Quality Control In Illumina Sequencing Workflows Using The .
Application NoteGenomicsQuality Control in Illumina SequencingWorkflows Using the TapeStationSystemAuthorsNicolle Diessl, Ute Ernst,Angela Schulz, andStephan WolfDKFZ Genomics andProteomics Core Facility,High Throughput SequencingUnit,Heidelberg, GermanyEva GrafAgilent Technologies,Waldbronn, GermanyAbstractThis Application Note describes quality control (QC) steps in various Illuminanext-generation sequencing (NGS) workflows provided as a service by the GermanCancer Research Center (DKFZ) Genomics and Proteomics Core Facility. TheQC steps can be performed using the Agilent 4200 TapeStation system with theScreenTape portfolio. Appropriate reference samples are included as positivecontrols to monitor the individual steps during library preparation. RepresentativeQC data of these reference samples serve as positive examples for successfulcompletion of critical steps of the most frequently used NGS library protocols.
IntroductionExperimentalThe DKFZ is the largest biomedicalresearch institution in Germany. TheHigh Throughput Sequencing Unit of theDKFZ Genomics and Proteomics CoreFacility provides sequencing servicesto national and international cancergenome projects using several Illuminapaired-end and single-read sequencingplatforms. The service includes QC ofstarting material, and library preparationincluding QC, clustering, sequencing,and data analysis. Various sequencingapplications are provided such aswhole genome sequencing (WGS),exome sequencing (WES), targetedresequencing, RNA sequencing(RNA‑Seq), and sequencing ofprotein‑binding regions (ChIP-Seq).MaterialsThe selected protocols are suitableto process high-quality DNA and RNAstarting material, as well as morechallenging samples with low integrityor concentration. The sequencingcore facility subjects all samples to anincoming QC upon receipt. During librarypreparation, additional QC steps areperformed to monitor critical passagesof the workflow. Lastly, the quality of finallibraries is assessed before pooling thesamples for sequencing. To verify thesuccess of library preparation, at leastone positive control is processed parallelper batch of samples at the DKFZsequencing core facility. This ApplicationNote describes representative QC data ofthese reference samples analyzed withthe 4200 TapeStation system.2The HiSeq 2000, HiSeq 2500,HiSeq 4000, HiSeq X, NovaSeq, MiSeq,and NextSeq systems and kits forsequencing were obtained from Illumina(San Diego, CA, USA). The Agilent 4200TapeStation system (G2991AA) incombination with Agilent genomicDNA ScreenTape (p/n 5067-5365)with reagents (p/n 5067-5366),D1000 ScreenTape (p/n 5067‑5582)with reagents (p/n 5067‑5583),High Sensitivity D1000 (HS D1000)ScreenTape (p/n 5067-5584) withreagents (p/n 5067‑5585), andRNA ScreenTape (p/n 5067‑5576)with reagents (p/n 5067‑5577) fromAgilent Technologies (Santa Clara,CA, USA) was used for samplequality control. TruSeq Nano andTruSeq Stranded mRNA kits fromIllumina (San Diego, CA, USA),Agilent SureSelectXT Human AllExon v5 kit (p/n 5190-6210) fromAgilent Technologies (Santa Clara, CA,USA), and NEBNext ChIP-Seq fromNew England Biolabs (Ipswich, MA,USA) were used for library preparation.The Qubit 2.0 instrument or FiltermaxF3 microplate reader obtained fromMolecular Devices (San José, CA, USA)was used for DNA quantification. TheCovaris E220 and LE220 instrumentsfrom Covaris (Woburn, MA, USA) wereused for shearing. The MastercyclerPro from Eppendorf (Hamburg,Germany), Thermocycler TProfessionalfrom Analytik Jena (Jena, Germany),and GeneAmp PCR Systems fromApplied Biosystems were used for PCRamplification.Reference samplesAs positive control samples, Human DNA(NA12891 and NA12878) was obtainedfrom Coriell Institute (Camden, NJ, USA),Human DNA for Whole-Genome VariantAssessment (RM 8398) from NIST(Gaithersburg, MD, USA), and HumanGenomic DNA from Roche DiagnosticsGmbH (Mannheim, Germany). UniversalHuman Reference RNA was obtainedfrom Agilent Technologies (Santa Clara,CA, USA), and First Choice Human BrainReference total RNA was obtained fromThermoFisher Scientific (Waltham, MA,USA).
