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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
Mitch Leslie Science 7 January 2011:
Vol. 331 no. 6013 pp. 24-26
News and Views Nature Biotechnology 24, 657 - 658 (2006).
Single-cell genomics Clyde A. Hutchison, III1 & J. Craig Venter1
Zhang, K. et al. Nat. Biotechnol. 24, 681–687 (2006).
Raghunathan, A. et al. Appl. Environ. Microbiol. 71, 3342–3347 (2005).
Penny Chisholm (Broad), Ramunas Stepanauskas (Bigelow Lab), Tonya Wyoke (JGI)
• Single cells labeled with 16S rRNA gene probes • Isolation by micromanipulation
• DNA amplified by MDA
Isolation of FISH Probed Bacterial Cells by Micromanipulation
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
DNA Amplification by Multiple displacement amplification (MDA)
Genotype and Sequence
Funded by DOE, 2001, at Molecular Staging, Inc.
Genomic DNA
>109 fold
amplification
Raghunathan, A., Ferguson, H.R., Bornarth, C.J., Driscoll, M., and Lasken, R.S. Applied and Environmental Microbiology (2005) Vol. 71, 3342-3347
Noise
B.subtilis
Noise
E.coli
Noise
B.subtilis
Noise
B.subtilis
Noise
E.coli
Noise
E.coli
Flow cytometry
Single Cell Sequencing
Genomic DNA
Multiple Displacement Amplification (MDA)
1. Random hexamer primers 2. Phi29 DNA polymerase
Strand displacing 3. Isothermal reaction (30 C)
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
12.0 kb
Alkaline agarose
TempliPhi, GenomiPhi (GE Healthcare/Amersham)
REPLI-g (Qiagen)
gDNA
600x, kmer=55
Great MDA 98% genome in contigs
top contig 67k N50 68k
Single Cell MDAs Vary in Quality
Illumina Sequencing Redundancy
Poor MDA top contig 14k
N50 45
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
T4 phage
Shewanella sp.
Syb
r Gre
en fl
uore
scen
ce
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)
Marine organisms
Stained Unstained
FACS: Scripps Pier Seawater 30µm Filtered DNA Stained with SYBR Green I
Marine Single Cell Genomics 2 papers accepted and 1 more planned
Genome
SAR86-2, 50%
SAR86-1, 50%
SAR86-3, 10%
Uncultured, highly abundant SAR86 clade
SAR86-4, >95% in closure
SAR86 single cell genomes
MDA shallow
sequencing deep
sequencing Single cell
de novo assembly annotation (closure)
Marine Single Cell Genomics
Uncultured SAR324 clade
MDA shallow
sequencing deep
sequencing Single cell
de novo assembly annotation (closure)
SAR324-2, in assembly
SAR324-1, 75%
SAR324 single cell genomes
SAR324-3, in assembly
SAR324-4, in assembly
Genome
Nycodenz or
Filtration Oral cavity sample Cell fraction
EtOH fixation Staining Sorting
Single Cell Amplified Genomes From Human Oral Microbiome
B A
Stained Unstained
Automation Design Challenges
• 5,000 Single Cells processed per week • <6 months to complete 100,000 cell screen
• 1 and only 1 cell per well • 384-well format • Integrated automation with 3rd party platforms • 4C incubation and reaction temperature control • Sophisticated timing mechanisms to meet assay
time restraints • Isolate cells from complex mixtures
• Air (water impaction) • Soil (Nycodenz) • Seawater (filtering) • Human gut (Nycodenz) • Oral biome (Nycodenz/filtration)
• Non-contact pipetting of reagents for amplification • Minimal waste from expensive reagents • Filtered tips for cross-contamination control • Adequate mixing of reagents during reaction assembly • Pipetting of viscous solutions • Contamination control
• Free DNA • Well-to-well cross-contamination • Amplicons-MDA, PCR, Sanger cycle sequencing • Highest quality reagents • Lot testing and validation • Sealed amplification reactions
• Centrifugation during reaction assembly • Turn-key operation with minimal staffing • Icon-driven software with minimal training and skill required
Key Requirements
Etc., etc., etc.
