaryomycetaceae Diphyllobothriidae Dolichopodid ae Drosop hilidae Eimeri idae Ente r Ent obacteria omo ceae p Eos hthora cea ent e o mid Fla Faba ae vo ba ceae Fo cter iac F rmi ea G usa cida e lo m rivid e er a e ac ea e



ae id at m ne idae ae lo e gy min lac on Ho acil ae G e b e a ce iac cto La Lam aeria ae h sp ace ezi pto Le alass eae M vac l eae Ma inac c r osa ceae han owia Met chnik ceae Mets cocca Micro ae Moronid Mrakiaceae Muridae

co c on cac ad ea e a So d lan iida cea e e ac Sc ea om e bri d Sci arid ae a e Scia enid Schis ae toso mati dae Scato psida e Scathop hagidae Sarcocystida e Salicaceae Saccharomycodaceae cetaceae Saccharomy ceae ia b o Rhiz ae adace omon e a Pseud e c a yrell eae Psath riac acte e ib n pio cea e Pro ula a Prim entrid c a e m e ea Po ea ac Po orac ae d p i i s o d e Ple smo cea a ia Pl h c Pi

Sp

go

Ph ry m a sia ace af fom Ph ni ae d ae os yce ae t ph Pe ae ace da ria ae l Pa ste iacea cea e ure e On cho llacea cer e cid Not ae othe niid Noca rdioid ae acea Neiss eriace e ae Nectriace ae Narnaviridae Muscidae

log.counts

2.5

St

re St

10.0

Adrianichthyidae Agaricaceae Amarylli daceae Antho myiid ae Aphid idae Apt eryg idae Are c Ba aceae be Ba cte siida r e B oid Bo latti acea da e lbi e B ti Br ov acea ac ida e on e id ae

4

Family



4 of 32

Yersiniaceae

Vitaceae ceae Verbena eae ginac Ustila e acea Ur tic ae ace Ulm ae e ace cea The ria ae sph ceae ae ato a e Ter lan riac e So la cea hu e ia rop ea tin c o Sc ga eae ler ra c xif ta Sa apo S Sc

10

0

Sa lic ac ea o m ch y aro co e da my Ru c c tac eta eae ce e Ru ae bia ae cea Ro e sac Rho eae dan oba cter ace Rhiz ae obia ceae Rham nacea e Retroviri dae Ranunculace ae Pseudomonadaceae cc h

ar

e Nu mi acea Ne e lum dida e bo My na xot rich ceae a c Myr icac eae Myc eae osph aere llace Morga ae nellac eae Morace ae Meliaceae Melastomataceae

ce

gin

cta

Ny

On

ag

ra c

ea

or ac

ha

si fl

nc

ba

Pa s

ro O

Cladosporiaceae Cleomaceae Comandrace ae Convolv ulaceae Corna ceae Cras sula ceae Cuc urbit ace ae En Ectob ter iida ob e act eri Er ace Er icac ae Er wini eae a Eu ysi c e ae ph pha ce or bi ac ae ea e


False positives: - Retroviridae (WF)

ae ce eae c ba e Fa nia cea a ria er G sne ceae a Ge fni ae Ha minid eae Ho geac n e dra dacea Hy n a l Jug iaceae Lam e acea Laur ae Liliace ae Loasace e Loranthacea Malvaceae

Proteaceae riaceae Propionibacte ceae Primula ae onace Polyg e a e Poac ae race e spo a o le P ace e n i g nta cea a a l c P lac ae yto ce Ph ma idae y r Ph sian ae ae a e ce Ph alia riac d e t e c P a ob ct Pe

e ea ac eae r te iac ae As on e ac gn e Bi ragin cea a Bo ssic ae a e r c B e cta cea Ca ula e pan a m e ac Ca nab Can eae c a li rifo Cap ceae phylla Caryo ae ce ra st Cela eae Chenopodiac

ea e ae

log.counts

5

Sa

Sa c

Acanthaceae Acetobactera ceae Adoxace ae Albug inace ae Amb orell acea Ana e card iace Ana ae tida A e Ap piace ae oc y Ap na ce te Ar rygid ae a As Ara cea ae e pe liac rg e illa ae ce ae

