next generation sequencing for personal exomes, stem cell...

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1:00 – 1:25 PM 15-Oct Pasadena

Next generation sequencing for personal exomes, stem cell allele specific RNAs, microbiomes, VDJomes

Co-PIs: Sherley, Mitra, GottliebTalks: Li (mC, RNA), Vigneault (miRNA), Dantas (microbes)Posters: Ball (mC), Sismour (ligation), Laserson (VDJ)

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Instrument System

Integration: consider entire ecosystem: academic/commerical/clinical/consumer

Hardware(Danaher)EM-CCDTDIFlow-cells

SoftwareImagingBase calls

Wetware(Enzymatics)ChemistryEnzymes

Applications

Haplotypes (CGI)Exomes (Agilent)Stem cell RNA

SoftwareTrait data Association (Broad)HT-SysBioInterpret (Knome)

ELSwareConsent(PGP)CLIA(HPCGG)Education(OppenheimerFoundation, PGED,23andme)

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Inherited Genomics

TRAITS(Phenome)

PERSONAL GENOME

Once in a life-time genome sequence

to Predictive Medicine

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Inherited + Environmental Genomics

VDJ-ome

TRAITS(Phenome)

Microbiome

Multi-tissue

Epigenome(RNA,mC)

PERSONAL GENOME1 to 98%

Once in a life-time genome + yearly ( to daily) tests

Public Health Bio-weather map : Allergens, Microbes, Viruses

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9K chem/drugs

Omic combinatorics

VDJ-ome1M receptors

4000 disorders + non-medical

(quant)traits

Microbiome1M species

>>250 tissues

epigenome(RNA,mC)

PERSONAL GENOME3M alleles

(Alleles^n * environments^m) vs. (lumping via pathways)

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Multiple hypothesis testingY= Number of Sib Pairs (Assocation)

X= Number of Alleles (Hypotheses) Tested

GRR=1.5, p= 0.5 (population frequency)

0

200

400

600

800

1,000

1,200

1,400

1,600

1E+4 1E+7 1E+10 1E+13 1E+16 1E+19 1E+22

|

= Genotypic relative risk

based on Risch & Merikangas (1996) Science 273: 1516

Pool some alleles by pathway & mutation type(not LD or chromosome position)

Allele &environmentcombinations

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Sequencing tracked Moore’s law (2X / 2 yr) until 2004-8 (10X / yr)

40X 98% genome $5K in 2009 ($50 for 1%?)

0.0000001

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

10

1990 1995 2000 2005 2010

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G

A

C

T

Multiplex Cyclic Sequencing by SynthesisPolonator: multiple chemistries: polonies on slides or beads

Polymerase -or- Ligase Shendure, Porreca, et al. 2005 Science

Illumina, IBS*AB-SOLiD*, CGI*

Mitra, et al. 2003 Analyt.

Biochem.1999NAR

Dae Kim Mike Sismour

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5+ Next Generation Sequencing Platforms

2G/2h2.8 G/2h0.3 G /4h0.2 G /2.6h.001G/0.03h$155K$1350K$690K$680K$500K

Polonator Helicos AB-SOLiD Illumina Roche

+

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Open-architecture hardware, software, wetware

Polonator

$150K - 2 billion beads/run

e.g.1981IBM PC

Rich Terry

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36 to 64 flowcells (+ DNA barcodes)

1 to 4 billion beads

8.5 μ thicksequence image

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Rearrangements detected using polony paired end reads Shendure et al Science Sep 2005

Deletion Insertion Inversion(rare in this clonal population)

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Selective genome sequencing

Shendure, et al. Science 309:1728 Porreca et al 2007 Nat Methods 4:931Nilsson et al. (2006) Trends Biotechnol 24:83.

Red=Synthetic; Yellow=genome/cDNA

How do we optimize >100K 100mers ?

3 ways to capture alleles from genomic or c-DNA

In vitro Paired-end-tags (PET)

Science 2005Science 2005

Hybridiz.selection

Zhang, Chou, Shendure, Li, Leproust, Dahl, Davis, Nilsson, Church

For rearrangements

2. 3.1.

GapFill

Nat Methods 2007

3.

