Genome Sequencer 20 System First To The Finish

PicoTiterPlate Devices and GasketsSequence more than 20 million bases in a single runwith the specially designed PicoTiterPlate device(Figure 9). Choose from two different plate sizesand five different gaskets to meet your specificsequencing throughput needs (Table 1). The PicoTiterPlate device is created fromfiberoptic bundles that are etched to produceindividual wells in picoliter format. Each well is only able to accept a single DNA bead. Signals generated by reactions in the wells arecaptured by the CCD camera. Gaskets are used to divide the PicoTiterPlatedevice into separate regions and create loadingareas for different throughput needs.Availableformats ofPicoTiterPlatedevice gaskets.GenomeSequencer20 SystemClose-up viewof a PicoTiterPlatedevice.Figure 9: PicoTiterPlate device and accompanyingaccessories.Loadingregion sizePicoTiterPlatedevice sizeNumber of regionsper PicoTiterPlatedevice70 x 75 mm2Large: 30 x 60 mmMedium: 14 x 43 mmRun throughput*per regionRun throughputper PicoTiterPlatedevice20 Mbp10 Mbp40 x 75 mm170 x 75 mm470 x 75 mm16Small: 2 x 53 mm10 Mbp3.3 Mbp13 Mbp10 Mbp0.63 Mbp40 x 75 mm85 MbpTable 1: Sequencing throughput with various combinations of PicoTiterPlate devices and gaskets.* minimum achievable throughputMbp million base pairs 11

SoftwareBenefit from a fully integrated software packagePost-runRun-timeThe combination of signal intensity and positionalinformation generated across the PicoTiterPlatedevice allows the Linux-based software to determinethe sequence of hundreds of thousands of individualreactions simultaneously, producing millions ofbases of sequence per hour from a single run(Figure 10).ImageAcquisition The signal intensity is proportional to the numberof nucleotides incorporated. The bases of theconsensus sequence are called by averaging allflowgram signals of the individual reads. Background subtractionNormalizationIdentification of wellsRaw-signal extraction for all active wells(for all images) SoftwareA Flowgram is the graphic representation of thesequence of flowgram signals from a single well ofthe PicoTiterPlate device, which will be translatedinto the nucleotide sequence for an individual read.Image capture for every flow(during sequencing run) ImageProcessingFlowgrams and Base CallingSignalProcessing Signal normalization across wellsFiltering by signal qualityTrimming low-quality and primersequence ends Base-callQuality Score Generation of flowgrams and base-calledsequences with corresponding quality scoresProbability that a measured signalcorresponds to an ideal model signal(calculation of a Phred-like quality score)Data output: FASTA, SFF file formats Applications De novo assemblyMapping (resequencing)Data output: FASTA andace file formatsAmplicon analysisFigure 10: Data processing and analysis output of the Genome Sequencer 20 System.12 GS RunBrowserSoftware Display of raw images Graphic representationsof metrics files Assessment of thegeneral quality of a run,(also for troubleshootingpurposes) Evaluation of the resultsof titration experiments

Data Processing and Analysis OutputResults AssessmentAfter the signal is captured by the CCD camera, itundergoes image processing. The data-processingoutput for mapping and assembly includesnormalized signals across the wells, flowgrams,and base-called sequences. In addition, a Phredlike quality score is calculated. The data-analysisoutput results in a consensus sequence based onthe flowgram information from all wells of the runor a pool of runs, followed by base calling of theconsensus sequence. All raw data is accessible, andthe file formats are compatible with publiclyavailable sequencing analysis tools (Figure 11).GS Run Browser software is part of the standardsoftware package provided with the GenomeSequencer 20 Instrument. It is an inter-active application that allows the user to view the results of aGS 20 sequencing run, and it displays raw imagesand graphic representations of various metrics files.ImagesGS Run Browser software can be used to assess thegeneral quality of a run, and therefore is a usefultool for troubleshooting if problems are observed.The application also facilitates evaluation of theresults of titration experiments. Most of the datagenerated by GS Run Browser software can also beexported to an Excel spreadsheet.SignalGS De Novo Assembler SoftwareGS Reference Mapper SoftwareReference GenomeSoftwareOverlap and ConsensusGeneratorContig 1Contig 2Contig Ordering(Paired End Assembler)PairedEnd ReadsConsensusGeneratorReference GenomeConsensus GenomeMutationDetectorReference GenomeConsensus GenomeFigure 11: Bioinformatics flow process. 13