Agilent Design: BioCel Single Cell Genomics
Process >14x384 well plates/week >5,000 single cells/week
BioRAPTR FRD Liquid Handler
ABI 7900HT qPCR
Direct Drive Robotic Arm
Bravo Liquid Handler
Liconic Chilled Incubator
PlateLoc Plate Sealer
Vspin Centrifuge
Plate Hub
Barcode Labeler
Vworks Workstation PC
Mecour Plate Tower
Agilent Design: BioCel Single Cell Genomics
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
Register Sample
Register Samples (either pre-barcoded or requiring new barcoding) with the sytem
Generate plate barcodesCell Sorting
Prepare the MDA plate
MDA Hit PlateCreate the MDA Hit Plate
Generate Plate Barcodes
MDA Prep
MDA Dilution
16s PCR
Melt Curve Analysis
16s Analysis
Create the hit plate based on Melt Curve scores
Run 16s Analysis pipeline
• Custom JCVI LIMS Software • Web driven • Designed for Sanger Pipeline at JTC • Modified for Single Cell Pipeline • Currently evaluating
(not implemented)
SCGP-Design Checklist
• 5,000 Single Cells processed per week • <6 months to complete 100,000 cell screen
• 1 cell and 1 cell only • 384-well format • Integrated automation with 3rd party platforms • 4C incubation and reaction temperature control • Sophisticated timing mechanisms to meet assay
time restraints • Isolate cells from complex mixtures
• Air (water impaction) • Soil (Nycodenz) • Seawater (filtering) • Human gut (Nycodenz) • Oral biome (Nycodenz/filtration)
• Non-contact pipetting of reagents for amplification • Minimal waste from expensive reagents • Filtered Tips for cross-contamination control • Adequate mixing of reagents during reaction assembly • Pipetting of viscous solutions • Contamination control
• Free DNA (although cannot always be ruled out) • Well-to-well cross-contamination • Amplicons-MDA, PCR, Sanger cycle sequencing • Highest quality reagents • Lot testing and validation • Sealed amplification reactions
• Centrifugation during reaction assembly • Turn-key operation with minimal staffing • Icon-driven software with minimal training and skill
required
Etc., etc., etc.
SCGP Process Validation
1. Single cell Fluorescence Activated Cell Sorting (FACS flow cytometry) a) FrameStar 2uL Plate Setup
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
BioCel Validation: Step 1a: Framestar Plate Setup
•Created routine to fill 64 x 384-well plates with 2uL TE using 1 box of tips
•Pipettes TE from a trough on the Bravo deck
•Eliminates the need for BioRAPTR to fill empty plates
•Poses a risk to contaminating FACS sorting plates with gDNA on surfaces or amplicons
1. Single cell Fluorescence Activated Cell Sorting (FACS flow cytometry) a) FrameStar 2uL Plate Setup
BioCel Validation-BioRAPTR Tartrazine Assay • Calibration: Curve Fit, Automated • All 8 tips tested in triplicate 384-well plates • Tartrazine Assay readout on Flexstation 3 fluorimeter • Tartrazine Assay results: 5.5% CV or less @ 1,2,7, and 12uL • Accurate 150nL to 12uL range tested • Random Access Pipetting 12uL = good
Tartrazine+H2O Average STDEV Min Max CV Date
water blank 47uL 0.111 0.005 0.102 0.125 4.190 11/30/2010
7uL+43uL 2.038 0.061 1.829 2.279 2.981 11/30/2010
12uL+38uL 3.459 0.056 3.296 3.824 1.610 11/30/2010
2uL+48uL 0.640 0.035 0.524 0.719 5.479 11/30/2010
2uL+48uL_1_new 0.644 0.027 0.518 0.709 4.250 11/30/2010
2uL+48uL_2_new 0.649 0.030 0.520 0.736 4.646 11/30/2010
2uL+48uL_3 0.658 0.022 0.571 0.731 3.271 11/30/2010
2uL+48uL_4 0.650 0.026 0.546 0.791 3.995 11/30/2010
2uL+48uL_5 0.656 0.021 0.561 0.740 3.270 11/30/2010
1uL+49uL_1_new 0.360 0.017 0.259 0.399 4.765 11/30/2010
1uL+49uL_2_new 0.369 0.017 0.292 0.423 4.549 11/30/2010
1uL+49uL_3_new 0.365 0.019 0.300 0.415 5.131 11/30/2010
1uL+49uL_4_new 0.370 0.014 0.317 0.419 3.872 11/30/2010
1uL+49uL_5 0.374 0.014 0.319 0.418 3.696 11/30/2010
2uL+48uL_6 0.592 0.021 0.522 0.657 3.469 12/1/2010
