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Transcript of Next Generation Sequencing - UCLA CTSI · PDF fileNext gen sequencing •Steps...
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Next Generation Sequencing• Huge improvements in the technology over the
past two years has resulted in the routine abilityto generate about 20-30 billion bases ofsequence information from each machine eachweek.
• Major technologies• Illumina (GAIIx)• ABI (SOLID V3plus)• 454/Roche• Helicos• Pacific Biosciences
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Next Generation Sequencing1) whole genome and whole exome sequencing,
economical large scale targeted sequencing forvariant discovery,
2) CHIP_SEQ for full access to genomic bindingtargets,
3) RNA_SEQ for more complete surveys oftranscription on a genome scale,
4) Methyl_SEQ providing genomic assessment ofmethylation status at base resolution.
5) Small_RNA SEQ
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Next gen sequencing
• Steps– Obtain genomic DNA (gDNA)– Fragment the genomic DNA– Repair the ends of fragmented DNA, ligate to
specific primers– Sort the fragments so that there is one
fragment per location– PCR amplify the ligated fragment– Sequence the PCR amplified DNA
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Illumina TECH OVERVIEWgDNA
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Illumina TECH OVERVIEW
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Illumina TECH OVERVIEW
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ABI SOLID Tech OVERVIEW
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ABI SOLID TECH OVERVIEW
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SOLiD Users Group Meeting |11/18/09
Bead Preparation and Deposition
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Sample Input and Fragmentation
Library Preparation
One Fragment = One Bead
emPCR (Emulsion PCR) Amplification
One Bead = One Read
Data Analysis
Roche454sequencing
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Technology comparisons• Tech• ABI 50b $680 ~30Gb/wk• Illumina 75b $700 20Gb/wk• 454 400b $>10,000 3-6Gb/wk
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SOLiD Users Group Meeting |11/18/09
How Many Reads Do I Need to Surveythe Transcriptome?
Application Complexity Reads Estimate mappablereads needed
SamplesSOLID 3
Small RNADiscovery
Low 35bp ~10M Up to 20/slide
SAGE Low 35bp 5M 40/ slide
Expression ofannotatedgenes
Mid 50bp Minimum 50M(human
Up to 4/slide
WholeTranscriptomeDiscovery
High (alternativetranscripts & splicing
50 bp Minimum 100million (human)
2/slide
Allele SpecificExpression
High (variants to bedefined)
50 bp > 150 million(human)
1/slide
The number of reads needed is dictated by the complexity of application
* Current best estimates from literature and internal research
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Acute myelogenous leukemia
• Clonal hematopoietic disease caused byinherited and acquired genetic alterations.
• Current AML classification and prognosticsystems incorporate genetic information,but are limited to known abnormalities thathave been identified by cytogenetics,CGH, expression profiling, and generesequencing.
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Acute myelogenous leukemia• Karyotype remains the most powerful predictor of
outcome.• Sub-cytogenetic lesions using CGH and SNP arrays
have been employed but remain investigational.• Expression profiling has revealed candidate genes which
are currently being examined.• Candidate gene resequencing has identified recurrent
mutations that can be used to stratify patients withnormal cytogenetic profiles to risk and to identifypatients for targeted therapy.
• These revised classification systems are imperfect,suggesting that important genetic factorsfor thepathogenesis of AML remain to be discovered.
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Acute myelogenous leukemia
• Within this background the authorsdescribe their 2nd patient with AML-M1(minimal maturation) for which the entireAML genome has been sequenced.
• The genes identified in their sequencingstudy were then examined in 187additional AML samples.
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The patient
• 38 yo male, european descent, who presentedwith fatigue, cough.
• WBC 39.8x103, Hg 8.9, and Plt 38x103
• Bone marrow revealed 90% cellularity, with 86%of the blasts staining for myeloperoxidase.
• Karyotype was normal.• Treatedwith cytarabine, daunorubucin, followed
by consolidation treatement with cytarabine.• Complete remission for 3 years.
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Genetic studies
• Paired bone marrow sample and skin biopsy.• Affymetrix Genome Wide Human SNP Array 6.0• Affymetrix U133 Plus 2.0 expression arrays• 69.9 billion bp sequencing of the bone marrow
sample• 63.9 billion bp sequencing of the skin sample• Characterized 4 tiers of single nucleotide
variants (SNV)
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Tiers of single nucleotide variants
• Tier 1- amino acid changes in coding regions ofannotated exons, consensus splice site regions,and RNA genes.
• Tier 2- changes in highly conserved regions ofthe genome that have regulatory potential
• Tier 3- mutations in the non-repetitive part of thegenome that does not meet criteria for tier 2.
• Tier 4- the remainder of the genome
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Conclusion• Next gen sequencing allows for full genome sequencing
of individual samples.• Becoming more affordable and accessible.• Allows for relatively complete genomic evaluation (98%
coverage), with fewer sequencing runs and fewer falsepositives (47%).
• 20,000 novel SNV described; of these 750 are found incoding or regulatory regions of the genome
• 12 nonsynonymous mutations specific to the tumor aredescribed, 3 of which are found in other AML samples.
• One of these SNV codes for IDH1, which previously hadnot been described in AML patients.
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Conclusion
• Available• Powerful• Expensive• Results are limited• Must be combined to other technologies
which interrogate other aspects of generegulation (chromatin).
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ABI SOLID TECH OVERVIEW
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SOLiD Users Group Meeting |11/18/09
Agenda Item0102 Agenda Item
03 Agenda Item
The SOLiD™ 3 Plus System
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SOLiD Users Group Meeting |11/18/09
SOLiD Kits
ChIP Kit & Antibodies (#1) LAUNCHED - Mar 15
Eco Mate-Pair Kit LAUNCHED - Mar 15
Super SAGE Kit LAUNCHED - May 15
Whole Transcriptome (WT) Kit LAUNCHED - June 8
LMP Mate-Pair Kit LAUNCHED - July 15
Fast Strips -1x35, 2x35 LAUNCHED - July 16
SOLiD 16 Barcode Kit
QC Tools
ChIP Kit & Antibodies (#2)
SOLiD Methylation kit
SOLiD 96 Barcode Kit
SOLiD 3.0
SOLiD 3 Plus 2009
SOLiD 3 Plus 2010
LAUNCHED – Sept 15