ApplicationsExpand your versatilityThe Genome Sequencer 20 System uses arevolutionary technology, deciphering more than20 megabases in 5.5 hours on a single instrument.This powerful system enables the followingapplications:Whole genome sequencing De novo whole genome shotgun sequencing andresequencing of microbial genomes and BACclonesOrganization of contigs into scaffolds by apaired-end assembly approachTranscriptome and gene regulationstudies High-throughput transcriptome analysis basedon short tags, ESTs, ChIP, or GIS-PET sequencing,or the genome-wide identification of miRNAsequences Investigation of gene regulation by studying DNAmethylation patternsAmplicon analysis ApplicationsUltra-deep sequencing of PCR products(resequencing for medical research) for— Identification of somatic mutations incomplex cancer samples— High-confidence SNP discovery on apopulation levelWhole Genome Mapping (Resequencing)IntroductionAn integrated software pipeline performs wholegenome mapping on the Genome Sequencer 20Instrument. This pipeline consists of threemodules — GS Reference Mapper software,ConsensusGenerator, and a high-confidenceMutationDetector (Figure 11).The GS Reference Mapper software allows users tomap individual reads to a reference genome up toone megabase in size. Runs can be combined: forexample, up to 200 million bases can be mapped,yielding up to 20x coverage of a 10-million-basegenome. The GS Reference Mapper software createsan ideal flowgram signal space genome from thereference genome for comparison with the reads.By operating in flowgram signal space, the mappingprocess is able to utilize the volume of informationcontained in the flowgram signals (such as theinformation inherent in the negative flows as well aspositive flows) that is partially lost after base calling(conversion to nucleotide space). After mapping theindividual reads, the ConsensusGenerator programcombines the individual read information to createa higher confidence base call using overlappingreads. The MutationDetector sorts through theconsensus base calls to list high-confidencedifferences from the reference genome.Resequencing – Applications— Comparative genomics Identify single base mutations Identify mutation hotspots and conservedregions Identify inserted or deleted genes Assess gene correlations or sequence deviations with observable traits (e.g., understandthe genetic basis for drug resistance)214

Study virulence prediction based on genesequence variation Perform epidemiological analysis Understand the genetic difference betweenindustrial producer strains and theircorresponding parental strains as the basisfor producer strain development Perform metagenomic analysis based onshotgun sequencing of environmental DNAand subsequent mapping against knownmicrobial genome sequences3, 4, 5 Sequence ancient DNA (e.g., shotgunsequencing of the woolly mammothgenome)6Example: Resequencing of a variety ofbacterial genomesgenome sequencing of bacterial strains up to anx-fold coverage, consensus contigs were generatedbased on mapping of raw reads against thecorresponding reference sequence. As the resultsdemonstrate, the Genome Sequencer 20 Systemachieves a high degree of coverage across thegenomes with a high degree of concordance withthe published genomes. This approach was used,for example, by Johnson & Johnson PharmaceuticalResearch and Development to find point mutationsin multiple bacteria.2 Breaks in the scaffold arethe result of incomplete coverage of the genomedue to random chance and repeat regions that arelonger than the raw sequence reads, and thus cannotbe uniquely anchored. The contigs generated bythe GS Reference Mapper software are provided instandard file formats and are readily incorporatedinto standard sequence viewers or assembly tools.Resequencing results obtained with the GenomeSequencer 20 System across a variety of bacterialgenomes are presented in Table 2. After wholeM. genitalium B. licheniformisE. coliS. pneumoniae S. coelicolor S. cerevisiaeGenome Size 12,070,820Coverage Depth (x-fold)20.92223.121.619.821.1Number of Contigs886119116349694Average Contig Size (kb)72.40448.5438.29317.7124.46816.547Size of Largest Contig l Genomic Coverage (%)99.8698.8898.2295.9598.5295.14Total Genomic Coverageof Non-Repeat Regions (%)100.00100.00100.00100.00100.00100.00Total Accuracy ofConsensus Sequence r of Runs0.32.53.01.56.011.0ApplicationsTable 2: The uniformity of coverage — achieved with the Genome Sequencer 20 System — on a number of bacterialgenomes. The results shown are mapping of reads to a known genome. As Coverage of Non-Repeat Regions demonstrates, theGenome Sequencer 20 System process achieves a high degree of coverage across the genomes. The repeats are excluded fromthe mapping results, as they are not uniquely mapped with 100 bp reads. 15