2uL+48uL_7 0.600 0.019 0.535 0.667 3.100 12/1/2010
2uL+48uL_8 0.601 0.025 0.507 0.681 4.232 12/1/2010
water blank 0.105 0.004 0.096 0.125 4.234 12/1/2010
2uL+48uL_Tip2_1 0.624 0.020 0.532 0.681 3.264 12/1/2010
2uL+48uL_Tip2_2 0.617 0.020 0.495 0.678 3.162 12/1/2010
2uL+48uL_Tip2_3 0.619 0.017 0.548 0.689 2.802 12/1/2010
2uL+48uL_Tip3_1 0.646 0.018 0.577 0.705 2.804 12/1/2010
2uL+48uL_Tip3_2 0.643 0.015 0.583 0.698 2.376 12/1/2010
2uL+48uL_Tip3_3 0.631 0.015 0.570 0.691 2.357 12/1/2010
2uL+48uL_Tip4_1 0.623 0.019 0.531 0.673 2.987 12/1/2010
2uL+48uL_Tip4_2 0.602 0.016 0.550 0.669 2.641 12/1/2010
2uL+48uL_Tip4_3 0.613 0.016 0.536 0.685 2.636 12/1/2010
2uL+48uL_Tip5_1 0.618 0.014 0.550 0.687 2.199 12/1/2010
2uL+48uL_Tip5_2 0.623 0.014 0.568 0.681 2.172 12/1/2010
2uL+48uL_Tip5_3 0.620 0.016 0.569 0.702 2.560 12/1/2010
2uL+48uL_Tip6_1 0.643 0.018 0.555 0.713 2.823 12/1/2010
2uL+48uL_Tip6_2 0.631 0.014 0.576 0.702 2.270 12/1/2010
2uL+48uL_Tip6_3 0.637 0.015 0.590 0.712 2.311 12/1/2010
2uL+48uL_Tip7_1 0.589 0.013 0.543 0.652 2.134 12/1/2010
2uL+48uL_Tip7_2 0.587 0.012 0.548 0.648 2.114 12/1/2010
2uL+48uL_Tip7_3 0.586 0.012 0.554 0.657 2.035 12/1/2010
2uL+48uL_Tip8_1 0.687 0.015 0.652 0.828 2.144 12/1/2010
2uL+48uL_Tip8_2 0.681 0.014 0.545 0.763 2.125 12/1/2010
2uL+48uL_Tip8_3 0.693 0.012 0.656 0.757 1.753 12/1/2010
EpMotion MDA dilution and 16S PCR Bravo dilution and 16S PCR, 1st experiment
Correct
Incorrect
BioCel Validation: Bravo Cross-Contamination Experiments • Checkerboard analysis of Bravo demonstrates improvement over EpMotion • Viscosity of MDAs needed to be addressed on Bravo
BioCel Validation: Bravo Optimization of Viscous MDA Pipetting
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A Shewanellaceae
Shewanellaceae
Shewanellace
ae
Burkholderiace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Burkholderiace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Burkholderiac
eae
Shewanellace
ae
Shewanellace
ae
Shewanellacea
e
B Shewanellace
ae Shewan
ellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Rhodocyclace
ae
Shewanellace
ae
Rhodocyclaceae
Shewanellaceae
Burkholderia
ceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellacea
e
C Shewanellaceae
Burkholderia
ceae
Shewanellaceae
Shewanellace
ae
Rhodobactera
ceae
Shewanellace
ae
Burkholderiac
eae
Shewanellace
ae
Shewanellace
ae
Bacteroidace
ae
Shewanellace
ae
Burkholderiace
ae
Shewanellaceae
Shewanellace
ae
Burkholderia
ceae
Shewanellace
ae
Comamonadaceae
Shewanellace
ae
Shewanellace
ae
Shewanellacea
e
D Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Rhodobactera
ceae
Shewanellace
ae
Rhodobactera
ceae
Shewanellaceae
Shewanellace
ae
Burkholderiace
ae
Shewanellace
ae
Burkholderia
ceae
Shewanellace
ae
Shewanellacea
e
E Shewanellaceae
Shewanellaceae
Burkholderiacea
e
Shewanellace
ae
Rhodocyclace
ae
Shewanellace
ae
Shewanellace
ae
Moraxellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Burkholderia
ceae
Shewanellace
ae
Enterobacteria
ceae
F
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Burkholderiacea
e
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellacea
e
G Shewanellaceae
Shewanellaceae
Burkholderiacea
e
Shewanellace
ae
Burkholderiace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Burkholderiace
ae Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Oxalobacterac
eae
Shewanellace
ae
Shewanellace
ae
H Shewanellace
ae Shewan
ellaceae
Moraxellacea
e
Shewanellace
ae
Shewanellace
ae
Burkholderia
ceae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae Shewan
ellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellacea
e
I Shewanellaceae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Comamonadaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Sphingomonada
ceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
J Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Burkholderia
ceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Paenibacillaceae
Shewanellace
ae
Burkholderia
ceae
Shewanellacea
e
K Shewanellaceae
Rhizobiacea
e
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Burkholderia
ceae
Shewanellace
ae
Chitinophagac
eae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
L Actino
mycetales
Shewanellace
ae
Shewanellaceae
Rhodobactera
ceae
Shewanellace
ae
Burkholderia
ceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Burkholderia
ceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Phyllobacteriaceae
Shewanellace
ae
Shewanellace
ae
Burkholderia
ceae
Shewanellacea
e
M Shewanellaceae
Actinomyceta
les
Shewanellaceae
Pseudomonada
ceae
Shewanellace
ae
Shewanellace
ae
Burkholderiac
eae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
N Shewanellace
ae
Sphingomonada
ceae Shewanellaceae
Carnobacteriac
eae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Pseudomonada
ceae Shewanellaceae
Aeromonadac
eae
Shewanellace
ae
Burkholderiace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellacea
e
O Shewanellaceae
Shewanellaceae
Chitinophagace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
P Burkholderiacea
e
Shewanellace
ae
Burkholderiacea
e
Shewanellaceae
Rhizobiaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellacea
e
Shew MDA gDNA 191/192 99.48% Shew contaminant 4/192 2.08% Other contaminants 55/192 28.65%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A Shewanellaceae
Burkholderiace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Burkholderiace
ae
Shewanellac
eae
Shewanellace
ae
Burkholderiacea
e
Shewanellaceae
Shewanellaceae
Shewanellaceae
Shewane
llaceae
Shewanellace
ae
Rhizobiaceae
B Burkholderiacea
e
Shewanellace
ae
Burkholderiace
ae Shewanellaceae
Rhodobacterace
ae
Shewanellace
ae Shewan
ellaceae
Burkholderiacea
e
Shewanellace
ae
Shewanellace
ae Shewan
ellaceae
Rhodobacterace
ae Shewane
llaceae
Shewanellace
ae
Rhodobacterace
ae
Shewanellace
ae
Comamonadaceae
Shewanellaceae
Shewanellace
ae
C Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellac
eae
Shewanellace
ae
Burkholderiacea
e
Shewanellaceae
Burkholderiaceae
Shewanellaceae
Shewanellaceae
Burkholderiace
ae
Shewanellaceae
Shewanellaceae
Shewanellace
ae
D Shewanellace
ae
Shewanellaceae
Flavobacteriace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Burkholderiacea
e
Shewanellace
ae
Shewanellace
ae
Burkholderiace
ae
Shewanellaceae
Shewane
llaceae
Shewanellace
ae
Shewanellace
ae
Burkholderiacea
e
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
E Shewanellaceae
Actinomycetal
es
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Burkholderiacea
e
Shewanellaceae
Shewanellac
eae
Shewanellace
ae
Shewanellaceae
Shewanellaceae
Burkholderiace
ae
Shewanellaceae
Shewane
llaceae
Shewanellace
ae
F Actinomycetales
Shewanellace
ae
Burkholderiace
ae
Shewanellaceae
Streptococcace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Burkholderiace
ae
Shewanellaceae
Shewanellaceae
Actinomycetales
Shewanellace
ae
Shewanellace
ae
Pseudomonadac
eae
Shewanellaceae
Shewanellace
ae
G Shewanellaceae
Shewanellace
ae
Rhodobacterace
ae Shewanellaceae
Shewanellace
ae Shewan
ellaceae
Shewanellac
eae
Shewanellace
ae
Rhodobacterace
ae Shewanellaceae
Shewanellaceae
Burkholderiace
ae Shewanellaceae
Burkholderiace
ae Shewane
llaceae
Comamonadace
ae
Shewanellace
ae
H Shewanellace
ae Shewan
ellaceae
Shewanellace
ae Shewan
ellaceae
Rhodobacterace
ae
Shewanellace
ae
Shewanellace
ae Shewan
ellaceae Shewane
llaceae
Shewanellace
ae
Shewanellace
ae
Rhodobacteraceae
Shewanellaceae
Shewanellace
ae
I Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellac
eae
Shewanellace
ae
Moraxellaceae
Shewanellaceae
Shewanellaceae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Burkholderiace
ae