Result Report

Family



7 of 58

Vitaceae

Tytonidae ae Trogonid aridae Toxoc ae nithid skior Thre idae aup e Thr tida don rao e Tet lida e Su a d rni idae e n Stu ea e o a ac thi ru cc ace eae St co c ac o c pt co teri re lo hy ac ap gob n hi St

St

Sp

10

0

he n lan iscid ac ae Sil e ur Sh ida ae ew e an ell Sc a olo pa ceae Sch c id isto som ae a Rhiz obia tidae cea e Ran idae Psitta cidae Pseudo monada ceae Propionibacte riaceae Procellariidae

co Nu m m Mu idid idae s tel ae Mu i da sop e ha Mu scic gidae api d a Mur idae e Mora xella ceae Metha nosarc inace ae Mesitorn ithidae Megapodiida e Malvaceae

id

xy

ist

ho

tid

O

O

Op

e da ni idae a C am ae ri tid e Ca thar ida ec a e C ith p ea iac rco Ce etom dae a i Ch aradri ae Ch niid e Cico acea icipit Clav ae id b m Colu itaceae Cordycip Corvidae

Corynebacteriaceae Cyclobacteriaceae Cynoglossida e Cyprinid ae Diom edeid ae Diph yllob othrii Dra dae cun culi D dae En rosop ter hilid ob ae act Es eri ace t r ild Eu ae i da ry e Fa pyg i b d Fa a a lco cea e e ni da e



ae ae iid ce av ella G e er m uida iidae lo G Gr othr e b ida plo vir Ha rpes dae He mini Ho dae eni ae Hya ace ocre e Hyp r tida Lace e Larida ae Leporid iaceae Leptosphaer Leptotrichiaceae

Polystomatidae Poaceae diidae Plasmo ae Piprid eae c Pina idae pter o ic en ae o d h P ni sia ae Pha ntid o e th e a ida Ph an dae i lec l l e P e ere ae ss da rid Pa iri Pa xov y m ra Pa

ur id a ae e

log.counts

5

Sp

So

Acanthisittidae Accipitridae Adrianic hthyidae Alliga torida e Anati dae Aph idid ae Apt ery Ard gidae As e ida pe e Bo rgill ac rre e l Bu Bo iace ae a C rkho vid al ae e lo lde rh ria in ch cea e id ae

Result Report

Family


6 of 16

Vespertilionidae

e Vermileonida e Tytonida idae Triops ae nithid e skior ea Thre cac coc e rmo tida The don rao ae Tet niid e Tae da rni ae Stu onid thi dae e ru i a St rig nid ae e id St ra hy nisc he Sp Sp

0

e ae da bi ecid e e C ith yida p h o rc icht e e C ann da nii e Ch Cico acea i d i r t s e o Cl liida Co ae mbid lu o C a id e Corv ae tt Co id e Cracida

Cricetidae Cucurbitaceae Cylindroleberididae Cynoglossida e Cyprinid ae Desu lfurom onada ceae Diom edeid Dip ae hyllo b o Dra thrii dae cun c En Emy ulidae ter d i d ob ae a Er cter Es ebid iacea ae e Fa trild ba ida e ce ae

Procellariidae



ae id e on lc da i Fa avi ae G uid e da Gr i e br He inida e lic ida He min Ho idae Hyl ae er tid Lac ae Larid ae nathid Leiog ae Leporid e Megapodiida Megophryidae

Pomacanthidae e Polystomatida e a id o n Poly e odiida Plasm ae id r Pip idae sian ae Pha erid ae g n a l cid a e Ph ora roc ntida lac o a z e y a Ph m d e tro cani Pe ida le ell r Pe e e ss rida Pa Pa

Pa ra m yx O Or x yu ovir nit rid id h o Op ae a rh yn e ist Op hoco chid ilio a mi ac da e a e Op hid ridae iida On cho cer e cid Nec ae triac eae Myc toph idae Musc icapid ae Muridae Microhylidae

log.counts

5

So la na rra cea nid e Sc ae op Sc olo idae pa c Sc inc idae i da Sci e nai ace Sch a isto som e atid Salm ae onid ae Ranid ae Pseudo monada ceae Propionibacte riaceae Se

Microcoleaceae

10

Accipitridae Adrianichthyid ae Anatida e Apter ygida e Arde idae Ar th role ptid Bor ae reli ace B ae Br eg ovida ma e B c Ca uce erot i d r llo oti ae r d Ca hinc ae C at nid hida ha a e rti e da e

