genotyping & sequencing technologies in cancer studies · • next generation sequencing –...

Epidemiology 243 - Molecular Epidemiology of Cancer May 13, 2008

Genotyping & SequencingGenotyping & SequencingTechnologies in Cancer StudiesTechnologies in Cancer Studies

Jeanette Papp

© 2008 Jeanette Papp


www.genoseq.ucla.edu


Core EquipmentCore Equipment• Roche GS FLX (454) Next Gen Sequencer• 3730 capillary sequencers• Taqman 7900 Real-time PCR instrument• CEQ 8000 sequencers• Roche LightCycler 480• PSQ96 Pyrosequencer• Liquid handling robots• PCR Machines

Human Genetics Department• Affymetrix• Illumina• Solexa


Genetic AssaysGenetic Assays• SNP Genotyping• DNA Methylation Analysis• Gene Expression• LOH (loss of heterozygosity)• siRNA/RNAi, microRNA• In/Del Analysis• Microsatellite Genotyping• Large Fragment Sizing• AFLP• RFLP

• BAC Fingerprinting• SAGE• HLA Typing• Conformation Analysis• Allele Quantification• Sequencing• Resequencing• Comparative Sequencing


Maximize DNA QualityMaximize DNA QualityAll genotyping methods are sensitive to DNA…

• Quantity – Do you have sufficient DNA for your assay?Should you consider Whole Genome Amplification(WGA)? Advantages of WGA can be quantity andconsistency. Disadvantages – amplification bias, expense.

• Quality – What is the quality of your DNA? Highconcentrations of poor quality DNA will not help you.

• Consistency – Even if the quantity and quality areadequate, if they vary widely from sample to sample, youare liable to get poor results

Is your technology more robust to DNA quality or quantity?What is the dynamic range of detection?


Sample CollectionSample Collection

• Where will you get your DNA?

• Blood or Buccal Swab / Saliva?• Quality• Consistency• Compliance

DNA / RNA CollectionDNA / RNA Collection

1 2 3 4 5 6 7


Next Generation GeneticNext Generation GeneticTechnologiesTechnologies

Strategy

• Automation• Parallelization• Miniaturization


Genetic Marker MapsGenetic Marker Maps


Genetic MarkersGenetic Markers• Variable Number Tandem Repeats

(VNTRs)• Short Tandem Repeats

(STRs)• Dinucleotide Repeats (and tri- and tetra-)• CA Repeats• Microsatellites• Single Nucleotide Polymorphisms

(SNPs)


Genotyping Gel AutoradiographGenotyping Gel Autoradiograph


Fluorescent Genotyping GelFluorescent Genotyping Gel


Capillary SequencingCapillary Sequencing

• Background

•Methods• Applications


SNPsSNPs• Single Nucleotide Polymorphism• Responsible for 90% of all human genetic variation• A SNP occurs every 100-300 base pairs• Currently almost 12 million SNPs in the NCBI SNP database• May be within genes (coding SNP, cSNP) or outside gene (non-

coding, the majority)• May cause amino acid changes or not. If it causes an amino acid

change it is called non-synonymous (nsSNP)• Most SNPs are not responsible for a disease.• Like microsatellites, they are used as markers for pinpointing a

disease on the genome map. SNPs make particularly goodmarkers because- They occur frequently throughout the genome.- They are older and more stable genetically.

SNP PlatformsSNP PlatformsStudy SizeStudy Size

• Pyrosequencing – few SNPs, few samples, low-throughput,labor intensive

• Taqman – fewer SNPs, fewer samples, moderate-throughput• SNPlex – moderate numbers of SNPs and samples, high-

throughput (hundreds of thousands per day)• Sequenom – moderate numbers of SNPs and samples,high-

throughput (hundreds of thousands per day)• Affymetrix – many SNPs, many samples, ultra-high-

throughput (millions per day)• Illumina – many SNPs, many samples, ultra-high-throughput

(millions per day)• Next Generation Sequencing – massively parallel,

ultra-ultra-high-throughput

SNP PlatformsSNP PlatformsTechnologyTechnology

• Pyrosequencing – Sequencing by synthesis. Addition ofdNTPs one at a time. Luciferase generates light whennucleotides incorporated. Gives you the neighboringsequence. Labor intensive.