J Burkholderiacea
e
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Rhodobacterace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
K Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Enterobacteriace
ae
Shewanellaceae
Shewanellac
eae
Shewanellace
ae
Rhodobacterace
ae
Shewanellaceae
Comamonadaceae
Shewanellaceae
Burkholderiace
ae
Shewanellaceae
Shewane
llaceae
Shewanellace
ae
L Shewanellace
ae
Shewanellaceae
Actinomycetal
es
Shewanellace
ae
Burkholderiace
ae
Shewanellaceae
Shewanellace
ae
Moraxellacea
e
Shewanellace
ae
Shewanellaceae
Shewane
llaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
M Shewanellaceae
Shewanellace
ae
Shewanellaceae
Burkholderiace
ae
Shewanellace
ae
Shewanellaceae
Shewanellac
eae
Shewanellace
ae
Burkholderiacea
e
Shewanellaceae
Shewanellaceae
Shewanellaceae
Shewane
llaceae
Burkholderiacea
e
N Shewanellace
ae Shewan
ellaceae
Shewanellace
ae
Burkholderiace
ae Shewanellaceae
Actinomycetal
es
Shewanellace
ae
Shewanellace
ae Shewan
ellaceae Shewane
llaceae
Shewanellace
ae
Shewanellace
ae Shewan
ellaceae
Shewanellace
ae
O Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellac
eae
Shewanellace
ae
Shewanellaceae
Shewanellaceae
Shewanellaceae
Shewane
llaceae
Shewanellace
ae
P Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewane
llaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shew MDA gDNA 191/192 99.48% Shew contaminant 4/192 2.08% Other contaminants 50/192 26.04%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A Shewane
llaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellaceae
Shewanellaceae
Shewanellacea
e
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
B Shewanellace
ae
Burkholderiace
ae
Shewanellace
ae
Burkholderiacea
e
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Comamonadace
ae
Shewanellace
ae
Oxalobacteracea
e Shewanellaceae
Fusobacteriaceae
Shewanellacea
e
Shewanellace
ae
Shewanellace
ae Shewanellaceae
C Shewane
llaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellaceae
Shewanellaceae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
D Shewane
llaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Moraxellaceae
Shewanellaceae
Shewa
nellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
E Shewane
llaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellaceae
Burkholderiacea
e
Shewanellaceae
Actinomycetal
es
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
F Carnobacteriacea
e
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae Shewanellaceae
Shewanellace
ae
Enterobacteriace
ae
Shewanellaceae
Shewanellacea
e Moraxellaceae
Shewanellace
ae
G Shewanellaceae
Shewanellace
ae Shewan
ellaceae
Shewanellace
ae
Shewanellace
ae Shewan
ellaceae
Shewanellace
ae Shewan
ellaceae Shewan
ellaceae Shewan
ellaceae
Shewanellace
ae
Shewanellace
ae
H Shewane
llaceae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Streptophyta
Shewanellace
ae
Burkholderiacea
e Shewanellaceae
Shewa
nellaceae
Actinomycetal
es
Shewanellace
ae Shewan
ellaceae
I Shewane
llaceae
Actinomyceta
les
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellaceae
Rhodobacterace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Staphylococcace
ae
J Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewa
nellaceae
Shewanellace
ae
Shewanellaceae
K Shewane
llaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellaceae
Shewanellaceae
Shewanellace
ae
Bacillaceae
Shewanellace
ae
L Shewanellace
ae
Burkholderiace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Comamonadace
ae
Shewanellace
ae
Shewanellace
ae
Burkholderiacea
e
Shewanellace
ae
Shewanellaceae
Shewa
nellaceae
Shewanellace
ae
Shewanellaceae
M Shewane
llaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellaceae
Shewanellaceae
Shewanellace
ae
Burkholderiace
ae
Shewanellace
ae
N Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Streptophyta