Result Report

Family


6 of 16

Sp

hin

ing

log.counts

2.5

go

Ph yc i

Ph

e ea ac e er ida t ac nad e b a o o ul m ace ae Ca erco mpi ce ga C ha a C oph ceae lla itin Ch hlore yxidae C m e o r lo cea Ch lina ae omu dace r a h n C mo pido le o e s a Chry Clavariace e stomida Climaco ceae da na mo ma Co Cordycipitaceae Cryomorphaceae Cryptosporid iidae Cynomo riaceae Cysto bacte racea Cyto e phag acea Dele e Din sseriac eae o Dip physia od cea a e sca Du cea g En Ei esii e da te me e ro r Fl bac iidae ab t e el lu riac e lid ae ae



e ea ac ae at m anid ae e em pl ce G eo era acea i G m o ytr e Gl och llida om ere idae Gr rub d er i G e t a lop ace Ha eni idae lym Ha nell n tma ae Har tiace Helo e mitida Hetero ae Hominid Hydruraceae Kofleriaceae

Rhodocyclaceae Rhodobacteraceae eae Rhizophydiac teridae p o ch ty P ae rdiace onoca e acea Pseud d a n o e dom u a e id s t P n rodo ceae Pro a por dae s o e ii Ple od acea m s et Pla yc idae e m m to ea nc sto tac ae Pla gio rma ace a l i r e P te od ac ys ob Ph l l y Ph

sp h as ae Pa r ia ra ni ace to m dae ae Ot ell op Os lan ida tre e id op sid ae Op ac itut e a ace Nu ae e cle arii dae Neo lect ace ae Myx oboli dae Muco racea e Methylo philacea e Lepetodrilida e Legionellaceae

0.0

Te tra b

ot m hriid on o ad ae ba Sp a c ha ero teria cea e So liba myx cea e id cte rac ae Sip e hov irid ae Sino ae bac tera cea Sarc e ocys tidae Sapro legnia ceae Salping oecidae Rhodospirilla ceae

Sp h

5.0

Verrucomicrobiaceae n3 bia subdivisio Verrucomicro dae Vannelli idae ampfi Vahlk ceae gina e Ustila a hiid e nyc tida Uro ma e oso a e pan llac Try me tidae Tre c e ore rida e Th pe dae ida e a eo ii ig er Th st atid a eil Th tom erm a d m to au as Th um a Th

7.5

Albuginaceae Anatidae Apteryg idae Apuso mona didae Atop ome lidae Bac il Bal laceae a Bd ntidiid ello ae Br Bod urid ae ad o n yr id Bu Bru hizo ae C rkh cell biac am o ac ea e pa lder eae nu iac ea la r ii da e e

Result Report

Family



7 of 33

au

Th

Verrucomicrobiaceae n3 bia subdivisio Verrucomicro dae Vannelli idae ampfi Vahlk dae ychii ae Uron atid som ano eae Tryp llac e me a Tre ctid ore ridae Th e e ae p eo riida atid Th e m e eil er a Th od iid t r as oth eae m b c ra tra Te ynu S

10.0

7.5

5.0

0.0

Sp hi

ng

om o Sp ba nad ha ero cter ace So my iac ae lib e ac ter xida ae Sip hov acea e Sin e i rid oba cte ae rac Sar e coc ystid ae Salp ae ingo ecid Sacch ae aromy codac eae Rhodosp irillacea e Rhodocyclac eae Rhodobacteraceae hin

go

e ea ae e ac pi gac am ha eae h p C ino lac eae e it c rel ea Ch Chlo ulina adac m on o r m e Ch pido ida ole e por rys Cincti cea a h d a C mon e a a e m c a Co ycipit e Cord llacea Coxie e a d Cunaxi ae Cynomoriace Cystobacteraceae Cytophagaceae Daphniidae Delesse riaceae Dinop hysia ceae Dipo dasc acea Dug e esii dae En Eime riid ter ob ae act Fl eri ace Ge abel l ae G Ge mm ulida eo ob a e de ac tace rm ter ae a at op cea e hi la ce ae



e ea ae ac ace i er ae m hytr ce lo a i ae G oc m men ellid ro G aly ann ae H tm ce r a i Ha elot idae H min eae Ho c rura aceae d Hyd ona hom eae Hyp phaerac Isos aceae Kofleri llaceae Legione e Lepetodrilida Malvaceae