• Taqman – Allelic discrimination. Fluorescent probe for eachSNP variant. Simple, robust chemistry.

• SNPlex – Multiplexed, fluorescently labeled oligonucleotideson capillary electrophoresis.

• Sequenom – Mass Spectrometry. Highly accurate andreproducible.

• Affymetrix – Microarray of probe DNA spots printed on a glass or plastic chip.

• Illumina – Microarray of tiny beads with boundoligonucleotides. Sample DNA binds to the bead oligo, and is detected by an optical fiber.


InstrumentsInstruments


PyrosequencingPyrosequencing


Allelic DiscriminationAllelic Discrimination

SNPlex SNPlex Chemistry OverviewChemistry OverviewEncoding

Generation of genotype (GT) specificproducts through multiplex

oligonucleotide ligation reaction (OLA)

AmplificationMultiplex PCR with universal primers

DecodingHybridization of universal ZipChute

probes to amplicons and identificationof eluted ZipChutes by CE


LigationLigation


Enzymatic PurificationEnzymatic Purification


PCRPCR

Hybridization and ElectrophoresisHybridization and Electrophoresis

SNPlex Raw DataSNPlex Raw Data

SNPlex Raw DataSNPlex Raw Datafrom the Corefrom the Core


ExamplesExamples

Good

Good Bad

Bad


ExamplesExamples

Bad

SNP MicroArraySNP MicroArray


SNP BeadArraySNP BeadArray


SNP PlatformsSNP PlatformsCostCost

• Affymetrix – $0.0006-??

• Illumina – $0.0002-??

• Sequenom – $0.05-$0.20

• SNPlex – $0.08-$0.14

• Taqman – $0.20-$0.70

• Pyrosequencing – $0.35-$1.60


When Choosing a TechnologyWhen Choosing a TechnologyPlatform, Consider:Platform, Consider:

• Conversion rate – How many of the SNPs youchose worked - in silico, in vitro?

• Reproducibility – Do you get the same result whenyou repeat the whole process?

• Error Rate – Compared to “true” result.

• Concordance – Agreement with some othermethod.

• Call Rate – How much missing data?

• Cost – What can you afford?


Aarray Aarray DataDataChip-based GenomicsChip-based Genomics

•• Each spot represents one genetic markerEach spot represents one genetic marker

•• New generation chips holdNew generation chips hold 2.3 million 2.3 million SNPsSNPs

•• To find genes for common, complex traits To find genes for common, complex traits ititmay require DNA from 2000 individualsmay require DNA from 2000 individuals

4,600,000,000 4,600,000,000 SNP datasetSNP dataset


Using High-Resolution MeltingUsing High-Resolution Meltingon the Roche on the Roche LightCycler LightCycler toto

Determine Determine CpG-siteCpG-siteMethylation Methylation statusstatus


Bisulfite Bisulfite ConversionConversion• Methyl-C and C are indistinguishable to

DNA polymerase• Methylation state is lost in PCR

amplification• Bisulfite treatment converts unmethylated

cytosine to uracil, which becomes thymineafter PCR

• Methylated cytosines are protected frombisulfite and thus unchanged


Principle of High ResolutionPrinciple of High ResolutionMeltingMelting

• C to T proportion significantly changes the meltingtemperature of the product

• Degree of DNA methylation gives different melting profiles

• The high resolution dye from Roche intercalates and saturatesevenly giving a sharp, precise melting profile

HRM Dye vs. SYBRGreen


Example of High ResolutionExample of High ResolutionMelting curves on Roche LC480Melting curves on Roche LC480

fluorescence

Temperature


Analysis using Tm CallingAnalysis using Tm Calling• Significantly different melting temperatures of the two species

allows quantitative analysis using the Tm calling

• By comparing the area of each peak relative to the sum of bothpeaks a quantitative percentage can be obtained


Analysis using Analysis using GeneGene Scan Scan

Method ComparisonsMethod Comparisons

Optimizationdependent

Accurate, scalable,low cost

High precision

One tube method

QuantitativeMethylationSensitive HighResolution Melting

High setup costHigh accuracy.

