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