Rhizophydiaceae ae Ptychopterid ceae ocardia n o d u Pse ae adace n o m o ae Pseud d ti n odo ae Pror tidid e cys ea c Poly ora ae osp diid dae Ple o i sm ad ceae Pla on om ceta ae n e Pla tomy mid ea nc sto rac Pla lagio hae P isp yc h P

P Pa has Os rato iani tre m da e e o Op psi llida d itu e a c ta Nu e cle cea ae e ar i Ne ida o l e Ne e cta oca ce llim ast ae Nan i g a noc ysta ceae cea Myx e oboli dae Myco bacte riacea e Microba cteriace ae Methylophilac eae Mamiellaceae

log.counts

2.5

Sp

Acetobacteraceae Anatidae Apteryg idae Apuso mona Arch didae aeog loba Bac ceae il Bal laceae ant idiid Bo ae do ni Br ad Bovi dae da Bu yr h C rkh izo e C am old bia au p ce er a a ia lo ba nula cea e ct rii e d er ac ae ea e

Result Report

Family



7 of 34

Result Report

rd

So

Sip

0

ac

ia

ae id e ec ea e th i p ac a co omi ace r ae t Ce ae hag dace h p a C ino n o it e m Ch ydo llacea lam hlore eae c h a C C lin e mu o r cea Ch omyxa c ae id Coc ig onos e Cod dacea mona Coma ae ce ita ip Cordyc ae Cucurbitace

ce

e ho Se vir ae rra ida sa e Se lm len ida ast e Sch r a c isto som eae Sar atid coc ystid ae Sapr ae oleg niac eae Salpin goecid ae Salmon idae Saccharomy cetaceae Rhodospirillaceae

ae

Rhodocyclaceae aceae Rhodobacter ceae ia b o iz h R ae adace omon eae Pseud c a ti o deur ae Pseu atid tom s ly ae Po d i i l ci Poe iridae dov ae o P e ae e ac Po odiid cea ta m e s c Pla my dae cto yuri n a x Pl O

O

Culicidae Cyprinidae Cytophagace ae Debary omyceta ce ae Diphy lloboth riidae Dros ophil idae Edw ards iida Eim e erii Eu d ph orb ae iac Fla Fab ea a vo e ba ceae c F t G em orm eriac ea in icid e ig er ae ac ea e



e ea ae ac at ace ae m ll e e em re iac G ome ter acea c l l G oba hiel l e ic Ha potr inida r e om da He i H e l l cea ale Hy tera bac ae eno robiace ic Hym hom ceae Hyp nada omo Hyph aceae apor th Magn ae Malvace e Mamiellacea Methanosarcinaceae

xa lo ba O ct Op pi t ut era hio ac ce s to On m eae ae ch a No ela ocer tace c a erh ab idae e Ne da ctr c ea iac Na e ea tria e lba cea Myo e virid ae Murid ae Mixia ceae Microba cteriace ae Methylophilac eae Methylobacteriaceae

log.counts

5

ar

lan

Adrianichthyidae Alcaligenacea e Apidae Aptery gidae Apus omo nadid Are ae Asp caceae Ba ergilla thy coc ceae c Br ad Bovi acea e yrh dae B i z ra B o C urk ssic biac au ho ea ac l e lo ba der eae ct iac er ea ac e ea e

Xanthomonadaceae Volvocaceae e Vitacea idae r tilion Vespe e fiida amp Vahlk eae inac dae ilag ti Ust ma ae oso ace pan ron Try spo idae e ho t hri Tric ida e p d e Te ea loi gy olac cea e a a ron b o St ad ce idi on ria or om cte Sp ng oba hi ng Sp hi Sp

So

10

Family



7 of 195

Xanthomonadaceae Volvocaceae e Vitacea idae r tilion Vespe e fiida amp Vahlk eae inac dae ilag ti Ust ma ae oso ace pan ron Try spo idae e ho t i a r Tric ph ae did Te loi ace e gy a e et ron ce ea yc a St ol dac om b pt a io re on rid St o om Sp ng hi Sp

10

So

So

ria ce a a ho cea e Se vir e rra ida sa e Se lm len ida ast e Sch r a c isto som eae Sar atid coc ystid ae Sapr ae oleg niac eae Salpin goecid ae Saccha romyce taceae Rhodospirilla ceae Rhodocyclaceae

