Closeandfinishyourmicrobialgenomewithtwolibraries

  • Flexibleinsertsizes:gel-based20kbandgel-free<8kb
  • Enablemicrobialgenomeclosureusing20kbprotocolwithyourexistingIlluminasequencer
  • IdealforNGSdenovogenomeassembly,closureandfinishing,chromosomalrearrangementdetection,haplotyping,andBACsequencing
  • IncreasedN50sandlargerscaffoldsfromyourassemblyinNextGenSequencing

TableofContents

  • Moreaccurateassembliestocloseandfinishyourgenome
  • Howdoesitwork?
  • ChimeraCode™sequences
  • Insertsizeflexibility
  • Indicesformultiplexinglibraries
  • NxSeqScriptsandSampleDataSet
  • NxSeqGenomeClosureServices

Moreaccurateassembliestocloseandfinishyourgenome.

Fragmentlibrariesarenotsufficienttofullyassemblegenomesduetorepetitiveelementsthatmakethecorrectorderandorientationofcontigsimpossibletodetermine. Longspanreaddata(obtainedthroughmatepairlibraries,jumpinglibraries,orinformativematepairs)canbecombinedwithfragmentlibrariestoproperlyassemblenextgenerationsequencingdataintolargescaffolds,enablingeasiergenomeclosureandfinishing. Theadditionofmatepairlibrariescanmakecost-effectivegenomeclosureareality,withlimitedmanualsequencingrequiredforsmallgenomes.


Figure1:DenovoassemblyandclosingThermusaquaticus

 JGIPermanent
DraftGenome
Fragmentlibrary
NxSeq®MatePairs
+fragmentlibrary
Manuallyfinished
Thermusaquaticusgenome
#Contigs>500bp225422NA
ContigN50106kb79kb144kbNA
Maxcontigachieved343,213167,687260,181NA
Genomescaffolds>5kb0011

Maxscaffoldachieved

343,21302,161,6782,158,963
Genomesize2,338,1932,256,9232,161,6782,338,240
Plasmidscaffolds?024
Plasmidsizes?NA14.5kb,70.3kb14,047bp,16,597bp
78,727bp,and69,906bp
 

Figure2:SingleScaffoldAssemblyofE.coliK12
Fragment+8kbMatePairLibrary

Fragmentlibrary

5,001,861reads–4,656,638mappable
Assembled2.5MreadsinSPAdeswithK45

  • Contigs>1kb: 163
  • Contigs>500bp: 184
  • N50: 56,903
  • Maxcontig:179,213bp

8kbNxSeqlibrary

Megaruptorsheared

  • Rawreads: 6,909,356 
  • Truematepairs: 3,288,275(48%ofraw)
  • Matepairdistance: 8,310bp

K12 Scaffold

DenovoassemblyofE.coliK12genome.2.5Mfragmentreadswereassembleddenovointo163contigsover1kbbySPAdes3.1.Scaffoldingwasperformedwithcommercialsoftwareusing3.2M8kbmatepairs.Thesinglescaffoldwascomparedtoareference genomewithMauve2.3.1.

 

Figure3:AssemblyofRepeat-RichMouseBACs
AssemblyFormsOne171kbScaffold

Assembly Repeat-Rich Mouse BACs

Sequenceassemblyfortworepeat-richmouseBACs.ThesequenceswereassembledwithDNAStarsoftwareusingIonTorrent400bpfragmentsand5kbNxSeqsequencedata.Despitehavingover50%repeatsequence,twoBACswereeachassembledintosinglescaffoldsof171kb(shown)and143kb(notshown).

 

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Howdoesitwork?

Lucigenhascreatedanewparadigminlongspanreadtechnologyviahighlyefficientmatepairlibrarypreptechnology. GenomicDNAisshearedtothedesiredsize(2-8kbforbead-basedmethodsand10-20kbforgel-basedsizingmethods),endrepaired,A-tailedandligatedtobarcodeadaptorspriortosizeselection.TheinsertisligatedtoauniquemultiplexcouplerwithencryptedChimeraCode™sequences.SamplesarethentreatedwithexonucleasetoremoveunwantedDNA,andfinallydigestedwithaselectionofendonucleasestoproducethecorrectsizeddi-tags.BiotincaptureallowsfortheremovalofunwantedDNAfragmentspriortotheadditionofaJunctionCodeadaptorandre-circularization. LibrariesarethenPCRamplifiedandsequencedonanIlluminasequencer.

Figure4. NxSeqLongMatePairLibraryWorkflow

NxSeq Long Mate Pair Workflow

 

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ChimeraCode™Sequences

Lucigen'spatent-pendingChimeraCodesequencesarethekeytoachievingultra-highfrequenciesoftruematepairs,ensuringthemostaccurateassemblypossible. Softwareanalysisoffinalsequencesfiltersoutfalsematepairsformedbychimerasduringthelibraryprepprocess. Asaresult,mostlibrariesachieve>90%truematepairefficiency.


Figure5.ChimericReadDetection
Chimera Code Sequence Prevents False Mates

 

Figure6.

 E.coliDH10B
2kb
E.coliDH10B
5kb
E.coliDH10B
8kb
RawReads6,377,7925,995,9746,851,682
TotalMates2,167,2862,242,9303,091,359
TrueMatePairs2,071,267
(96%)
2,094,413
(93%)
2,938,426
(95%)
ChimericReads96,019
(4%)
148,517
(7%)
152,933
(5%)

Avg.ReadLength(aftersplit)

170b161b159b
TotalMatePairBases352,115,390337,200,493467,209,734
MappedMatePairDistance2,5435,1456,191
 

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InsertSizeFlexibility–YouChooseYourLibrarySize

TheNxSeqLongMatePairLibraryKitcanaccommodateawiderangeofinsertsizestofityourneeds. Bead-based,gel-freefragmentsizingprotocolsenablelibrariesupto8kbinsertsize,whilegel-basedsizingprotocolswillaccommodate10-20kbinsertsize.
Theresultistightsizingofyourmatepairs,enablingaccurateandcompletebioinformaticassembly.

Figure7.LongMatePairLibraries

Long Mate Pair Libiraries

An8kbNxSeqLongMatePairlibrarywasconstructedusingbead-based,gel-freemethods,anda10-20kbmatepairlibrarywasconstructedusinggelisolation. Resultingtruematepairsweremappedagainsttherespectivereferencegenometodeterminetheresultingmatepairdistances.

 

 

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Indicesfor
MultiplexingLibraries

Wanttomultiplexupto12librariesatonetime? LucigenofferstheNxSeqLongMatePairLibraryIndexkitwith12differentindexedamplificationprimersets(Illuminacompatible). Seetheorderinginformationtabformoredetails.

NxSeqScripts&
SampleDataSet

ToperformbioinformaticanalysisofyourIlluminaruns,scriptsmustberuntoconfirmChimeraCodeandJunctionCodesequencesaswellasfilteroutthesesequencespriortofinalassembly. Thesescripts,alongwithasampledatasetfortrialanalysiscanbefoundhere. 

NxSeqGenome
ClosureServices

WouldyouliketohaveaNxSeqLongMatePairlibrary,butdon'twanttodoityourself?ContactourCustomGenomicServicesgroupandwe'llprovideano-obligationquoteforarangeofservicesofferedbyLucigen.

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ORDERINFORMATION

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.