emPCR454

CSB2008August2008 UCSCSequencingCenter

1)PrepareAdapterLigatedssDNALibrary(A‐[insert]‐B) 2)EmPCR:ClonalAmplificaJonon28µbeadsfollowedbyenrichment

4)Performsequencing‐by‐synthesisonthe454Sequencer

3)LoadbeadsandenzymesinPicoTiterPlate™

OverviewofThe454SequencingSystem


MixDNALibrary&capturebeads(limiteddilu8on)

EmulsionBasedClonalAmplificaJon

“Breakmicro‐reactors”IsolateDNAcontainingbeads

•  GeneraJonofmillionsofclonallyamplifiedsequencingtemplatesoneachbead•  Nocloningandcolonypicking

Create“Water‐in‐oil”emulsion

+PCRReagents

+EmulsionOil

PerformemulsionPCR

AdaptercarryinglibraryDNA

A

BMicro‐reactors


Centrifuge Step

LoadEnzymeBeads

44μm

LoadbeadsintoPicoTiter™Plate

DeposiJngDNABeadsintothePicoTiter™Plate


SequencingBySynthesis

DNACaptureBeadContainingMillionsofCopiesofaSingleClonalFragment

AATCGGCATGCTAAAAGTCA

AnnealPrimer

  SimultaneoussequencingoftheenJregenomeinhundredsofthousandsofpicoliter‐sizewells

  PyrophosphatesignalgeneraJon

T

PPi

ATP

Light+oxyluciferin

Sulfurylase

Luciferase

APS

luciferin

Sequencing‐By‐Synthesis


SequencingWorkflowOverview

Clonal amplification of fragments bound to beads in microreactors

One Bead

One Read 400,000 reads per run

One Fragment

Generation of small DNA fragments via nebulization

Ligation of A/B-Adaptors flanking single-stranded DNA fragments

Emulsification of beads and fragments in water-in-oil microreactors

Sequencing and base calling

Sample input: Genomic DNA, BACs, amplicons, cDNA


SequencingWorkflowLibrary Preparation

8.0 h 7.5 h 4.5 h and 10.5 h DNA library preparation and titration emPCR Sequencing

sstDNA library created with adaptors A/B fragments selected using streptavidin-biotin

purification

GenomefragmentedbynebulizaJon


SequencingWorkflowEmulsion PCR


Emulsion-based clonal amplification

Anneal sstDNA to an excess of DNA Capture Beads

Emulsify beads and PCR reagents in water-in-oil microreactors

Break microreactors, enrich for DNA-positive beads

Clonal amplification occurs inside microreactors


SequencingWorkflowLoading of PicoTiterPlate Device

•  Welldiameter:averageof44µm•  >400,000readsobtainedinparallel•  AsingleclonallyamplifiedsstDNA

beadisdepositedperwell

Depositing DNA beads into the PicoTiterPlate device

Quality filtered bases Amplified sstDNA library beads


SequencingWorkflowSequencing by Synthesis

•  Bases(TACG)areflowedsequenJallyandalwaysinthesameorder(100JmesforalargeGSFLXrun)acrossthePicoTiterPlatedeviceduringasequencingrun.

•  AnucleoJdecomplementarytothetemplatestrandgeneratesalightsignal.

•  ThelightsignalisrecordedbytheCCDcamera.

•  ThesignalstrengthisproporJonaltothenumberofnucleoJdesincorporated.


Flowgram Key sequence


GSFLXDataAnalysisFlowgram Generation

Flowgram

Key sequence = TCAG for signal calibration

FlowOrder

1‐mer

2‐mer

3‐mer

4‐mer

TACG

TTCTGCGAA


GSFLXDataAnalysisOverview

GS De Novo Assembler

GS Reference Mapper

GS Amplicon Variant Analyzer

Image capture

Image processing

Signal processing GS Run Browser


GSFLXSystemPerformanceRead Length


TheGenomeiscomprisedofrepeatregions

•  DependinguponthespecificgenomecharacterisJcs,microreads(~25bp’s)coveronlyaporJonofthegenome–  Inhuman–25basepairreadscanonlybemappeduniquelyto80%ofthegenome

•  ShortreadsarelimiJnginknowngenomes –Whataboutunknowngenomes?– Mappingversusdenovoassemblies– Mappingwillmissgenomerearrangements– Mappingisonlyasgoodasthereference


WhyDoesLengthMaVer?

