Illumina Sequencing Overview

Illumina SequencingOverviewPart # 15045845 Rev.C 2013 Illumina, Inc. All rights reserved.Illumina, IlluminaDx, BaseSpace, BeadArray, BeadXpress, cBot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium,iSelect, MiSeq, Nextera, NuPCR, SeqMonitor, Solexa, TruSeq, TruSight, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarksof Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

Session ObjectivesBy the end of this training, you will be able to:– List the major steps in the Illumina sequencing workflow– Describe cluster generation– Discuss the sequencing by synthesis process2Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Illumina Sequencing WorkflowLibrary PreparationCluster GenerationSequencingData Analysis3Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Illumina Sequencing WorkflowLibrary PreparationCluster GenerationSequencingData Analysis4Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Library Prep is Critical for Successful SequencingDual Index Library shownThe aim of library prep is to obtain nucleic acidfragments with adapters attached on both ends5Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Illumina Sequencing WorkflowLibrary PreparationCluster GenerationSequencingData Analysis6Part # 15045845 Rev.DFOR RESEARCH USE ONLY

What is a Flow Cell?Cluster generation occurs on aflow cellA flow cell is a thick glass slidewith channels or lanesEach lane is randomly coated witha lawn of oligos that arecomplementary to library adapters7Part # 15045845 Rev.DFOR RESEARCH USE ONLY

What is a Cluster?Clusters are bright spots on animageEach cluster represents thousandsof copies of the same DNA strand ina 1–2 micron spotCycle 1, G ChannelSingleDNALibrary8Part # 15045845 Rev.DAmplifiedClonalClusterFOR RESEARCH USE ONLY

terSequencercBot9Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Hybridize Fragment & ExtendAdaptersequenceSingle DNA libraries arehybridized to primer lawnBound libraries are thenextended by polymerasesSurface of flow cellcoated with a lawnof oligo pairs3’extension10Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Denature Double-Stranded DNADouble-strandedmolecule is denaturedOriginal template scardNewly synthesized strandis covalently attached toflow cell surface11Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Single-Stranded DNANOTE:Single molecules bindto flow cell in arandom pattern12Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Bridge AmplificationSingle-stranded molecule flips overand forms a bridge by hybridizing toadjacent, complementary primerHybridized primer isextended by polymerases13Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Bridge AmplificationDouble-stranded bridge is formed14Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Denature Double-Stranded BridgeDouble-stranded bridge isdenaturedResult:Two copies of covalently boundsingle-stranded templates15Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Bridge AmplificationSingle-stranded molecules flip overto hybridize to adjacent primersHybridized primer isextended by polymerase16Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Bridge AmplificationBridge amplification cycle isrepeated until multiplebridges are formed17Part # 15045845 Rev.DFOR RESEARCH USE ONLY

LinearizationdsDNA bridges aredenatured18Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Reverse Strand CleavageReverse strands are cleavedand washed away, leaving acluster with forward strands only19Part # 15045845 Rev.DFOR RESEARCH USE ONLY

BlockingFree 3’ ends are blocked toprevent unwanted DNApriming20Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Read 1 Primer HybridizationSequencing primer is hybridizedto adapter sequence21Part # 15045845 Rev.DSequencingprimerFOR RESEARCH USE ONLY

Illumina Sequencing WorkflowLibrary PreparationCluster GenerationSequencingData Analysis22Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Sequencing By Synthesis (SBS)Add 4 Fl-NTP’s PolymeraseIncorporated FINTP imagedTerminator & fluorescentdye cleaved from FI-NTPX 36–25123Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Four Channel SBS Chemistry: GA, HiSeq, MiSeqEach of the four DNAbases emit an intensity of aunique wavelengthCollects four images: During each cycle, eachcluster appears in onlyone of four images26Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Two Channel SBS – NextSeq 500Two channel SBS uses two imagesClusters appearing in green only are TClusters appearing in red only are CClusters appearing in both images are AClusters not present in either green norred are GCluster intensities are plotted and basesare called accordingly27Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Paired-End Sequencing28Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Paired-End SequencingBlocked3’-endsSequenced strand isstripped off3’-ends of templatestrands and lawn primersare unblocked30Part # 15045845 Rev.DFOR RESEARCH USE ONLYSequencedstrand

Paired-End SequencingSingle-stranded templateloops over to form abridge by hybridizing witha lawn primerBridgeformation3’-ends of lawn primerare extended3’ extension31Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Paired-End SequencingDoublestrandedDNA32Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Paired-End SequencingBridges are linearized andthe original forward templateis cleavedOriginalforwardstrand33Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Paired-End SequencingBlocked3’-endsFree 3’ ends of thereverse template andlawn primers are blockedto prevent unwanted DNAprimingSequencingprimerSequencing primer ishybridized to adaptersequence34Part # 15045845 Rev.DReversestrandtemplateFOR RESEARCH USE ONLY

Sequencing By Synthesis 2nd ReadAdd 4 Fl-NTP’s PolymeraseIncorporated FINTP imagedTerminator & fluorescentdye cleaved from FI-NTPX 36–25135Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Illumina Sequencing WorkflowLibrary PreparationCluster GenerationSequencingData Analysis40Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Data Analysis OverviewControlSoftware41Part # 15045845 Rev.DAnalysisSoftwareFOR RESEARCH USE ONLYVisualizationSoftware

Analysis OverviewAnalysis TypeSoftwareOutputsControl SoftwareICS/MCS/RTAImages, Intensities and Base CallsCASAVAAnalysis SoftwareHiSeq AnalysisSoftwareMiSeq ReporterAlignments, Variant DetectionVisualizationSoftwareAnnotation, Filtering, Reports42Part # 15045845 Rev.DFOR RESEARCH USE ONLY

SummaryLibrary PreparationCluster GenerationSequencingData Analysis43Part # 15045845 Rev.DFOR RESEARCH USE ONLY

Questions?44Part # 15045845 Rev.DFOR RESEARCH USE ONLY

2 Part # 15045845_Rev.D FOR RESEARCH USE ONLY By the end of this training, you will be able to: –List the major steps in the Illumina sequencing workflow –Describe cluste