Sip

0

ae id e ec ea e th i p ac a co omi ace r ae t Ce ae hag dace h p a C ino n e it mo Ch ydo llacea e cea lam hlore a h d C C na e a mo e a ac m Co dycipit ae e c Cor a rbit Cucu ae Culicid ae id n Cypri ae Cytophagace

Debaryomycetaceae Diphyllobothriidae Drosophilida e Edward siidae Eimeri idae Euph orbia ceae Fab ace Fla ae vob act eria F cea Ge ormi e cid mi n Ge ige ae rac Gl mm ea ata Ha om e e c lo ba rella eae ct er cea e ia ce ae



e ea ae e ac ac llac e er of chi ae i d al H potr ini ae e r om llid ea He H le rac a e e Hy bact cea bia no e icro me cea a om Hy h p nad Hy homo eae riac Hyp Kofle ceae nella Legio aceae apor th Magn ae Malvace e Mamiellacea Methylobacteriaceae

Rhodobacteraceae Rhizobiaceae aceae Rhamn ae adace o om n eae Pseud c a ti o deur ae Pseu atid tom s ly ae Po d i i l ci Poe iridae dov ae o P e ae e ac Po odiid cea e ta ea m e s c c ia Pla my er cto ct an oba l P yll Ph

Pa O p xa lo ilion ba id Op c te ae Op i hio tuta rac ea sto ce On ma ae e ch tac oc No e e ela erh rcida ae ab Ne da e ctri cea ace Nat ae e rialb a cea Myx e ococ cace Myco ae bacte riacea e Muridae Microbacteria ceae Methylophilaceae

log.counts

5

rd a

lan

Adrianichthyidae Alcaligenacea e Apidae Aptery gidae Apus omo nadid Are ae Asp caceae Ba ergilla thy coc ceae c Br ad Bovi acea e yrh dae B i z r B a o C urk ssic biac au ho ea ac l lo e ba der eae ct iac er ea ac e ea e

Result Report

Family



7 of 178

Xylonomycetaceae

Vitaceae ilionidae Vespert ae Ursid iidae Tupa ae ulid e Trag rida e oca ida Tox en ym rah dae ae i Tet Su oidid e l gy da i l ron ngy ae e id ro a ce na St

St

ric

So

So

12

la

Sc Sc iu his to rida so e Sa m lm on atid Sa i Sa lica dae ae cch ce aro ae my Rh cet ino ace lop ae hid Rhin ae oce rotid ae Retr ovirid ae Ptero podid ae Pseudo monada ceae Pontoporiidae Polystomatidae

0

yc

M

M



ae id e ic a r m id Fo alag idae ae ff ce G a a r r Gi acte ae ob svirid lic ae e He erp mid H ota p e po ida Hip min Ho ae batid Hylo dae Indrii ae Ixodid e bracida Lateola Lemuridae Leporidae

Cricetidae Cyprinidae Dasypo didae Delph inidae Diph yllob othrii Dra dae Ech cunculi dae i n ost En om ter atid ob ac ae Eq teria cea Fa uida e e b a Fl av Fe cea e ob lid ac ae te ria ce ae

Poaceae e Plasmodiida e a d ri te Physe nidae e o c o Ph idae Phoc idae hthy eae c li a r c a P tera ae bac did ae alo o x p O id ero ch e yct r yn a O orh rcid h t e ni ae ae oc d Or i h n c id be chi On o n Od bra o th No

ob ac t u st eria M el us cic ida cea e Mu api e d Mo rida ae e r o Mo rga nidae ne l Mo nod lacea e ont Mon ida aca e nthid a Mixia e ceae Metha nosarc inace ae Manida e Malvaceae

log.counts

4

e da e ni a Ca viid e Ca bida dae i Ce ithec p rco vidae e C Cer ae eid gal eiro ae d li Ch il ch Chin ceae rella Chlo ceae a Cleom iaceae d ri st lo C Clupeidae Coccomyxaceae

Lipotidae

8

Adrianichthyidae Angiostrongy lidae Aotidae Aptery gidae Asca ridid ae Ate li Bab dae esi ida B e Ba alae nid lae Ba nop ae ter thy ida Ca Bo ergi e llo vid dae a r h C am inc e el hid id ae ae

