Validation of the Agilent BioCel Automated Platform for Single Cell Genomic Analysis Mark Novotny, Project Lead Joyclyn L. Yee-Greenbaum Jeffrey S. McLean Shino Ishii Mary-Jane Lombardo Todd Hughes Robert Vandenberg Roger S. Lasken (PI)
Single Cell Genomics • What is it? The study of a genomic sequence obtained from a single cell • Why is it useful? Most bacterial cells are uncultivable • What benefits can be achieved by automation? High-throughput access to numerous unknown genomes Raghunathan, A. et al . Appl. Environ. Microbiol. 71 , 3342–3347 (2005). Zhang, K. et al . Nat. Biotechnol. 24 , 681–687 (2006). News and Views Nature Biotechnology 24 , 657 - 658 (2006). Single-cell genomics Clyde A. Hutchison, III 1 & J. Craig Venter 1 Penny Chisholm (Broad), Ramunas Stepanauskas (Bigelow Lab), Tonya Wyoke (JGI) Mitch Leslie Science 7 January 2011: Vol. 331 no. 6013 pp. 24-26
Isolation of FISH Probed Bacterial Cells by Micromanipulation • Single cells labeled with 16S rRNA gene probes • Isolation by micromanipulation • DNA amplified by MDA Lasken R, Raghunathan A, Kvist T, Ishøey T, Westermann P, Ahring B, Boissy R In Whole Genome Amplification: Methods Express Edited by Hughes S., Lasken, R. Oxford: Scion Publishing Ltd. 2005
Single Cell Sequencing Funded by DOE, 2001, at Molecular Staging, Inc. Raghunathan, A., Ferguson, H.R., Bornarth, C.J., Driscoll, M., and Lasken, R.S. Applied and Environmental Microbiology (2005) Vol. 71, 3342-3347 E.coli E.coli E.coli B.subtilis B.subtilis B.subtilis Flow cytometry Noise Noise Noise Noise Noise Noise DNA Amplification by Multiple displacement amplification (MDA) >10 9 fold amplification Genomic DNA Genotype and Sequence
Multiple Displacement Amplification (MDA) Dean FB, Nelson JR, Giesler TL, Lasken RS (2001) Genome Res. 11, 1095-9 Dean FB, Hosono S, Fang L, et al. (2002). Proc Natl Acad Sci USA;99:5261-6 REPLI-g 12.0 kb TempliPhi, GenomiPhi (Qiagen) (GE Healthcare/Amersham) Genomic DNA Alkaline agarose 1. Random hexamer primers 2. Phi29 DNA polymerase Strand displacing 3. Isothermal reaction (30 C)
Single Cell MDAs Vary in Quality Illumina Sequencing Redundancy Great MDA gDNA Poor MDA 98% genome in contigs top contig 14k top contig 67k N50 45 N50 68k 600x, kmer=55
Single Cell Genomics Pipeline Discovery platform for producing sequenced genomes from single cells
11 Major Steps in the Process 1. Single cell Fluorescence Activated Cell Sorting (FACS flow cytometry) 2. MDA 3. 16S or 18S PCR or qPCR 4. PCR analysis: melt curve assay 5. PCR hit picking 6. PCR SAP/Exo cleanup 7. Sanger sequencing 8. 16S or 18S characterization 9. MDA hit picking and re-amplification 10. Whole genome sequencing 11. MDA archival
High Throughput Fluorescence Activated Cell Sorting on BD FACS Aria II Shewanella sp. Sybr Green fluorescence T4 phage Forward scatter • 488nm 100mW laser • FSC-PMT detector • SSC PMT detector • 3 color detection (2 Green, 1 Red) • One 384-well plate with 384 single cell in 8-10mins • Capable of detecting up to 70,000 events/sec • >97% accuracy of 1 bead/well • Sort cells into 2uL of TE (10:0.1)
FACS: Scripps Pier Seawater 30µm Filtered DNA Stained with SYBR Green I Unstained Stained Marine organisms
Marine Single Cell Genomics 2 papers accepted and 1 more planned shallow deep de novo Single MDA (closure) annotation sequencing sequencing assembly Genome cell Uncultured, highly abundant SAR86 clade SAR86 single cell genomes SAR86-1, 50% SAR86-2, 50% SAR86-3, 10% SAR86-4, >95% in closure
Marine Single Cell Genomics shallow deep de novo Single MDA (closure) annotation sequencing sequencing assembly Genome cell Uncultured SAR324 clade SAR324 single cell genomes SAR324-1, 75% SAR324-2, in assembly SAR324-3, in assembly SAR324-4, in assembly
Single Cell Amplified Genomes From Human Oral Microbiome EtOH fixation Nycodenz Staining or Sorting Filtration Cell fraction Oral cavity sample A B Unstained Stained
Automation Design Challenges Key Requirements • 5,000 Single Cells processed per week • Non-contact pipetting of reagents for amplification • <6 months to complete 100,000 cell screen • Minimal waste from expensive reagents • 1 and only 1 cell per well • Filtered tips for cross-contamination control • 384-well format • Adequate mixing of reagents during reaction assembly • Integrated automation with 3 rd party platforms • Pipetting of viscous solutions • 4C incubation and reaction temperature control • Contamination control • Sophisticated timing mechanisms to meet assay • Free DNA time restraints • Well-to-well cross-contamination • Isolate cells from complex mixtures • Amplicons-MDA, PCR, Sanger cycle sequencing • Air (water impaction) • Highest quality reagents • Soil (Nycodenz) • Lot testing and validation • Seawater (filtering) • Sealed amplification reactions • Human gut (Nycodenz) • Centrifugation during reaction assembly • Oral biome (Nycodenz/filtration) • Turn-key operation with minimal staffing • Icon-driven software with minimal training and skill required Etc., etc., etc.