Shewanellaceae
Shewa
nellaceae
Shewanellace
ae Shewan
ellaceae
O Shewane
llaceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellaceae
Shewanellaceae
Streptococcace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Comamonadace
ae
P Shewanellace
ae
Shewanellace
ae
Carnobacteriacea
e
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellaceae
Enterobacteriace
ae
Shewanellacea
e
Burkholderiace
ae
Shewanellace
ae
Shewanellaceae
Shew MDA gDNA
191/192 99.48%
Shew contaminant 20/192 10.42% Other contaminants 28/192 14.58%
Correct
Incorrect
16S PCR Replicate 1 16S PCR Replicate 2
BioCel Validation: Reagent QC for Automated PCR
•384-well checkerboard process reveals contamination •Green wells are sequences from PCR reagents alone
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A Shewanellacea
e Burkholderia
ceae Shewanellac
eae
Shewanellaceae
Shewanellac
eae
Shewanellaceae
Burkholderiaceae
Shewanellaceae
Shewanellac
eae Burkholderiac
eae Shewanellacea
e
Shewanellaceae
Shewanellace
ae Shewanellaceae
Shewanellaceae
Rhizobiaceae
B Burkholderiace
ae Shewanellace
ae
Burkholderiaceae
Shewanellaceae
Rhodobacteraceae
Shewanellaceae
Shewanellacea
e
Burkholderiaceae
Shewanellaceae
Shewanella
ceae
Shewanellace
ae
Rhodobacteraceae
Shewanellaceae
Shewanellace
ae
Rhodobacteraceae
Shewanellaceae
Comamonadaceae
Shewanellaceae
Shewanellac
eae
C Shewanellacea
e
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellacea
e
Shewanellaceae
Shewanellac
eae Burkholderiac
eae Shewanellacea
e Burkholderiacea
e Shewanell
aceae
Shewanellaceae
Burkholderiaceae
Shewanellaceae Shewanellace
ae Shewanella
ceae
D Shewanellace
ae
Shewanellaceae
Flavobacteriaceae
Shewanellaceae
Shewanellaceae
Shewanellaceae
Burkholderiaceae
Shewanellaceae
Shewanella
ceae Burkholderia
ceae Shewanellace
ae Shewanellaceae
Shewanellaceae
Shewanellac
eae Burkholderiace
ae Shewanellace
ae Shewanella
ceae Shewanellac
eae
E Shewanellacea
e Actinomycet
ales Shewanellac
eae
Shewanellaceae
Shewanellac
eae Burkholderiace
ae Shewanellacea
e
Shewanellaceae
Shewanellac
eae
Shewanellaceae
Shewanell
aceae Burkholderia
ceae Shewanellace
ae Shewanellaceae
Shewanellaceae
F Actinomycetale
s
Shewanellaceae
Burkholderiaceae
Shewanellaceae
Streptococcaceae
Shewanellaceae
Shewanellaceae
Shewanellaceae
Shewanellaceae
Burkholderiaceae
Shewanellaceae
Shewanellaceae Actinomy
cetales
Shewanellaceae
Shewanellaceae
Pseudomonadaceae
Shewanellaceae
Shewanellaceae
G Shewanellacea
e
Shewanellac
eae
Rhodobacteraceae
Shewanellaceae
Shewanellac
eae
Shewanellacea
e
Shewanella
ceae
Shewanellac
eae
Rhodobacteraceae
Shewanellaceae
Shewanell
aceae
Burkholderiaceae
Shewanellaceae
Burkholderiaceae
Shewanellaceae
Comamonadaceae
Shewanellaceae
H Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Shewanellacea
e
Rhodobacteraceae
Shewanellaceae
Shewanella
ceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellac
eae
Rhodobacteraceae
Shewanellaceae
Shewanellac
eae
I Shewanellacea
e
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellacea
e
Shewanellaceae
Shewanellac
eae Moraxellaceae
Shewanellaceae
Shewanell
aceae
Shewanellaceae
Shewanellaceae
Shewanellaceae Shewanella
ceae
Burkholderiaceae
J Burkholderiace
ae Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellac
eae
Shewanellaceae
Shewanellace
ae Rhodobactera
ceae Shewanellaceae
Shewanellaceae
Shewanellac
eae
Shewanellaceae
Shewanellac
eae
K Shewanellacea
e
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Enterobacteriaceae
Shewanellaceae
Shewanella
ceae
Shewanellaceae
Rhodobacteraceae
Shewanellaceae
Comamonadaceae
Shewanellaceae
Burkholderiaceae
Shewanellaceae
Shewanellaceae Shewanella
ceae
L Shewanellace
ae
Shewanellaceae
Actinomycetales
Shewanellaceae
Burkholderiaceae
Shewanellaceae
Shewanellac
eae Moraxellac
eae Shewanella
ceae
Shewanellaceae