Result Report

Family

Figure 3: Graphical representation of read counts for the 100 most abundant families. In order to represent all detected superkingdoms, the displayed families are selected from the complete result applying the HighestAverages algorithm from the R package SciencesPo. Families are sorted alphabetically, color-coded according to superkingdoms. Note that the graph is in log-scale. lib02099.IonXpress-020-R-2017-03-08-10-32-04-user-SN2-129-lauf121-resultprotocol.pdf

6 of 14

e

h Sc

e da ni e Ca bida idae c Ce ithe op idae c r e v Ce Cer ida ale og ae eir d i l h l C hi inc ae Ch iace strid lo e C a id e Clup ae adace n o m Coma ae ce ita rb Cucu dae Cynocephali

Cyprinidae Dactyloidae Dasypodidae Delphin idae Dixon iellace ae Dros ophil idae Elep han Equ tidae Ery ida thr e ob Es acter ace tril d ae Fa i ba dae ce F Fo elid ae a rm ici e da e

Pipridae

O

er

Lingulidae

M

el a No



ae id ae ag d e i al G iraff da e ini ea G ar ac eg ter tidae Gr bac ma o so e lic He ligmo virida s He ae r pe riid He spe idae He otam pop e Hip erida osid Hipp idae Homin ae Hylobatid Indriidae Lacertidae

Physeteridae omidae Phyllost e enida o Phoc e a id Phoc idae sian ae a h P ace c o c e c o t ida Pep tom e a nta d e ae ri P e yu did Ox opo hida ter ync ae c d y i h r c Or ho cer nit o Or nch O

be ni d bd ae yo v a irid Mu ce Mu steli ae ae d sc ica ae pid Mu a rid ae e Mo ron ida Mim e ivirid ae Man idae Malva ceae Lutjanid ae Lipotidae

0

do

log.counts

5

Sa lp in m ino goe Rh co cid a c ino lop cace e Rh ino hid ae ce a e rot Re i tro viri dae da Pyt e hon id Pte ropo ae dida Pseu e dom onad acea Ponto e poriid ae Polysto matidae Poaceae Plasmodiidae Ru

ha

10

Aotidae Apteryg idae Arena virida e Asca ridid ae Bab esii dae Bac illac B eae Ba alae nid lae Ba nop ae ter thy ida Bu B er e rk ov gida ida h e C old e am er el iac e id ae ae

Vitaceae

Vesper tilionida

Se

Ursidae e chida Triony iidae Tars ae thid gna Syn dae ae Sui id oid gyl idae on l Str gy e on ea e Str ac cea lan a e So astr ae da len urid ati i Sc som to is

15

Adrianichthyidae

Result Report

Family


lib02100.IonXpress-027-R-2017-03-08-10-32-04-user-SN2-129-lauf121-resultprotocol.pdf

7 of 14

Yersiniaceae

aceae Xanthomonad ceae Waddlia idae r tilion Vespe ceae ae Thea atid er m e stod tida uma Tha don rao e Tet ida ae Su did ae loi e gy ac eae on cc c o a Str toc occ ae c rep id e o l St sc hy ni cea he a Sp lan So ap

St

10

Sc

Sc

0

to

Loricidae

e da ni idae a C tor s ae Ca ebid cidae C ithe p e rco ida Ce Cerv e ida n i r a Ch idae r e f li Che idae haer losp e Coe dacea mona Coma e a d ili ch Cono eae Cordycipitac

Coronaviridae Corvidae Cricetidae Crocod ylidae Culicid ae Cypr inida Dicr e oglo ssid Dic ae tyn ida Dip Didel e ph log ida a Dr e ste ac r En Ech unc oidid ae uli i te da ro nod e e ba ct rida er e ia ce ae


False positives: - Retroviridae (WF) - Flaviviridae (RS) - Orthomyxoviridae (RS) - Poxviridae (RS)

e da ui eae c Eq ba ae Fa elid ae F irid e viv a Fla laxiid ae Ga mydid oe ae did Ge nia Go ae inid Hom ae Hylid iidae desm Hyme e Lauriida Leporidae Lipotidae

Poxviridae Pontoporiidae idae Polyclin dae ii il c Poe eae e Poac cea rella a h e p e s a o t id odi Plec sm e Pla cea e a n a Pi nid tidae a i a as rm ae Ph e d llid ae no e a nn ulid Ph Pe c di Pe