MethodsQuality control of starting material:The 4200 TapeStation system withthe genomic DNA ScreenTape assayand RNA ScreenTape assay was usedfor sample integrity assessment ofDNA and RNA starting material. Thequantification of DNA or RNA samplesat the sequencing core facility wasperformed using the Qubit assay basedon fluorescence detection.Library preparation: In general, theAgilent SureSelectXT protocol1 was usedfor WES from genomic DNA startingmaterial. The D1000 ScreenTape assaywas used for intermediate QC steps ofthe SureSelectXT protocol. The TruSeqNano protocol2 was used to generateWGS libraries. The High SensitivityD1000 ScreenTape assay was usedto analyze sheared DNA of the TruSeqNano protocol. The NEBNext ChIP-Seqprotocol3 was used for ChIP‑Seq ofprotein-linked DNA. The TruSeq StrandedmRNA protocol4 was used for RNA‑Seq.Custom WGS from WES: WGS librarieswere processed according to theSureSelectXT protocol1, as described inChapter 3: Sample Preparation. Insteadof hybridization and capturing, 100 ngper sample were amplified with sixPCR cycles using appropriate indexprimers and the postcapture PCR cyclingprogram according to Chapter 5, step 1of the SureSelectXT protocol. The indexedlibrary was purified as described instep 2, using 1.8x magnetic beads.Custom TruSeq Nano for FFPE samples:The TruSeq Nano DNA protocol2was slightly modified to processformalin‑fixed paraffin‑embedded(FFPE)‑derived samples. Briefly, 100 ngof gDNA were sheared to 150–200 bpfragments by prolonged incubationat Covaris (when using LE220, FFPEsamples were ultrasonicated for480 seconds instead of 130 seconds;when using E220, shearing for FFPEsamples was increased to 480 secondsinstead of 45 seconds). End repair,adenylation of 3’ ends, and ligation ofadapters were performed as describedin the standard protocol. Adapter-ligatedDNA samples were enriched with 10 PCRcycles instead of eight cycles.Custom ChIP-Seq: The generationof adaptor-ligated libraries wasperformed in accordance with theNEBNext ChIP‑Seq protocol3. The PCRenrichment was modified, using 10 µL ofadapter‑ligated DNA sample and 10 µL ofHigh-Fidelity 2X PCR Master Mix for thePCR reaction.QC and sequencing of generatedlibraries: Final libraries were analyzedwith the D1000 ScreenTape assayaccording to the assay guide to evaluatethe library size. The molarity wascalculated by the maximum peak sizeof the Agilent TapeStation system andthe concentration measured by Qubitor Filtermax microplate fluorometer. Ingeneral, libraries of customer sampleswere normalized to 10 nM, equimolarpooled, transferred to a sequencing flowcell, and loaded onto the appropriateIllumina sequencer. Reference standardswere used to control successful librarypreparation, and were only sequencedfor testing, validation, or troubleshootingpurposes.3
1000Size(bp)29,584erwLo 9001,5002,000200Sample intensity (normalized FU)300acceptance criteria to ensure successfullibrary preparation with DNA samples6.The sequencing core facility cannotguarantee a high coverage rate for DNAmaterial with DIN below 7, and processesthese samples only after approval by theclient. When processing FFPE DNA, otherfacilities observed good sequencingresults using starting material with aDIN 3 and optimized protocols1.DIN 7.35003,0004,0007,00015,00048,500Sample intensity (normalized FU)400ererwLoDIN 8.9500100250400600900Sample intensity (normalized FU)DIN 9.7 60,000QC of genomic DNA (gDNA): gDNAis used as the starting materialfor SureSelectXT and TruSeq Nanoprotocols. The integrity of the gDNAcritically affects the success of librarypreparation and sequencing. TheAgilent gDNA ScreenTape assay offersan objective DNA Integrity Number (DIN)wDNA sequencingfor the assessment of gDNA integrity(Figure 1), resulting in a numerical valuefrom 1 (degraded) to 10 (intact)5. gDNAoriginating from FFPE tissue is typicallypartially degraded, resulting in DIN valuesbelow 6 (Figure 2). Optimized protocolsallow for successful preparation of FFPEmaterial, and usually require adaption ofinput amount, shearing incubation time,and increased PCR cycles dependenton sample integrity. Empirical studiesaccounted for a DIN threshold of 7.0 asLoResults and 3,0004,0007,00015,00048,500100Sample intensity (normalized 00400DIN 3.3500100250400600900Sample intensity (normalized FU)Low9497DIN 4.2500100Sample intensity (normalized FU)DIN 6.1erFigure 1. Electropherograms of reference gDNA analyzed with the gDNA ScreenTape assay. With a DIN above 7.0, the samples all qualify as starting material forDNA library preparation workflows.Size(bp)Figure 2. Electropherogram patterns of gDNA with various degradation levels. The DIN of all samples is below the general quality threshold of 7.0, which is typicalfor FFPE-derived DNA.4
