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Fragmentlibrariesarenotsufficienttofullyassemblegenomesduetorepetitiveelementsthatmakethecorrectorderandorientationofcontigsimpossibletodetermine. Longspanreaddata(obtainedthroughmatepairlibraries,jumpinglibraries,orinformativematepairs)canbecombinedwithfragmentlibrariestoproperlyassemblenextgenerationsequencingdataintolargescaffolds,enablingeasiergenomeclosureandfinishing. Theadditionofmatepairlibrariescanmakecost-effectivegenomeclosureareality,withlimitedmanualsequencingrequiredforsmallgenomes.
Figure1:DenovoassemblyandclosingThermusaquaticus
| JGIPermanent DraftGenome | Fragmentlibrary | NxSeq®MatePairs +fragmentlibrary | Manuallyfinished Thermusaquaticusgenome | |
| #Contigs>500bp | 22 | 54 | 22 | NA |
| ContigN50 | 106kb | 79kb | 144kb | NA |
| Maxcontigachieved | 343,213 | 167,687 | 260,181 | NA |
| Genomescaffolds>5kb | 0 | 0 | 1 | 1 |
Maxscaffoldachieved | 343,213 | 0 | 2,161,678 | 2,158,963 |
| Genomesize | 2,338,193 | 2,256,923 | 2,161,678 | 2,338,240 |
| Plasmidscaffolds | ? | 0 | 2 | 4 |
| Plasmidsizes | ? | NA | 14.5kb,70.3kb | 14,047bp,16,597bp 78,727bp,and69,906bp |
5,001,861reads–4,656,638mappable
Assembled2.5MreadsinSPAdeswithK45
Megaruptorsheared
DenovoassemblyofE.coliK12genome.2.5Mfragmentreadswereassembleddenovointo163contigsover1kbbySPAdes3.1.Scaffoldingwasperformedwithcommercialsoftwareusing3.2M8kbmatepairs.Thesinglescaffoldwascomparedtoareference genomewithMauve2.3.1.
Figure3:AssemblyofRepeat-RichMouseBACs
AssemblyFormsOne171kbScaffold
Sequenceassemblyfortworepeat-richmouseBACs.ThesequenceswereassembledwithDNAStarsoftwareusingIonTorrent400bpfragmentsand5kbNxSeqsequencedata.Despitehavingover50%repeatsequence,twoBACswereeachassembledintosinglescaffoldsof171kb(shown)and143kb(notshown).
Backtotop
Lucigenhascreatedanewparadigminlongspanreadtechnologyviahighlyefficientmatepairlibrarypreptechnology. GenomicDNAisshearedtothedesiredsize(2-8kbforbead-basedmethodsand10-20kbforgel-basedsizingmethods),endrepaired,A-tailedandligatedtobarcodeadaptorspriortosizeselection.TheinsertisligatedtoauniquemultiplexcouplerwithencryptedChimeraCode™sequences.SamplesarethentreatedwithexonucleasetoremoveunwantedDNA,andfinallydigestedwithaselectionofendonucleasestoproducethecorrectsizeddi-tags.BiotincaptureallowsfortheremovalofunwantedDNAfragmentspriortotheadditionofaJunctionCodeadaptorandre-circularization. LibrariesarethenPCRamplifiedandsequencedonanIlluminasequencer.
Figure4. NxSeqLongMatePairLibraryWorkflow
Backtotop
Lucigen'spatent-pendingChimeraCodesequencesarethekeytoachievingultra-highfrequenciesoftruematepairs,ensuringthemostaccurateassemblypossible. Softwareanalysisoffinalsequencesfiltersoutfalsematepairsformedbychimerasduringthelibraryprepprocess. Asaresult,mostlibrariesachieve>90%truematepairefficiency.
Figure5.ChimericReadDetection
Figure6.
| E.coliDH10B 2kb | E.coliDH10B 5kb | E.coliDH10B 8kb | |
| RawReads | 6,377,792 | 5,995,974 | 6,851,682 |
| TotalMates | 2,167,286 | 2,242,930 | 3,091,359 |
| TrueMatePairs | 2,071,267 (96%) | 2,094,413 (93%) | 2,938,426 (95%) |
| ChimericReads | 96,019 (4%) | 148,517 (7%) | 152,933 (5%) |
Avg.ReadLength(aftersplit) | 170b | 161b | 159b |
| TotalMatePairBases | 352,115,390 | 337,200,493 | 467,209,734 |
| MappedMatePairDistance | 2,543 | 5,145 | 6,191 |
Backtotop
TheNxSeqLongMatePairLibraryKitcanaccommodateawiderangeofinsertsizestofityourneeds. Bead-based,gel-freefragmentsizingprotocolsenablelibrariesupto8kbinsertsize,whilegel-basedsizingprotocolswillaccommodate10-20kbinsertsize.
Theresultistightsizingofyourmatepairs,enablingaccurateandcompletebioinformaticassembly.
Figure7.LongMatePairLibraries
An8kbNxSeqLongMatePairlibrarywasconstructedusingbead-based,gel-freemethods,anda10-20kbmatepairlibrarywasconstructedusinggelisolation. Resultingtruematepairsweremappedagainsttherespectivereferencegenometodeterminetheresultingmatepairdistances.
Backtotop
Indicesfor
MultiplexingLibraries
Wanttomultiplexupto12librariesatonetime? LucigenofferstheNxSeqLongMatePairLibraryIndexkitwith12differentindexedamplificationprimersets(Illuminacompatible). Seetheorderinginformationtabformoredetails.
NxSeqScripts&
SampleDataSet
ToperformbioinformaticanalysisofyourIlluminaruns,scriptsmustberuntoconfirmChimeraCodeandJunctionCodesequencesaswellasfilteroutthesesequencespriortofinalassembly. Thesescripts,alongwithasampledatasetfortrialanalysiscanbefoundhere.
NxSeqGenome
ClosureServices
WouldyouliketohaveaNxSeqLongMatePairlibrary,butdon'twanttodoityourself?ContactourCustomGenomicServicesgroupandwe'llprovideano-obligationquoteforarangeofservicesofferedbyLucigen.
Backtotop
Forafulllistofreagentsandcomponentsincludedinthisproduct,refertotheusermanual. TheNxSeqLongMatePairLibrarykitincludestwoboxes,eachofwhichcanbeorderedseparately. Box1containsallreagentsnecessaryforendrepairandtailingoffragmentedDNA,ligase,andaninternaladaptersequence. Box2containsreagentsforligationtothecouplerandJunctionCode™sequence,exonucleasedigestion,biotincapture,andamplification.
TheNxSeqLongMatePairLibraryIndexkitcontains5reactionseachof12separateindexprimersets,foratotalof60indexreactions. Thekitmaybeorderedincombinationwiththelibrarykitorasaseparateitem.
Forresearchuseonly. Notforhumanordiagnosticuse.
1Thiskitcontainsthereagentsnecessarytogenerate10librariesof8kborless. Largerlibraries,10-20kb,willusemorereagentsandgeneratefewerlibrariesperkit. Instructionstogeneratematepairlibrariesusing10-20kbinsertsaredescribedinSP001:NxSeq20kbMatePairProtocol.