O xy O r th sto om m in Or ec yxo ida v t Oo olob irid e a no i On pi dae e ch oc dae e r Nu mid cida No e ida tho e bra nch Myx ii idiid dae a Murid e ae Morax ellace ae Microco ccaceae Malasseziace ae

log.counts

5

yli

or hi Sa so nida m rco at e cy Sa sti idae lm on dae Sa i lica dae cea Rin e gic ulid Ret ae rovir idae Pyxic epha lidae Ptero podid ae Pseudo monada ceae Propionibacte riaceae Proctophyllodidae his

Acipenseridae Acrocirridae Adrianic hthyidae Alliga torida e Anati dae Aot id ae Apt ery Ara gidae As pe ceae rg Bo illac Br vida eae B a C ras mid e al lo sica ae rh in cea ch e id ae

Result Report

Family



7 of 16

10.0 St

Su re id pt a oc oc e ha ca ero the cea So lan r e ac iidae Sc ea iur Sch ida e isto e som Sar atid coc ystid ae ae Salm onid ae Salica ceae Salangid ae Rhodobacter aceae Rhabditidae

Sp

5.0

0.0

as

ec

Ph

Pl

Pl

ae id e ec th lida i p u e co rat r da b Ce ere onii C hth iidae e C us ea Cla ipitac e c i a v d i Cla cculin Co opidae e Con acea ycipit Cord e lida Cordy viridae Corona Cricetidae

Culicidae Cyprinidae Delphinidae Dicroglo ssidae Didym ellace Diph ae yllob o Ecto thriidae biid ae Ele ctri E dae l e En ter phan t ob ac idae t Et Equ eriac ida m ea e Fa opte e rid ba ae ce ae


False positives: - Coronaviridae (RS) - Flaviviridae (RS) - Orthomyxoviridae (RS) - Poxviridae (RS)

ae e lid da e Fe iri ea v c i lla av Fl ere ae e rid ida om t b a e Gl m H so mo nidae e lig o ida He Hesi tom ren spe inidae He e Hom hiida prop Lam e a d Lepori ae Malvace Mimetidae Muridae

Reoviridae ae Pyxicephalid hyidae Pycnop ae adace omon Pseud ae id il r d ae Proto iace cter niba e io a p e d Pro viri ida Pox nch rhy idae o h nth e mp a ca Po ma enid Po lyx eae ae o P tid ac Po nec ro u e Pl

to sp h

log.counts

2.5

ae Ph mo rell d a yll iro iida cea ida e as e i Pa an e ss ere idae Os l ph ron lidae Or t hom emid ae y x Orn ovi r itho rhyn idae c hida Opit e utac eae Nymp honid ae Nectriace ae Mycoplasmata ceae Muscidae

7.5

Xanthomonadaceae Waddliaceae e Vitacea idae r tilion Vespe ridae Vene ae onid tryg e Uro ida bor ae Ulo nid ula e Tub hiida e a c Tri rid e ca cea e xo a a To e tid ini gn don ilida To o h tra op Te ing r Sy

12.5

Adrianichthyidae Ammotheida e Anatida e Aporo cotylid ae Apte r Bala ygidae mut hiid ae Bra Bovi d d Bu yrhiz ae rkh ob iac ol ea C deri e Ca anid acea e a s e C ep Ce torid ha bid ae lo ba ae en id ae

Result Report

Family



7 of 15

hi

st

os om at ha rom ciac idae Rh op e a alo yce tac e so Pte rom matid eae Pse ae alid udo a mo nad e Pleo a c spo race eae Plas ae mod iidae Plagio rchiid ae Phytose iidae Phasianidae Pediculidae

Sa

cc

cc

ot

ae ce dae i ra te astr s ae A cig rid e a l ae d i u r iid A oth ad B hycl e ida ac Br racon nidae e B oba e dyn Bra rentida B ae d ti s e Bupr llidae e n re Bu ae acteriace Cardiob e Cephida

he

ph ae re s lla m M u sc ata cea Mu ida ce sc ae e Mo ic e rga apid Me n ae ell tsc a hn iko ceae Me tha wia noc cea Met occ han ace e oba ae cter Leuc iace ospo ae ridia c e ae Latrid iidae Lactoba cillacea e Iflaviridae Ichneumonidae la

op

M

yc

M

Cerambycidae Chironomidae Chrysomelid ae Cladosp oriacea e Clavic ipitac eae Cler idae Clo strid iace C ae Cu orvida cur e bita C cea Cu ulici e da rc e De Cy ulio ba nid p r ry ae om inida e yc et ac ea e



ae rid vi ae tro ilid is ph ae ic D oso riid e e Dr ime ida ea E pid riac te e Em c a cea a ob occ ter e En teroc cea En inia Erw didae il Estr ceae Faba icidae Form idae Hirundin Hominidae Hybotidae