Examples for poor-quality librariesgenerated during SureSelectXT workflowsand possible corrective actions wererecently 0019502pererwSample intensity (normalized FU)3LoUp173pererwLo425Sample intensity (normalized FU) 1035Figure 4. Precapture DNA of the SureSelect XTworkflow analyzed with the D1000 ScreenTapeassay with a maximum peak size of 264 bp. Theexpected maximum peak size ranges from 225 to275 bp.WGS allows identification of unknownconserved alterations of the genome,and serves as a reference sequencefor selected samples that require deepsequencing of the exome by WES.The standard SureSelectXT protocol1is designed to generate WES librariesby target enrichment. The sequencingcore facility created a modified protocolthat allows splitting samples duringthe SureSelectXT workflow into WESand WGS libraries. With this procedure,whole genome and whole exomesequencing data can be achieved froma single aliquot of starting material.The final libraries are qualified usingthe 4200 TapeStation system (Figure 6)with the same requirements for size andquantity as in the standard protocol.43210UpLo286wSample intensity (normalized FU) 103pererIllumina TruSeq Nano: The TruSeqperUpLo54321Figure 5. Final library of a SureSelect XT workflowanalyzed with the D1000 ScreenTape assay. Theelectropherogram shows a peak with a maximumat 289 bp, within the acceptable size range of250 –350 00500200100500Size(bp)25 1035289werFigure 3. Size evaluation of a sheared DNA sampleduring the SureSelect XT workflow with the D1000ScreenTape assay. The sheared DNA shows amaximum peak size of 173 bp, which matches theexpected size range of 150 to 200 bp.25The last QC step of the SureSelectXTprotocol implies the qualification of thefinal library with the D1000 ScreenTapeassay prior to pooling (Figure 5). Due tothe addition of index sequences, anothersize shift is expected between precapture(Figure 4) and final library (Figure 5).The peak maximum of the final library isexpected to be positioned between 250and 350 bp. A minimum concentrationof 2 ng/μL is expected for successfullygenerated final libraries7. High-qualitysamples display a symmetric peak withinthe expected size range of the precaptureproduct or final library without additionalpeaks in the electropherogram. 103Sample intensity (normalized FU)SureSelectXT: NGS target enrichmentenables a deep analysis of specificregions to identify causal geneticvariants of complex conditions. TheSureSelectXT protocol1 is designed tocreate libraries with enriched targetedregions of the genome for sequencingwith Illumina paired-end platforms.For each sample to be sequenced, anindividual indexed library is prepared. Thesequencing core facility implementedfour QC steps7, starting with gDNA inputmaterial (Figures 1 and 2). The twointermediate QC steps include evaluationof smear size after shearing as well asbefore capturing, and are carried outfor all samples in the sequencing corefacility using the D1000 ScreenTapeassay. The expected size range of themaximum peak of sheared DNA is150–200 bp (Figure 3). For precaptureDNA, a larger maximum peak size of225–275 bp is expected due to adapterligation (Figure 4). In addition to sizing,the concentration is evaluated during thesame analysis. This is essential, as thesubsequent hybridization step requires aDNA concentration above 221 ng/μL.Size(bp)Figure 6. Final library of the SureSelect XT workflowmodified for WGS sequencing analyzed with theD1000 ScreenTape assay. The electropherogramshows a peak with a maximum at 286 bp, whichmatches the acceptable size range of 250–350 bp.Custom whole genome sequencing5