•  LongersequencingreadsmeanmoreapplicaJons

–  IdenJfyandcharacterizesmallandshortRNA’s–  FulllengthcDNAsequencingforexpressionlevelsandvariaJons–  AmpliconresequencingforgeneJcvariaJonincludingsomaJcmutaJons–  Sequencingofmicro‐organismsinasingleinstrumentrun–  Sequencingofcomplexgenomes–mammalian&plant–  Sequencingofcomplexsamples–Metagenomics,AncientDNA


Longersequencingreadsmeanmoreapplica8ons

–  HIVStudies(3)–  ChIP‐Sequencing(8)

•  Boyleetal,Cell:MixedtechnologiesformappingopenchromaJn–  Metagenomics(12)

•  Palaciosetal,NewEnglandJournalofMedicine,PathogenicVirusDetecJon–  WholeGenomeSequencing(30)

•  Velascoetal,PLoS:PinotNoirGenome–  Paired‐Endsequencing

•  DetecJngStructuralVariaJonsacrosstwohumangenomes–  TechnologyandBioinformaJcs(11)

•  Meyeretal,NAR,:UsingPicogramquanJJesofsample–  Transcriptomestudies–cDNA(17)–  SmallRNA(32)–  AmpliconandMethylaJonStudies(9)


ApplicaJonsofWholeGenome,UltraBroadandUltraDeepHT‐Sequencing

HT‐ Sequencing Technology Applica8ons

WholeGenome Sequencing

Virus Bacteria Fungus HigherEukaryotes Human

UltraDeep Sequencing

Popula8onBiology

HIV

Bacterial16S

ResistanceTropism

Amplicons

UltraBroad Sequencing

SmallRNA

Metagenomics

Expression

NovelstrainID

Transcriptome

HLATyping


ThepowerofMetagenomics

•  HowtoIdenJfyanenvironmentbaseduponthemicrobialorganismsthatarepresent–  MicrobialPopulaJonStructuresintheDeepMarineBiosphere

•  Huberetal.,Science,318,p97,2007

•  Determiningthestateofanenvironmentbaseduponthepresenceandmixtureofmicrobialorganisms

–  TheinterdependenceofCoralandit’smicrobialenvironment•  Wegleyetal.,EnvironmentalMicrobiology,9,p2707,2007

•  DetecJngviralpathogens–quicklyandaccurately–  Lessthan12monthsfromfirstidenJficaJonofaffectedhivestopossiblepathogen

•  Cox‐Fosteretal.,Science,2007–  TransplantvicJmsfromAustralia

•  Palaciosetal,NewEnglandJournalofMedicine,2008


TranscriptomeAnalysisWorkflow Comparison

GS FLX (clonal sequencing ensured through emPCR)

Sanger (E. coli cloning, often concatemerization)

cDNA libraries (short tag library, EST library)

Concatemerization, insert fragments into vectors and clone into bacteria

Grow, pick colonies

Template Generation

Sequencing

Time: Weeks

emPCR

Sequencing Time: Days

cDNA libraries (short tag library, EST library)


•  Sequencingofapproximately400,000smallRNAsfromC.elegans

•  Another18unknownmiRNAgenesweredetected

•  ThousandsofendogenoussiRNAsacJngpreferenJallyontranscriptsassociatedwithspermatogenesisandtransposonswereidenJfied

•  AnewclassofsmallRNAswasidenJfied:21U‐RNAs.TheyallbeginwithanUandareprecisely21ntlong.


MulJplexIdenJfierBasics •  Whatisit?

–  Twonewkits,eachwith6differentlibraryadapters(totalof12adapters)–  EachMIDlibraryadapterhasanadded,speciallyencoded10‐baseregion–  Usedto“bar‐code”upto12differentgenomiclibrarysamplestoberuninthe

sameregionofasinglesequencingrun

PrimerA MID1 Key Libraryfragment PrimerB

Seq.primer Read

#bases:15410

PrimerA Key Libraryfragment PrimerB#bases:404

MIDLibrary

StandardLibrary Seq.primer Read

PrimerA MID2 Key Libraryfragment PrimerB

PrimerA MIDn Key Libraryfragment PrimerB


Paired‐EndApplicaJons

•  ~100bpsequencingtagsseparatedby3kbspacing•  Usefordenovoassembly

–  OrderconJgs•  UseforStructuralVariaJonstudies

–  Inversions,DeleJons,InserJons…–  HighresoluJondetecJon–3kbspacingvs10to40kb


Paired‐Endsworkflow


TargetedEnrichmentofHumangDNA

gDNAExon1 Exon2 Exon3 Exon4 Exon5

FragmentandhybridizetoNimbleGencapturearray

HT‐SequencingAnalyze

ExonSequences

Elute


Sequencingalltheknownexonsfromthehumangenome

•  “DirectselecJonofhumangenomiclocibymicroarrayhybridizaJon,”

–  Albertetal.,NatureMethods,(4)11,903‐905,2007•  ~6,700gDNAlociselected•  BRCA1region

2 MB Region


AnotherSequence‐CaptureExample

•  19KbregionfromChromosome4

GSFLXSeqReads

SequencingCoverage

Seq‐CapArrayProbes

TargetedExons

emPCR454

Documents

Transcript of emPCR454