Shewanellaceae Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
M Shewanellacea
e
Shewanellaceae
Shewanellace
ae Burkholderia
ceae Shewanellac
eae
Shewanellaceae
Shewanella
ceae
Shewanellaceae
Burkholderiaceae
Shewanellaceae
Shewanell
aceae
Shewanellaceae
Shewanellaceae Burkholderiac
eae
N Shewanellace
ae
Shewanellace
ae
Shewanellace
ae
Burkholderiaceae
Shewanellaceae
Actinomycetales
Shewanellaceae
Shewanella
ceae
Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellac
eae
Shewanellace
ae
Shewanellac
eae
O Shewanellacea
e
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellacea
e
Shewanellaceae
Shewanellac
eae
Shewanellaceae
Shewanell
aceae
Shewanellaceae
Shewanellaceae Shewanella
ceae
P Shewanellace
ae
Shewanellaceae
Shewanellace
ae
Shewanellaceae
Shewanellac
eae
Shewanellaceae
Shewanellace
ae Shewanellaceae
Shewanellaceae
Shewanellac
eae
Shewanellaceae
Shewanellac
eae
Shew MDA gDNA 191/192 99.48%
Shew contaminant 4/192 2.08%
Other contaminants 50/192 26.04%
BioCel Validation-Bravo Viscosity Test and Hit Picking
BioCel Validation-HMP Fecal Sample-1 Plate
Automation tasks to be validated in single 384-well plate run:
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 (outsourced to JCVI-JTC) 8. 16S or 18S characterization 9. MDA hit picking and re-amplification 10. Whole genome sequencing (outsourced to JCVI-JTC and others) 11. MDA archival
Human fecal sample (from Marty Blaser lab, NYU)
• SybrGreen staining
• FACS single cell sorting
from multiple gates
• BSL2 facility aerosol containment
Flow Cytometry of Bacteria
Example of Taxonomic Analysis: OTUs Single cell Clostridiales MDAs fall into several taxonomic groups
Red - 16S fecal sequences (Eckburg)
Blue – 16S from sequenced genomes
BioCel Validation-Step 8: 16S Characterization of Fecal Plate
• 119 single cell amplified bacterial genomes obtained • Confirmation that bacteria were derived from GI tract
100/119 have >99% identity to known 16S rRNA gene sequences
(Eckburg fecal 16S library, Science, 2005)
Taxonomic diversity observed: Firmicutes (Clostridium sp., Eubacterium sp., Lactobacillus sp.), Bacteroidetes (B. fragilus group,
Flavobacteriales), Proteobacteria, Verrucomicrobia
• Confirmation that many genomes are from novel uncultured species
21%: >99% identity to sequenced genomes
57%: 90-99% identity to sequenced genomes
22%: 80-90% identity to sequenced genomes
BioCel Validation-HMP Fecal Sample-1 Plate
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 (outsourced to JCVI-JTC) 8. 16S or 18S characterization 9. MDA hit picking and re-amplification 10. Whole genome sequencing (outsourced to JCVI-JTC and others) 11. MDA archival
BioCel Validation-Step 9 and 11: MDA Hit Picking, Re-amplification and Archiving
• Hit Pick MDAs from original plate according to their 16S OTU’s
• Protocol validated during SAT/FAT • Meets requirements • Slow, but works
• Not currently validated with real MDAs, but based on hit pick colormetric data, no problems are anticipated
• Matrix 2D barcoded tubes currently in use for hand picked MDA archival
BioCel Validation-HMP Fecal Sample-1 Plate
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 (outsourced to JCVI-JTC) 8. 16S or 18S characterization 9. MDA hit picking and re-amplification 10. Whole genome sequencing (outsourced JCVI-JTC and others) 11. MDA archival
X
X
Jonathan Badger JCVI | Todd DeSantis LBL | Dirk Gevers Broad | Anthony Fodor UNC | Ashlee Earl Broad
PHYLOGENY-BASED SELECTION OF STRAINS FOR THE HMP REFERENCE GENOME COLLECTION
100 MOST BACTERIA!
HMP: Most Wanted Phyla
HMP: Body Region OTU’s
Oral Microbiome: Oral Cavity OTU’s
BioCel SCGP Multi-Plate Validation: Human Microbiome Oral Samples
Total of 35 plates run on human oral microbiome Full automation scaling from 6, 6, 11, 12 plates on the BioCel SCGP
How’d we do?