6broad peak at the expected size range.High‑quality final libraries are normalizedand pooled before the hybridization tothe sequencing flow 0030050050Sample intensity (normalized FU)400LoUp326BerNEBNext ChIP-Seq: ChromatinpererwLo50025Sample intensity (normalized 50Sample intensity (normalized FU)5LoB 10325perUp545wLoA 103erFigure 7. Fragmented DNA generated with the TruSeq Nano protocol using a Covaris Ultrasonicator andanalyzed with the High Sensitivity D1000 ScreenTape assay. A) Sheared gDNA sample with a maximumpeak size of 326 bp. B) Sheared DNA derived from FFPE tissue with a maximum peak size of 174 bp.25Effectively fragmented samples ofeither gDNA or FFPE starting materialare end‑repaired, and, in the case ofgDNA, size selected. After adenylationof 3’ ends and adapter ligation, thelibraries are enriched to generate the finalproduct. The final libraries are evaluatedwith the D1000 ScreenTape assay toverify the expected size shift. A sizeshift of 120 bp for single-index adaptors,and 130 bp for dual-index adaptorsin comparison to the sheared DNA isexpected. Consequently, for 350 bplibraries from gDNA with high integrityas starting material, the expectedmaximum peak size of the final library isapproximately 470 bp for single‑indexedlibraries, or 480 bp for dual-indexedlibraries, respectively (Figure 8A). Withrespect to FFPE starting material, theexpected maximum peak size of thefinal library is approximately 320 bp(Figure 8B). Electropherogram profiles ofhigh-quality end products show a singleSample intensity (normalized FU)Nano DNA workflow2 generates WGSlibraries, and was designed for sampleswith limited available DNA. The workflowcomprises three QC steps, the firstbeing quality assessment of gDNAstarting material, as shown in Figures 1and 2. After initial QC, 100 ng of gDNAare used for ultrasonic fragmentationto create a 350 bp insert size. The sizedistribution of the fragmented DNA isevaluated using the High SensitivityD1000 ScreenTape assay (Figure 7),which is suited for QC of low sampleamounts. A maximum peak sizebetween 280 and 480 bp indicatessuccessful fragmentation of intactgDNA (Figure 7A). For the processingof FFPE‑derived DNA samples, thesequencing core facility uses anoptimized protocol including prolongedshearing and more PCR cycles. Thecustom workflow generates shearedFFPE DNA with a maximum peak size of150–200 bp (Figure 7B).Size(bp)Figure 8. End-products of the TruSeq Nano protocol analyzed with the D1000 ScreenTape assay. A) Finallibrary of a gDNA reference sample with a maximum peak size of 545 bp. For 350 bp libraries, the expectedmaximum peak size of the final libraries ranges from 460 to 600 bp. B) Final library of a DNA sampleoriginating from FFPE material with a maximum peak size of 313 bp. A size shift of 120 bp resp. 130 bp forthe final library compared to the sheared DNA sample is expected.
The final library includes inserts andadapters; therefore, a size shift of 120 bpfor single-index adaptors, and 130 bpfor dual-index adaptors is expectedcompared to the starting material(Figure 10A). Figure 10B shows anexample of a final library with 494 bp,generated from starting material withapproximately 300 bp. Larger librariesmay show an increased size shift, sincethe bead‑based size selection is not asaccurate. The sizing result is used tocalculate the molarity of the libraries,which are then normalized, pooled, andsequenced.Sample intensity (normalized B 1035Loper255UperwLo325Sample intensity (normalized e intensity (normalized FU)6494 103LoBperUp318wLoA 103erFigure 9. Size determination of starting material for ChIP sequencing with the D1000 ScreenTape assay.A) Example for a sample with an insert size of 255 bp, which was used for the protocol optimized for250 bp. B) Example for a dsChIP-Seq DNA with an insert size of 324 bp, assigned to the protocol optimizedfor 300 bp.25End products of the ChIP workflow areanalyzed with the D1000 ScreenTapeassay to verify the expected total librarysize according to the size selection step(Figure 10).A 1035Sample intensity (normalized FU)immunoprecipitation (ChIP) sequencingis an NGS method combining ChIPwith massive parallel sequencing toreveal binding sites of DNA-associatedproteins. The starting material forthe NEBNext ChIP-Seq workflow3 ischromatin‑immunoprecipitated DNAunlinked from protein. The workflowrequires two QC steps, the first beingquality assessment of the unlinked DNA,and the second the quality assessmentof the final library. After incoming QCof the unlinked DNA starting material(Figure 9), the samples are end-repaired,followed by dA-tailing and adaptorligation. The adapter-ligated librariesundergo a bead-based size selection.The conditions for the size selection areoptimized for specific fragment lengths,which are 150, 200, 250, 300, or 400 bp.The insert size of the starting material isdetermined (Figure 9), and samples arecollated to the closest available fragmentlength for the size selection step.Size(bp)Figure 10. Final library of the ChIP-Seq workflow analyzed with the D1000 ScreenTape assay. Successfullibraries show a narrow library distribution with a peak size of 120–130 bp larger than the starting material.A) Example for a final library with a maximum peak size of 318 bp, correlating to an insert size of 200 bp.B) Example of a final library with a maximum peak size of 494 bp, generated with starting material with anapproximate size of 300 bp.7
001,0002002502500131,00024200Sample intensity (normalized FU)3w28S18S4LoerwB 1035perUp296wLo 103543217001,0001,50030040050020010050025followed by second-strand cDNAsynthesis and subsequent ligation ofthe adapter. The products are purified,and PCR-amplified to create the finalcDNA library (Figure 12), which isanalyzed with the D1000 ScreenTapeassay4. A high-quality library showing asingle symmetric peak with a maximumbetween 250 and 350 bp is suitablefor subsequent cluster generationand sequencing. The exact library sizedepends on the sample. The molarityof the end products is calculated usingthe concentration result of the Qubitfluorometer or the microplate reader.erFigure 11. RNA integrity analysis of RNA reference samples with the RNA ScreenTape assay. A) This RNAsample with a RINe of 8.1 qualifies for RNA-Seq library preparation. B) RNA sample with a RINe of 7.8,missing the RINe threshold of 8.0.Sample intensity (normalized FU)TruSeq Stranded mRNA: RNA-Seq isapplied for transcriptome sequencingand gene expression analysis. TheTruSeq Stranded mRNA workflow4requires total RNA for starting material.It is essential to monitor the integrityof total RNA as starting material inRNA-seq for reliable sequencing data(Figure 11). During the first step in theworkflow, poly-A RNA molecules arecaptured by poly-T oligo magnetic beads.Fragmentation of mRNA is achievedby cleavage with divalent cations.Fragmented mRNA is transcribedinto first-strand cDNA using reversetranscriptase and random primers,A 1035Sample intensity (normalized FU)QC of RNA: RNA is even more subjectto degradation than DNA due to theubiquitous presence of RNase and itsmore fragile single-stranded structure.Therefore, monitoring the integrity ofstarting material is indispensable, and itis highly advisable to process a referencesample as positive control throughoutthe library preparation and sequencing.With the Agilent RNA ScreenTape assay,the RNA integrity number equivalent(RINe) delivers an objective assessmentof the integrity of RNA starting material.RINe has been proven to be equivalentto the widely accepted quality metricRIN9. The fragmentation conditionsof RNA‑seq protocols used by thesequencing core facility are optimizedfor high-quality RNA; more precisely, aRINe of 8.0 or higher is recommendedfor successful library preparation4.Figure 11 shows two samples closeto this threshold, one passing and onefailing the quality requirement. The useof degraded RNA can result in low yield,over-representation of 3’ ends of the RNAmolecules, or failure of the protocol.LoRNA sequencingSize(bp)Figure 12. Final cDNA library product of a TruSeqStranded mRNA workflow analyzed with the D1000ScreenTape assay. The maximum peak size istypically at approximately 300 bp, as shown in thiselectropherogram of a reference sample.
