Workstation for High Throughput Sequencing Workflows. Jeremy Lambert, Nate Cosper, Raymond Basherad, Bill Thompson, Kevi
Development of an Automated Sample Processing Workstation for High Throughput Sequencing Workflows Jeremy Lambert, Nate Cosper, Raymond Basherad, Bill Thompson, Kevin Keras, Nicholas Lin, Michael Benway Caliper Life Sciences, Hopkinton, MA USA
TruSeq RNA Sample Prep Process Map and Deck Layout
Plate Parking / Storage
RNA Purification Beads
80% EtOH
Magnet
Water
22°C / 16°C Shaker
Tip Waste
150 µl barrier tips
150 µl barrier tips
150 µl barrier tips
Stacked PCR Plates x4
150 µl barrier tips
150 µl barrier tips
65°C / 80°C / 94°C
150 µl barrier tips
HeLa Cell Total RNA
Figure 7. QC of 96 Total RNA starting samples.
Agilent
SureSelect
4°C-95°C
4
18
Applied Biosystems
SOLiD Fragment Library Prep
4°C-22°C
2
14
Applied Biosystems
SOLiD Total RNA-Seq Kit
4°C-70°C
3
34
Epicentre Biotechnologies
Nextera DNA Sample Prep Kit
4°C-55°C
3
12
Illumina
TruSeq RNA Sample Prep
4°C-95°C
4
46
Illumina
TruSeq Small RNA Sample Prep
4°C-72°C
4
16
Illumina
TruSeq Exome Capture
4°C-42°C
3
24
New England Biolabs
NEBNext DNA Sample Prep Master Mix Set 1
4°C-37°C
3
22
New England Biolabs
NEBNext mRNA Sample Prep Master Mix Set 1
4°C-94°C
4
28
Nimblegen
SeqCapEZ
4°C-47°C
4
18
NuGEN
Encore NGS Library System I
4°C-70°C
3
24
NuGEN
Ovation RNA-Seq System
4°C-98°C
3
32
Pacific Biosciences
Sample Prep (250 bp)
4°C-37°C
3
24
Library Preparation
Figure 1. Sciclone NGS Workstation. The Sciclone NGS workstation has been engineered with a total of 24 deck locations including three thermal locations for heating and cooling and one heated shaker. The 96 channel pipetting head and integrated plate gripper allows for unattended processing of a wide range of NGS sample processing applications. An environmental enclosure (not shown in photo) provides a clean and low maintenance workspace.
500
800
400
600 400 Average = 601 SD = 67
200
300 200
Empty Box / Plate Waste
150 µl barrier tips
150 µl barrier tips
150 µl barrier tips
4°C Master Mix Plate 1
4°C Master Mix Plate 2
Liquid Waste Plate
150 µl barrier tips
150 µl barrier tips
150 µl barrier tips
Plate Parking / Storage
Ampure XP Beads
80% EtOH
150 µl barrier tips
150 µl barrier tips
150 µl barrier tips
Stacked PCR Plates x3
Magnet
150 µl barrier tips
150 µl barrier tips
150 µl barrier tips
30/37°C
150 µl barrier tips
22°C Shaker
Tip Waste
A-Tailing
Adapter Ligation
Average = 262 SD = 11
100 0
Sample Number
Sample Number
PCR Enrichment Figure 8. mRNA Seq Library QC Metrics. Analysis of post-PCR mRNA Seq libraries was performed using the LabChip GX High Sensitivity DNA Assay. All 96 samples met the quality control metrics defined in the TruSeq RNA Sample Prep User Guide.
Summary
Figure 4. Deck Layout for Library Construction. The Sciclone NGS is easily reconfigured between different applications. A simple GUI guides the operator through the setup of the reagents and consumables.
The Sciclone NGS is a robust and flexible solution for sequencing sample prep, enabling between 8 and 96 samples to be processed in parallel, covering a wide range of applications from basic paired end library preparation, to more complex methods including sequence capture, RNA-seq and ChIP-seq all on a single platform. Paired with the Zephyr Genomics Workstation for DNA and RNA extraction, LabChip DS for high-throughput UV/Vis absorbance, LabChip GX instrument for high-throughput analytical electrophoresis and LabChip XT for automated size fractionation, the Sciclone NGS completes a suite of highperformance automated sample processing technologies designed to meet the growing needs of high-throughput sequencing laboratories.