Agilent Design: BioCel Single Cell Genomics
Agilent Design: BioCel Single Cell Genomics BioRAPTR FRD Liquid Handler Bravo Liquid Handler ABI 7900HT qPCR Direct Drive Robotic Arm Mecour Plate Tower Barcode Labeler Plate Hub Liconic Chilled Incubator Vspin Centrifuge Vworks Workstation PC PlateLoc Plate Sealer Process >14x384 well plates/week >5,000 single cells/week
BioCel Subsystems: BioCel 1200 and 2 Device Tables ~7’ L x 7’ W x 7’ H
BioCel Subsystem: Agilent DDR • Fast • Accurate • Precise • Compact • Easy to program • Efficient movement • Large range of movement • Portrait and landscape
BioCel Subsystem: Agilent Bravo • Fast • Accurate (750nL-50uL) • Precise <5% CV • Easy to program • Efficient movement • 384-tip pipeting • Disposable Filter tips (no washing) • Low retention in tips • Hit picking • Chilled platform • Gripper • Weigh station (reservoirs) • Easy to clean/decontaminate
BioCel Subsystem: Beckman BioRAPTR • Fast (30sec -1min/plate) • Accurate (150nL-60uL) • Precise <5% CV • Non-contact pipeting • Integration with BioCel • Easy to program • Controllable through Agilent VWorks • Efficient movement • Random access to wells • 8-Tip dispenser head • 8 Sealed Reagent Reservoirs • 8 Removable Chillers (Cooling Nest) • Volumes compatible (150nL-50uL) • Low vol. retention in tips and lines • Easy to clean/decontaminate
BioCel Subsystem: ABI 7900HT • qPCR Taqman chemistries • Syto9 melt curve capability • Sybr Green qPCR • 96-and 384-well formats • Automation capable (but not friendly) • Custom compatible with Vworks
BioCel Subsystems: Incubators and Temperature Controls • BioRAPTR Cooling Nest • Liconic and Liconic plate trays • Polyscience chiller • Chills Mercour tower • Chills Cooling Nest • Thermocube chiller • Chills Bravo deck 3-plate platform
BioCel Subsystems: Vspin, Vcode, and PlateLoc • Vspin • Meets g requirements • Fast • Counterbalanced • PlateLoc plate sealer • Compatible with all plates • Seal works with ABI 7900HT • Warms up fast • Vcode barcode labeler • JLIMS compatible • Thermostable @ -80C
BioCel Subsystems: Plate Hub, Barcode Reader, Lid Tower, Lid Tool • Plate Hub • Capacity for 14 plate runs • Holds tip boxes • Holds Framestar and Greiner plates • Barcode Reader • Vworks compatible • Lid Tower • Holds lids for 14 plate runs • High performance and precision • Low error rate • Compatible with Greiner low profile lid • Lid Tool
ABI 9700 Thermocyclers • Dual 384-well ABI 9700’s • 12 total in the lab • 24 plate capacity • MDAs, PCRs, Lysis
BioCel Software: VWorks • Controls all major components and subsystems • Incredibly sophisticated event-driven software • Efficient task control with error recovery • Icon-driven programming • Ease of re-programming • Tons of features
BioCel Software: JLIMS • Custom JCVI LIMS Software • Web driven • Designed for Sanger Pipeline at JTC • Modified for Single Cell Pipeline • Currently evaluating (not implemented) Register Register Samples (either pre-barcoded or Sample requiring new barcoding) with the sytem Cell Sorting Generate Plate Generate plate barcodes Barcodes MDA Prep MDA Dilution Prepare the MDA plate 16s PCR Melt Curve Create the hit plate based on Melt Curve scores Analysis Run 16s Analysis pipeline 16s Analysis MDA Hit Plate Create the MDA Hit Plate
Recommend
More recommend