Whole Genome SequencingExpand your versatilityDe Novo Assembly of Whole GenomesGS De Novo Assembler software is the new de novoassembly software for use with the GenomeSequencer 20 Instrument. Exploiting the inherentadvantages of the GS 20 Instrument’s performance,the GS De Novo Assembler software operates inflowgram signal space, as opposed to the standardnucleotide space. By operating in flowgram signalspace, GS De Novo Assembler software is able toutilize the abundant information stored in theflowgram signals that is lost after base calling(conversion to nucleotide space).GS De Novo Assembler software has three mainfunctions: overlap generation, contig layout, andconsensus generation. The overlap generatoraligns raw reads in flowgram signal space using aproprietary algorithm. Consensus generation isbased on signal averaging where all alignedflowgram signals at each position are averagedand the final base call is performed on the averagedsignal. The signal averaging allows higher qualityconsensus base calls (Figure 11).Applications16 De novo Sequencing – Applications— Unknown microorganisms up to 50 Mb Generate an overview of the genomestructure Study DNA sequence organization,distribution, and information content Conduct gene surveys: novelty, locations,and functions Compare to other organisms and correlatewith observable traits— BACs, YACs Sequence BAC clones, for example, as thebasis for whole genome sequencing of plantsand animals— Unknown viruses— Paired End AssemblyExample: De novo sequencing of a variety ofbacterial genomesThe results for de novo sequencing of a variety ofgenomes are shown in Table 3, page 17. The genomeassemblies are nearly as comprehensive as themapping results shown in Table 2, page 15, with thevast majority of bases in the assemblies correct (asmeasured by their concordance with the publishedreference genome). As with the mapping results,the breaks in the contigs occur as a result ofrandom chance and at the boundaries of repeats.The contigs generated by the GS De Novo Assemblersoftware are provided in standard file formats andare readily incorporated into standard sequenceviewers or assembly tools.

M. genitalium B. licheniformisE. coliS. pneumoniae S. coelicolor S. cerevisiaeGenome Size 12,070,820Coverage Depth (x-fold)20.6821.9823.522.3720.525Number of Contigs201361392291013717Average Contig Size (kb)28.00830.65732.6038.8028.38315.817Size of Largest Contig (kb)154.741200.162163.59559.57870.53498.656Total Genomic Coverage (%)96.57%98.63%97.45%92.99%96.96%92.86%Total Genomic Coverageof Non-Repeat Regions (%)99.42100.0000100.0000100.0097.9097100.00Total Accuracy ofConsensus Sequence r of Runs0.32.531.5611Misassemblies02343616Table 3: The performance of GS De Novo Assembler software, the Genome Sequencer 20 System de novo assembler,for sequencing of several bacterial genomes. The genome sequences from all six bacteria are publicly available in GenBank.Using the Genome Sequencer 20 System, de novosequencing of more than 100 bacterial artificialchromosomes (BACs) per month is feasible. Thismakes the system a perfect platform for performinglarge-scale BAC sequencing projects within theframework of metagenomics projects; resequencing portions of eukaryotic genomes;or whole genome sequencing based on a BAC-toBAC approach (e.g., plant genomes) (Table 4).BAC 1BAC 2BAC 3BAC 4Number of Contigs 500 bp(all contigs)6 (9)8 (18)4 (9)8 (12)Total Contig Size (kb)139.794119.438123.051150.849Average Contig Size (kb)23.29914.92930.76218.856Largest Contig Size (kb)102.35635.37578.30551.957Bases PHRED 40 (%)99.999.999.999.8Coverage (%)29.740.234.820.6ApplicationsTable 4: Performance of the Genome Sequencer 20 System — de novo sequencing of Brassica napa BACs. 17