Can be scaled up

QuantitativeMethyLight

Labor intensive.

3rd primer necessaryfor extra specificity

Sequence contextQuantitativePyrosequencing

Labor intensive, highper sample cost.

Time consuming

Sequence context.Gives preciseinformation for entiresequence

Qualitative/semi-quantitative

BisulfiteSequencing

Extensive PCRoptimization

Low cost.

Variable precision

QualitativeMethylationSpecific PCR

ConsProsQuantitative/

Qualitative

Technique


Sequencing TechnologiesSequencing Technologies


A Brief History of SequencingA Brief History of Sequencing……

• A adenine

• C cytosine

• T thymine

• G guanine


Sanger SequencingSanger SequencingChain TerminationChain Termination


Sequencing Gel AutoradiographSequencing Gel Autoradiograph


Dye TerminationDye Termination


Fluorescent GelFluorescent Gel


DNA SequencingDNA Sequencing


Capillary SequencingCapillary Sequencing

• Background

•Methods• Applications


Human Genome ProjectHuman Genome Project

3 Billion Base Pairs

• 1990 - Begin: estimate 15 years, $3 billion• 1998 - Craig Venter starts new companyCelera, “will sequence human genome in 3years for $300 million”

• 2000 - Working draft• 2003 - Complete in 13 years, $2.7 billion


NIH $1000 Genome InitiativeNIH $1000 Genome InitiativeRELEASE DATE: February 12, 2004:

The purpose of this Request for Applications (RFA) is to solicitgrant applications to develop novel technologies that willenable extremely low-cost genomic DNA sequencing. Currenttechnologies are able to produce the sequence of amammalian-sized genome of the desired data quality for $10to $50 million; the goal of this initiative is to reduce costs byat least four orders of magnitude, so that a mammalian-sizedgenome could be sequenced for approximately $1000.Substantial fundamental research is needed to develop thescientific and technological knowledge underpinning such amajor advance. Therefore, it is anticipated that the realizationof the goals of this RFA is a long-range effort that is likely torequire as much as ten years to achieve.


Next Generation SequencingNext Generation SequencingTechnologiesTechnologies

Strategy

• Automation• Parallelization• Miniaturization


Commercially Available NowCommercially Available Now• 454 (now Roche GS FLX) - 2005

~300 base pair reads. Good for novel genomes 100 million base pairs in 8 hour run

• Solexa - 2006 Short reads, ~ 23-35 bp. Best when there is a reference

sequence (“resequencing”) or for analyzing small moleculeslike RNA.

4 gigabases per 3 day run - the equivalent of one-third of thehuman genome

• SOLiD - 2007 Short reads, ~35 bp 3 gigabase per 3 day run


TechnologyTechnology• 454 (now Roche GS FLX)

DNA is fragmented, bound to beads, amplified till each bead has100,00 copies of the DNA fragment

Pyrosequencing - When a base is added to the DNA strandpyrophosphate is released, used as a substrate for luciferase, lightis emitted and detected by a camera

• Solexa Sequencing by synthesis with fluorescently labeled bases and

DNA fragments bound to a slide.

• SOLiD Sequencing by ligation. Bead-bound DNA molecule is interrogated

with each of the 16 possible 2 base pair combinations in afluorescently labeled oligonucleotide.