Sciclone NGS Workstation 96 channel pipetting(1-150 µl barrier tips) Left Gripper Four sided enclosure w/access doors 24 deck positions 4-70°C Shaking Locator Three 4-110°C Temp Locators Tip waste chute Tip box / plate waste chute Maestro/GUI software
1000
0
End Repair
Table 1. Representative survey of high-throughput sequencing sample prep requirements considered for developing specifications for automated workstation.
mRNA Seq Fragment Length
mRNA Seq Library Yield
96
38
84
4
72
4°C-72°C
60
cDNA Rapid Library Prep
Figure 3. Detailed Process Map for mRNA Purification and cDNA Synthesis. User intervention steps are shown in yellow boxes. Less than 30 minutes hands-on time is required to process anywhere from 8-96 samples per day. The software integrates directly with MS Excel worksheets for sample normalization prior to mRNA purification.
48
454
Figure 8. Analysis of final purified library samples.
36
Estimated Number of Tip Boxes for 96 Samples
Rat Brain RNA Seq Library
24
Number of Temp. Controlled Positions
HeLa Cell RNA Seq Library
0
Require d Temp. Range
Rat Brain Total RNA
96
150 µl barrier tips
84
150 µl barrier tips
72
150 µl barrier tips
60
Liquid Waste Plate
48
4°C Master Mix Plate
36
4°C
48 replicate samples of 1 µg total RNA obtained from HeLa Cell and Rat Brain was processed through the automated TruSeq RNA Sample Prep method. A total of 96 samples were processed in parallel over a 2-day workflow. QC of total RNA samples was performed using the Caliper LabChip HT RNA Assay and LabChip GX instrument. All samples reported RNA Quality Scores (RQS) between 7.1-8.6.
24
150 µl barrier tips
Experimental Design
12
150 µl barrier tips
1st and 2nd Strand Synthesis
0
Empty Box / Plate Waste
150 µl barrier tips
mRNA Purification and Fragmentation
Post-PCR Yield (ng)
Kit or Protocol Name
Total RNA Normalization and mRNA Capture
Figure 2. Deck Layout for mRNA Purification and cDNA Synthesis. The figure illustrates the deck configuration for automated processing of up to 96 samples through the Illumina TruSeq RNA Sample Prep protocol in under five hours.
Survey of Protocol Requirements Vendor
mRNA Purification and cDNA Synthesis
12
Recent advances in data generation capacity for high throughput sequencing technologies has resulted in sample preparation bottlenecks previously observed in other market applications, such as small molecule drug efficacy screening. Taking advantage of best practices from other industries, Caliper has developed an automated platform that is appropriate for a wide range of applications. In addition to supporting reagents kits from the sequencer instrumentation vendors, methods are available for kits provided by several leading third party reagent providers including multiple sequence capture protocols. Recognizing that sequencing technology and methods rapidly change, deck accessories with wide operating ranges have been chosen for this platform to create a robust and flexible solution. A detailed workflow for mRNA sequencing is presented along with performance data demonstrating reproducibility and quality of resulting libraries with a throughput of as many as 96 libraries per day.
Results from Automated Processing of 96 RNA Seq Samples
Peak Size (bp)
Abstract
Isolate Sample Isolation/Purification DNA and/or RNA
Fragment Sample
Generate Library
Quant. Sample
Shear/Fractionate Size select fragments
Library Generation - Enzymatic reaction - Purification
Quantitation & Sample Analysis Purity and integrity
Sequence
Sequencing Seq. chemistry
Zephyr Genomics Workstation
Figure 5. Image of Sciclone NGS deck configured for library preparation. The gripper mounted on the left of the pipetting head provides access to all 24 deck locations for automated tip box management.
Figure 6. Detailed Process Map for Library Construction from cDNA. User intervention steps are shown in yellow boxes. A simple MS Excel spreadsheet is used to define the indexing plate map for workflows utilizing indexing and pooling of multiple samples. Less than 30 minutes of hands-on time is required for processing up to 96 samples from End Repair to PCR purification.
DNA and RNA Isolation
LabChip DS 96-channel microvolume UV/VIS
Sciclone NGS Sample prep including mRNA-Seq and Sequence Capture
LabChip XT Automated Nucleic Acid Fractionation
LabChip GX Library QC