Whole Genome SequencingExpand your versatilityPaired-End AssemblyFacilitate finishing of the high-quality draftsequenceStandard whole genome de novo assembly uses theGS De Novo Assembler software to assemble readsinto contigs. Thereafter, paired-end reads are usedto order and determine the relative positions ofcontigs produced by de novo shotgun sequencingand assembly.The GS Paired End Adaptor Kit provides reagentsfor the creation of a paired-end library of fragmentsfrom a DNA sample. The generated paired-endreads are DNA fragments that have a 44-meradaptor sequence in the middle flanked by a 20-mersequence on each side. The two flanking 20-mersare segments of DNA that were originally locatedapproximately 2.5 kb apart in the genome ofinterest (Figure 13). The ordering and orientingof contigs generates scaffolds which provide ahigh-quality draft sequence of the genome andsimultaneously facilitate finishing of the genome(Figures 12 and 14).The genomes of three different organisms wereshotgun sequenced. The number of assembledcontigs was reduced by adding paired-end data fromadditional sequencing using the Genome Sequencer20 System. This resulted in a higher coverage of theentire genome (Table 5).Genome SizeE. coliB. licheniformis4.6 Mb4.2 Mb12.2 MbOversampling 222723Number ofContigs(unoriented)14098821Number ofPaired-EndReads112,000255,000395,000Number ofScaffolds249153Coverage ofGenome (%)98.699.293.2Table 5: Paired-end data of different organisms.E. coli140BenefitsApplicationsPrepare a paired-end library to order andorient the contigs from your de novosequencing project. Generate a paired-end DNA library for manysequencing runs. Facilitate finishing of the high-quality draftsequence.120Number of Scaffolds S. 0300,000318,357Number of Paired ReadsFigure 12: Incremental assembly from paired-end readsand whole genome shotgun reads.The graph shows thenumber of reads achieved versus the number of scaffolds thatwere obtained. Using more than 200,000 paired-end readsdoes not result in further reduction of the number of scaffolds.18

Figure 13: Generation of a paired-end library. Intact genomic DNA is fragmented to yield an average length of 2.5 kb usinghydroshearing). The fragmented genomic DNA is methylated with Eco RI methylase to protect the Eco RI restriction sites. The endsof the fragments are blunt-ended, polished, and an adaptor DNA oligo is blunt-end ligated onto both ends of the digested DNAfragments. Subsequent digestion with Eco RI cleaves a portion of the adaptor DNA, leaving sticky ends. The fragments are circularized and ligated, resulting in 2.5 kb circular fragments. The adaptor DNA contains biotin tags and two Mme I restriction sites;after treatment with Mme I, the circularized DNA is cleaved 20 nucleotides away from the restriction sites in the adaptor DNA.This digestion generates small DNA fragments that have the adaptor DNA in the middle and 20 nucleotides of genomic DNA thatwere once approximately 2.5 kb apart on each end. These small, biotinylated DNA fragments are purified from the rest of thegenomic DNA using streptavidin beads. The purified paired-end fragments are processed using the standard library-preparationprotocol for the Genome Sequencer 20 System (see page 6, Figure 3).ApplicationsFigure 14: Schematic view of paired-end reads that are used to orient and order contigs and build scaffolds. 19

Transcriptome and Gene Regulation StudiesExpand your versatilityIntroductionThe Genome Sequencer 20 System enables the studyof transcriptomes with outstanding depth of coverageand sensitivity. This is due to the system’s massivelyparallel sequencing technology which generates ahigh number of sequence reads (minimum of200,000 ESTs per five-hour run). As a result,sequencing of transcriptomes is now possible up toa previously unattainable sequence coverage in avery short period of time, facilitating the identification of previously unknown transcripts.7 Anotherapplication using Genome Sequencer 20 technologyis the genome-wide identification of small noncoding RNAs. Several publications prove that thisnew technology offers a straightforward method forgenome-wide identification of completely unknowngroups of small non-coding RNAs.3, 8, 9In addition, the Genome Sequencer 20 technologyfacilitates gene expression studies. Ditags or longertags (e.g., EST tags) can be sequenced very rapidlyat a very high throughput, providing informationon the types of genes expressed and alternativestart or termination sites, as well as revealinginformation about expression levels.Binding sites of DNA-binding proteins, such astranscription factors, can now be identifiedwithout the use of microarrays7; DNA fragmentsthat include binding-site sequences can be isolatedafter immunoprecipitation with their associatedtranscription factors and characterized usinghigh-throughput sequencing.Overview of Transcription and Gene Sequencing Applications using the Genome

sequencing-by-synthesis on a PicoTiterPlate device image and signal processing whole genome mapping or assembly Comparison of high-throughput Sanger technology to the 454 technology used by the Genome Sequencer 20 System, in whole genome sequencing 7 days * Weeks ** 2.5 days 1 day † De novo s