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2nd-Gen Synthesis: off chips

8K Xeotron Photo-Generated Acid12K Combimatrix Electrolytic120K Roche, Febit Photolabile 5'protection244K Agilent Ink-jet standard reagents

Tian et al. 2004 NatureCarr & Jacobson 2004 NAR Smith & Modrich 1997 PNAS

$500 per 15Mbp

Amplify pools of 50mers using flanking universal PCR primers &

3 paths to 10X error correction

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Aug 2007 R= .53 Jan 2008 R=.986

Zhang, Li et al. unpublished

Gapfill

r = 0.986 Between Exome Replicates

Increase oligo concentration * time 1800X

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Inherited + Environmental Genomics

VDJ-ome

TRAITS(Phenome)

Microbiome

Epigenome(RNA,mC)

PERSONAL GENOME1 to 98%

One in a life-time genome + yearly ( to daily) tests

Public Health Bio-weather map : Allergens, Microbes, Viruses

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RNA/epigenome challenge: Multiple cell types from adults

3mm skin sample

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Induced Pluripotent Stem Cell Generation & Transdifferentiation (Oct4/Sox2/Myc/Klf4)

Retroviral Infection

Tissue Culture on a Mouse Feeder Layer

ES Cell Colony Identification

Clonal Isolation and Propagation

Embryoid Body Induction&

Guided Differentiation

Adenoviral Infection

Mixture of differentiated cell types&

Guided Differentiation

2 monthsMultiple integration sites

1 weekNo genomic integration

Yamanaka, Daley(Park), ThomsonHochedlinger, Jaenisch labs Lee & Church

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Reprogramming reproducibility

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Cell-type & inter-individual differences

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Association studies using 3M point & CNV variants

vs1M LD surrogate SNPs

vsQuantitative measures per gene

(per cell type and condition)

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G

A

TC

Allele‐specific expression (ASE)

Combine all cis element variants

GA

AAAAAAAAAAAAAAAAAAAA

TC

TT

& eliminate environmental & trans-acting variation among individuals.Cis: Copy number, enhancer, promoter, splicing, polyA, termination, transport, decay.

G

A

GG

Allele‐specific transcription factor 

binding

TF

ChIP‐Seq

Digital RNA allelotyping

Zhang, Li, Church unpublishedForton et al. Genome Res. 2007

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Genomic DNA

Lymphocyte

cDNA

Lymphocyte

cDNA

Fibroblast

cDNA

Keratinocyte

rs1264899, ATP5F1, ATP synthase

T/C = 0.51 T/C = 3.47T/C = 3.73

Tissue specific & allele specific gene expression confirmatory assays

Kun Zhang & Alice Li

24Zhang et al. Nature Genet. Mar 2006

Haplotyping methods #1: ‘in situ’#2: Chromsome dilution libraries

153Mbp

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• Ultra-clean to reduce background amplification + Real-Time monitoring

• Post-amplification chip hybridization distinguishes alleles

• Amplification variation random & easily filled by PCR

• error rate <1.7 10–5

Haplotyping #2: Single-chromosome or fragment dilution

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Inherited + Environmental Genomics

VDJ-ome

TRAITS(Phenome)

Multi-tissue

Epigenome(RNA,mC)

PERSONAL GENOME1 to 98%

One in a life-time genome + yearly ( to daily) tests

Public Health Bio-weather map : Allergens, Microbes, Viruses

Microbiome

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Antibody VDJ regions

Lefranc, The Immunoglobulin FactsBook; Janeway, Immunobiology

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Maintaining clonal VDJ (H & L) mRNA phase

water‐in‐oil emulsion4 Encapsulation approaches 

Science 309: 1728

Nature Methods 3: 551 NAR 20: 3831 Anal. Biochem. 320: 55

2 Chain co‐amplification approaches

Dantas, Sommer, 

Agresti, Rowat

index

NAR 20: 3831  Embleton et al. In-cell PCR from mRNA: amplifying and linking heavy and light chain V-genes within single cells.

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Human B &T lymphocyte cDNA : VDJ Polonies

http://www.infobiogen.fr/services/chromcancer/Genes/TCRBID24.html

2-4 E6 / ml * 5L = 1E10 cells (blood) 46*23*6*67*5 = 2M combinations (24 bits vs 750 bp)

25-4-6 TRG

1435TRD/A

213239-46 TRB

150-45-47 TRA

4-54-5-29-32IGL

15-31-35 IGK

962338-46 IGH

CJDV

Uri Laserson, Francois Vigneault

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VDJ(H) 16 antigens &3 EBV-B cellscombinations

24x86

ImMunoGeneTics database http://imgt.cines.fr/

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Personal genome cost trade-offs

2*3 Gbp Genome 12*30 Mbp protein exons 100Pair-end 500+/-50 b 10Pair-end 50 +/-5 kb 1k20K full RNA 4-logs 0.220K RNA allelotype 1-log 20k750 bp VDJ-VJs 90M 24 bit VDJ-VJs 2G

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Inherited + Environmental Genomics

VDJ-ome

TRAITS(Phenome)

Microbiome

Multi-tissue

Epigenome(RNA,mC)

PERSONAL GENOME1 to 98%

One in a life-time genome + yearly ( to daily) tests

Public Health Bio-weather map : Allergens, Microbes, Viruses