ConclusionsSample QC enables monitoring ofthe success of library preparation forvarious Illumina NGS applications,minimizing the risk of producingunreliable sequencing data due to poorsample quality. The DKFZ sequencingcore facility successfully implementedthe 4200 TapeStation system togetherwith the ScreenTape portfolio forquality control at key steps of multipleIllumina sequencing workflows. Forthe core facility as a service provider,it is essential to determine whetherthe input material provided by clientsis fit for purpose. At the sequencingcore facility, DNA or RNA startingmaterial sample integrity is assessedwith the 4200 TapeStation system, andquantification is currently performedusing the Qubit assay. However, thegDNA and RNA ScreenTape assaysalso provide quantitative data, revealingthe sample concentration within thesame QC step. The intermediateQC steps are specifically usefulduring the establishment of a newlibrary preparation protocol and fortroubleshooting established protocols.Reference samples as positive controlsare used to identify deviations, whichallows for timely implementation ofcorrective actions in case of failure.To create optimum cluster densitiesduring sequencing, it is crucial towww.agilent.com/chemFor Research Use Only. Not for use in diagnostic procedures.This information is subject to change without notice. Agilent Technologies, Inc. 2018Printed in the USA, October 23, 20185994-0327ENaccurately quantify the final libraries ofall workflows. Currently, the molaritydetermination of final libraries isvalidated in the sequencing corefacility with an external calculationstep using the sizing results of theD1000 ScreenTape assay togetherwith the concentration results of theQubit fluorometer or microplate reader.However, the TapeStation Analysissoftware also provides molarity datausing the region function, which allows todirectly evaluate library size and molaritywithin a single QC step.System and the Agilent GenomicDNA ScreenTape Assay,Agilent Technologies ApplicationNote, publication number5991‑5258EN, 2015.6.Use of the Agilent 4200 TapeStationSystem for Sample QualityControl in the Whole ExomeSequencing Workflow at theGerman Cancer Research Center(DKFZ), Agilent TechnologiesApplication Note, publication number5991‑7615EN, 2016.7.The DNA Integrity Number (DIN)Provided by the Genomic DNAScreenTape Assay Allows forStreamlining of NGS on FFPE TissueSamples, Agilent TechnologiesApplication Note, publication number5991-5360EN, 2017.8.Sample Quality Control in AgilentNGS Solutions, Agilent TechnologiesApplication Note, publication number5994-0127EN, 2018.9.Comparison of RIN andRINe algorithms for theAgilent Bioanalyzer and theAgilent 2200 TapeStation systems,Agilent Technologies TechnicalOverview, publication number5990‑9613EN, 2016.References1.Agilent SureSelectXT TargetEnrichment System for IlluminaPaired-End Multiplexed Sequencing,Agilent Technologies User Manual,publication number G7530-90000,2017.2.TruSeq Nano DNA Library PrepReference Guide, publication number#15041110 Rev. D, 2015.3.NEBNext ChIP-Seq Library PrepMaster Mix Set for IlluminaInstruction Manual, publicationnumber #E6240S/L, 2016.4.TruSeq Stranded mRNA SamplePreparation Guide, publicationnumber #15031047 Rev. E, 2013.5.DNA Integrity Number (DIN) withthe Agilent 2200 TapeStation
quality control. TruSeq Nano and TruSeq Stranded mRNA kits from Illumina (San Diego, CA, USA), Agilent SureSelect. XT. Human All Exon v5 kit (p/n 5190-6210) from Agilent Technologies (Santa Clara, CA, USA), and NEBNext ChIP-Seq from New England Biolabs (Ipswich, MA, USA) were used for l
sequencing. By modifying the primer constructs to include the Illumina adapters and a unique barcode, the amplified region of the 16S rRNA gene can be identified in a pooled sample and the sample is prepared for sequencing with the Illumina MiSeq platform. Figure 2. Protocol for barcoded Illumina sequencing. A target gene is identified, which .
(Next-Generation Sequencing) 9/12/16 10 Impact of Next-Generation Sequencing Illumina NextSeq Flow Cell 400,000,000 DNA fragments X 300 nucleotides 120,000,000,000 nucleotides in 29 hours . 9/12/16 11 Illumina Sequencing Benchmarks NextSeq 500 1 Technician 29 hours 4000
Also referred to as Next-Generation Sequencing Parallelize the sequencing process, producing thousands or millions of sequences concurrently Lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods. In ultra-high-throughput sequencing as many as 500,000 sequencing-by-synthesis operations may
The mRNA library was prepared using an Illumina Truseq Stranded mRNA High Throughput Prep kit and samples were sequenced using an Illumina NextSeq 500 Mid-Output Sequencing Reagent kit (v2, 150 cycles), 132 M reads on an Illumina NextSeq 5
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Next generation sequencing (NGS) Nature Methods 2011 Sequencing technology defies Moore's law. 2009: Illumina, Helicos 40-50K 4 2010: 5K , a few days Sequencing the Human Genome Year Log 10 (price) 2000 2005 2010 10 8 6 60K , 2 weeks 4 2 2014: 1000 , 24 hrs 2008: ABI SOLiD 2007: 454
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
the Tiny seed Sequencing Use sensory trays/sequencing cards to explore the story the Tiny seed Sequencing Use sensory trays/sequencing cards to explore the story . Using sequencing cards to plant sunflower seeds Make butterfly Plant peas and carrots as part of a food farm Create habitats using a variety of
