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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)
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Page 1: Validation of the Agilent BioCel Automated Platform for ......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

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)

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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)

http://www.sciencemag.org/search?author1=Mitch+Leslie&sortspec=date&submit=Submit
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• 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

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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

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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)

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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

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Single Cell Genomics Pipeline

Discovery platform for producing sequenced genomes

from single cells

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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

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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)

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Marine organisms

Stained Unstained

FACS: Scripps Pier Seawater 30µm Filtered DNA Stained with SYBR Green I

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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)

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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

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Nycodenz or

Filtration Oral cavity sample Cell fraction

EtOH fixation Staining Sorting

Single Cell Amplified Genomes From Human Oral Microbiome

B A

Stained Unstained

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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.

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Agilent Design: BioCel Single Cell Genomics

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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

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BioCel Subsystems: BioCel 1200 and 2 Device Tables

~7’ L x 7’ W x 7’ H

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BioCel Subsystem: Agilent DDR

• Fast • Accurate • Precise • Compact • Easy to program • Efficient movement • Large range of movement • Portrait and landscape

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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

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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

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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

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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

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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

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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

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ABI 9700 Thermocyclers

• Dual 384-well ABI 9700’s

• 12 total in the lab

• 24 plate capacity

• MDAs, PCRs, Lysis

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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

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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)

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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.

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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

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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

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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

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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

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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

Page 34: Validation of the Agilent BioCel Automated Platform for ......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

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%

Page 35: Validation of the Agilent BioCel Automated Platform for ......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

BioCel Validation-Bravo Viscosity Test and Hit Picking

Page 36: Validation of the Agilent BioCel Automated Platform for ......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

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

Page 37: Validation of the Agilent BioCel Automated Platform for ......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

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

Page 38: Validation of the Agilent BioCel Automated Platform for ......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

Example of Taxonomic Analysis: OTUs Single cell Clostridiales MDAs fall into several taxonomic groups

Red - 16S fecal sequences (Eckburg)

Blue – 16S from sequenced genomes

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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

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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

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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

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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

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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!

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HMP: Most Wanted Phyla

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HMP: Body Region OTU’s

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Oral Microbiome: Oral Cavity OTU’s

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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

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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

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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

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BioCel in Action: Video Questions and Discussion


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