SCGP Process Wells % # Single cell wells Status as of 8/24/11 Notes
Total oral microbiome single cells to be analyzed 12,144 100% 12144 Design Completed 33 single cell plates, 1 partial plate, 1 NTC plate
In process on SCGP (La Jolla) - 0% 0 Completed
Completed SCGP (La Jolla) 12,144 100% 12144 Completed
In process 16S Sanger (JTC-Maryland) - 0% 0 Completed
Completed 16S Sanger (JTC-Maryland) 12,144 100% 12144 Completed
BLAST Complete, 16S IDs obtained 12,144 100% 12144 Data Analysis Completed
SCGP Oral Microbiome Analysis Results (as of 8/24/10) # Single cell wells
Successful Oral Single Cell MDAs 3,231 26.6% 12144
Sequenced Oral Single Cell MDAs* (Oral genomes already sequenced) 2,621 21.6% 12144
Novel** (Oral Microbiome db BLAST) 610 5.0% 12144
Novel-Good*** (Oral Microbiome db BLAST, good chromatogram) 509 4.2% 12144
100 Most Wanted Matched 99% **** 139 1.1% 12144
100 Most Wanted Matched 99% **** and non-redundant 11 0.1% 12144
100 Most Wanted Matched 97% ***** 303 2.5% 12144
100 Most Wanted Matched 97% ***** and non-redundant 16 0.1% 12144
Total Oral Single Cell MDAs already sent to sequencing centers 181 (JTC - Methe)
Total Oral Single Cell MDAs ready to be sent to sequencing centers 328
* Sequenced Oral Single Cell MDAs defn: BLAST >97% identity to the sequenced genomes database
** Novel defn: BLAST >97% identity to the oral 16S database and <97% identity to the sequenced genomes database
*** Novel-Good defn: BLAST >97% identity to the oral 16S database and <97% identity to the sequenced genomes database and good chromatagram quality
**** 100 Most Wanted Matched 99% defn: Defined as "Novel-Good", BLAST >99% identity to the 100 Most Wanted database, >100 bp hit
***** 100 Most Wanted Matched 97% defn: Defined as "Novel-Good", BLAST >97% identity to the 100 Most Wanted database, >100 bp hit
Oral Microbiome Single Cell Genomics Pipeline (SCGP) Summary Table as of 8/24/11
BioCel Validation: Summary Checklist: 8 processes completed, validated, and operational
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 (TBD) 5. PCR Hit Picking 6. PCR SAP/Exo cleanup 7. Sanger Sequencing (outsourced to JCVI-JTC) 8. 16S Characterization 9. MDA Hit picking and re-amplification 10.Whole genome sequencing (outsourced to JCVI-JTC and others) 11.MDA Archival
Acknowledgements
FUNDING This work was supported by the Alfred P. Sloan foundation, grant NIH-2 R01 HG003647
from the National Human Genome Research Institute. Genome Sequencing Centers (GSC) award funded in whole or part with federal funds
from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract number
HHSN272200900007C. HMP project supported by Award Number U54 AI-084844-01 administered by the
National Institute of Allergy and Infectious Diseases on behalf of the NIH Roadmap Human Microbiome Project.
JCVI Staff Joyclyn L. Yee-Greenbaum Jeffrey S. McLean Shino Ishii Mary-Jane Lombardo Jonathan Badger Dana Busam (JTC) Tamara Ryabtseva (JTC) Indresh Singh Barb Methe Roger S. Lasken (PI)
Agilent Staff Robert Vandenberg
Todd Hughes Noël Ruppert Steve Lappin
Ginger Cooper Bill Rust
Russ Berman Denise Williams Barb Nowaczyk
Derek Young Hanzel Lawas
BioCel in Action: Video Questions and Discussion
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