Paridae Papilionidae ceae rdycipita Ophioco idae rc e c o Onch idae metr halo e Omp d li a pha e Nym tida a sem idae No r i dav dae e No li ea idu Nit gitac ae y e z c ae o ia id Ne ctr yet e h N p no Na

yc os

log.counts

0

Yersiniaceae

5

Sc

Sa

Vitaceae e Vespida tidae Tephri idae brion Tene ae phid Syr ae e rnid ida S tu oid ae gyl ce on ae ca Str oc ce ca e toc oc ep ida Str loc hy nisc ae e ap e a he St ac ce Sp lan a ni So roti le Sc

10

Acanthaceae Acar tophthalm idae Acetoba cterace ae Aerom onada ceae Agao nida Ana e plas Ana matace ae xye Ap lida he e Ap linida hid e id Ap Apid ae ae As ter co yg i i As dea dae ilid ce ae ae

Result Report

Family



7 of 77

7.5

5.0

0.0

Sp or Sp hin idio bo go Sip bac lace te Se ae h r ov len irid iace om Sa a a o cch e e aro nada c my Ru cet eae min a oco cca ceae Ros cea ace e a e Rike nella ceae Rhod obacte racea e Rhizopo daceae Rhizobiaceae

ae id er ae e b a ea Bl ovid biac e B izo ea c yrh deria d l e a Br rkho mida e cea Bu aria C eria t c a e b a o id rn Ca ntropom eae Ce ellac n o tt eae Cha nellac tense Chris ae ce ita p Clavici e Clostridiacea

Pseudomonadaceae

O

Columbidae Comamonadaceae Cordycipitac eae Corioba cteriace ae Coryn ebacte Desu riacea e lfovib riona ceae Ec Egg tobiida e er th En ell te En robac aceae ter Er tero iac ys co ea e Eu ipelo ccac ea tric ba e c h t a Fa er c e i a ba ae ce cea e ae


False positives: - Togaviridae (RS)

ae ce ria e te a id ae ac ob mic ace av For iger ceae Fl in tera e m c da Ge oba nchi Ge emo itidae e Ha xam ida He der i pos ae Hip inid Hom eae triac ochy Hyph eae creac Hypo ae spirace Lachno eae Lactobacillac Lobulomycetaceae

Propionibacteriaceae ae Prevotellace e Poacea eae rellac sphae Plecto idae ir v a birn Pico idae eae sian cac Pha coc ae o t p e e r t cac ae tos e coc Pep pto rellac e P teu idae e s e a r Pa Pa chid cea i a ytr cter x O ba lo xa O

O sc ph i io llos p Od cor dy irac or iba cip ea c ita e Nu t mi erac ce did Ne ea ae ctr a e iac e Na e tria lba ae Mu cea riba e cul Mor ace gan ella ae cea Mora e xella ceae Micro cocca ceae Microba cteriace ae Methanobacte riaceae Malasseziaceae

log.counts

2.5

Yersiniaceae

10.0

Vibrionaceae llaceae dis Veillone e Se iridae cer ta Togav IV. In mily s Fa e terale iida obac e igon naer Tett moa cea Ther ella ae ner e lac ceae Tan rel na tte rio ae Su e vib d i n ini ea e o cc thi ccac cea Su ru a o e St oc cc ea pt co ac re ylo us St h om ap St or Sp

12.5

Accipitridae Actinomyceta ceae Akkerm ansiace ae Alliga torida Alloio e nem atida Ana e Apt tidae e ryg As ida p e Ato ergilla c p Ba obia eae Ba ce cil ae la B c Bi arn tero cea e i fid d e ob sie ace ae ac lla c te ria eae ce ae

Result Report

Family



6 of 24

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A Versatile Sample Processing Workflow for

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