Next Gen SequencerNext Gen SequencerRoche 454 GS FLXRoche 454 GS FLX


Current CostCurrent Cost

• ~$100,000 for research

• $350,000 from Knome personalgenomics company

Cost to Sequence Human GenomeCost to Sequence Human Genome

$1000Coming Next???

$60,000Applied BiosystemsSOLiD

< $100,000Illumina Solexa

$100,000Roche GS FLX (454)

$3 millionCurrent CapillarySequencer

$2.7 billionHuman GenomeProject


Coming NextComing Next…… Single Molecule Sequencing -Single Molecule Sequencing -without amplificationwithout amplificationNanoporeA single-stranded DNA molecule is driven through a pore so tinythat it partially blocks the pore as it moves through. Each baseblocking the pore has different electrical properties and is read offas it passes through the pore.

Nano-knife edgeA single-stranded DNA molecule is stretched and immobilized overa nano-edge probe. Each base in turn is excited by electrons, andthe base’s unique vibration pattern is read.


ApplicationsApplications

As the introduction of the personalcomputer created new applications incomputing, whole genome sequencingtechnology has generated newapplications


Whole Genome SequencingWhole Genome Sequencing

De novo sequence of novel genomes

• Mycobacterium tuberculosis

• Vibrio cholerae

• Streptococcus pneumoniae

• Haemophilus influenzae

• Helicobacter pylori


Genomic Diversity andGenomic Diversity andMetagenomicsMetagenomics

Microbial diversity in

• Human microbiome• Microbes in honey bee colony

collapse disorder• Deep sea microenvironments• Deep mine microbial ecology• Environmental sampling


Comparative Genomics,Comparative Genomics,PaleogenomicsPaleogenomics,,

Ancient DNA AnalysisAncient DNA Analysis• Neanderthal Genome

• Mammoth

• Ancient wolves

• Mitochondria from ancient hair shafts


Chromosome StructureChromosome Structure• Deletions

• Duplications

• Copy number variation

• Insertions

• Inversions

• Translocations

• Methylation Analysis


Transcriptome Transcriptome AnalysisAnalysis

• Use massively parallel sequencing toquantitatively measure gene expressionacross tens of thousands of samples

• Develop a genomic signature of

Cell differentiation state Disease state Therapeutic response Tumor signature


Deep Deep ResequencingResequencing

Sequencing against a reference genome

• Identify rare disease-causing variants

• Cancer, HIV mutation detection

Research Field Application Method

De Novo

Sequencing

Whole Genomes of Plants, Yeasts,

Fungi, Bacteria, Viruses

Shotgun Sequencing,

Paired-End Sequencing, De Novo Assembly

Whole Genomes of Humans, Plants,

Yeasts, Fungi, Bacteria, Viruses

Shotgun Sequencing,

Mapping to Reference

Sequence

Genomic rearrangements, Copy

number variation Paired-End Sequencing

Resequencing

Indels, SNPs, Somatic Mutations Amplicon Sequencing

Full-length transcripts

Multiplex Sequencing of Paired-End Ditags (Singapore MS-PET)

Transcriptome

Analysis

Serial Analysis of Gene Expression

(SAGE)

Small non-coding RNAs Gene Regulation

Studies Transcription Factor Binding Sites (ChIP-Sequencing)

cDNA fragment sequencing

DNA Methylation Pattern Amplicon Sequencing Epigenetic

Changes Nucleosome Modifications DNA fragment sequencing

Analysis of Environmental DNA Shotgun Sequencing Metagenomics &

Microbial

Diversity 16S rRNA Amplicon Sequencing

Paleogenomics Whole Genome Sequencing of Ancient

DNA Shotgun sequencing


DiagnosticsDiagnostics

• $1000 genome will allow individualsequence as diagnostic. Will it come toosoon to be useful?

• In genomic research technology anddata have often come before our abilityto extract information and knowledgefrom them.

genotyping & sequencing technologies in cancer studies · • next generation sequencing –...

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