and Standard PCR Library Amplification/Illumina series (KAPA Biosystems, Code ... Quantification Kit (KAPA Biosystems, Code KK4835) and pooled together at ...
Supplemental Materials Supplemental Methods DNA Sequencing. DNA Library preparations for WGS were performed using the KAPA Library Preparation Kits with SRPI Solution and Standard PCR Library Amplification/Illumina series (KAPA Biosystems, Code KK8232) with the following modifications. The adapters and 8bp index oligos purchased from IDT® (Integrated DNA Technologies, San Diego, CA) based on Kozarewa and Turner, 2011, were used in place of those supplied in the KAPA preparation kit. Quality and quantity of genomic DNA was evaluated by 0·7% agarose gel analysis. DNA per sample (~1 µg) was fragmented using a SonicMan (Matrical) with the following parameters: 75·0s pre chill, 16 cycles, 10·0s sonication, 100% power, 75·0s lid chill, 10·0s plate chill, and 75·0s post chill. The fragmented DNA was size selected to target 600-650bp by fragment separation using the Agencourt AMPure XP beads (Beckman Coulter, Code A63882) and eluted into 42·5µl of Elution Buffer. During the enzymatic steps, half the enzyme volume was used with the full volume of buffer as described in the KAPA Library Preparation protocol. For library preparation, three separate reactions for each sample were carried out as follows: 1) End repair: 2·5 µl Enzyme, 5 µl Buffer, 1·6X AMPure XP, Elution buffer 43·5 µl, 2) A-Tailing: 1·5 µl Enzyme, 5 µl Buffer, 1·6X AMPure XP, Elution buffer 36·5 µl and 3) Quick Ligation: 2·5 µl Enzyme, 10 µl Buffer, 0·9X AMPure XP, Elution buffer 30 µl. The adapter ligation step used 1 µl of the 10 µM adapter oligo mix (Kozarewa and Turner, 2011). The PCR was optimized to improve yield and genome coverage. Two µl of DNA, two µl of each 10 µM indexing primer, 25 µl of KAPA 2X HIFI PCR Master Mix (KAPA Biosystems), and 19 µl of molecular grade water were combined. The following PCR parameters were used: initial denaturation 2 min at 98°C, 8 cycles of 30 sec at 98°C, 20 sec at 65°C, 30 sec at 72°C, with a final extension of 5 mins at 72°C. The libraries were purified with a 0·9X AMPure XP bead cleanup and eluted into 50.0µl of Elution Buffer. The final libraries were quantified using the KAPA ABI Prism Library Quantification Kit (KAPA Biosystems, Code KK4835) and pooled together at equimolar concentrations. The pool was quantified using the KAPA ABI Prism Library Quantification Kit, and the quality of the pool was assessed with a Bioanalyzer DNA 1000 chip (Agilent Technologies, Code 5067-1504). The samples with 1% PhiX were sequenced on a MiSeq using the 600-cycle MiSeq Reagent Kit V3 (Illumina, Code MS-102-3003) with the standard Illumina procedure. The appropriate sequencing primers were added to the MiSeq kit as in Kozarewa and Turner, 2011. VNTR analysis: Multi Locus Variable number of tandem repeat Analysis (MLVA) for B. anthracis was performed as previously reported 1, 2. Where possible, in silico VNTR information was extracted from genome data sets and combined with results from MLVA-PCR. melt-MAMA PCR for SNP genotyping. We designed 31 melt-MAMA real-time assays following design guidelines published in Birdsell et al 2012. Primers were designed using Primer Express 3·0 software (Applied Biosystems, Foster City, CA) or a free on-line primer design software by Premier Biosoft (http://www.premierbiosoft.com/netprimer/). Each Melt-MAMA reaction mixture (7 µl or 10 µl) contained 1x SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) and amplified on ABI 7900 instruments (Applied Biosystems, Foster City, CA). Thermocycling parameters were 50°C for 2 min, 95°C for 10 min, and then cycled at 95°C for 15s and 55°C - 60°C (dependent on assay; see Table S3) for 1 min for 34 cycles. Four negative water controls were included for every assay. End-point PCR products were subject to melt analysis using a dissociation protocol comprising 95°C for 15 s, followed by incremental temperature ramping (0·2°C) from 60°C to 95°C. SYBR Green fluorescent intensity is measured at 530 nm at each ramp interval and plotted against temperature. For one clinical sample (P35 A4569), we used a nested PCR strategy to SNP genotype. We designed external primers (unpublished) targeting a 300 bp fragment encoding the SNP loci of interest. We then diluted this 300 bp fragment 1/100 – 1/1000 with molecular grade water and SNP genotype the diluted fragment using relevant Melt-MAMA SNP-assays (Table S3).
Supplemental Results and Discussion VNTR Analysis. MLVA is a valuable and commonly used tool for differentiating B. anthracis strains 1, 2. Whereas whole genome sequences are becoming more common for this task, large databases from past MLVA work exist and coordinating these with the increasing popularity of WGS is valuable for bridging these different genotyping formats. We have used a mixture of in silico VNTR genotyping and with PCR directly on the specimens to generate MLVA data for these strains (Table S4). As with previous reports 3, we found that the two IA Groups differed at a single VNTR locus on the pXO2 plasmid. Group-I (G-I) has a 7-repeat allele, whereas most of Group-II (G-II) has the 8-repeat allele. The exception in G-II is the single Norwegian isolate with its 9-repeat allele. The non-IA members of the subclade carry the 7-repeat allele, suggesting that this is the ancestral allelic state. This dinucleotide “AT” repeat locus is highly diverse in global strain studies 1, 4 and undoubtedly has a high mutation rate 5. The single-repeat differences imply a stepwise mutational process where the G-II ancestor mutated to 8 repeats and the Norwegian isolate ancestor once more to 9 repeats.
Fig S1. IA-Group
I and IA-Group II Isolate Diversity and Country of Origin.
Each IA group is dominated by an ancestral genome type (genomes within the circles) with minor variants derived from them and differentiated by SNPs. SNPs are represented by red hash marks on the radiating lines. Group I (N=49) contains 27 ancestral genomes and 22 derived genomes, 16 of which were unique. Shared SNPs (synapomorphic) identify 6 derived pairs, with 4 of the pairs from the same 4 IA victims (P1, P12, P18, P20). However, two of the pairs were observed from different IA victims (P5/10 and P12/22) arguing for diversity in the original B. anthracis spore populations rather than just mutations generated within the victims. Group II has six ancestral and five derived genomes. One clinical sample, for which an isolate was not obtained, was phylogenetically placed in G-II by PCR and remains collapsed in the ancestral base. Two genomes are singularity unique by one SNP but are from the same IA case and the other four genomes are derived from unique IA cases.
Figure S2. The lack of positively correlated temporal signal between branch length and date of isolation.
Substitutions/Site
Maximum likelihood and Bayesian phylogenies were reconstructed in MEGA6.0 and BEAST v1.8.1, respectively. A linear regression of root to tip distances (y-axis) plotted over time (x-axis) indicates a lack of positive linear correlation, which is a fundamental assumption in all BEAST models. As such, this data set could not be used to construct a time-calibrated phylogeny.
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Bayesian Skyride
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y = -0.003x + 6.1693 R² = 0.1989
Maximum Likelihood
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