Closeandfinishyourmicrobialgenomewithtwolibraries
- FlexIBLeinsertsizes:gel-based20kbandgel-free<8kb
- Enablemicrobialgenomeclosureusing20kbprotocolwithyourexistingIlluminasequencer
- IdealforNGSdenovogenomeassembly,closureandfinishing,chromosomalrearrangementdetection,haplotyping,andBACsequencing
- IncreasedN50"sandlargerscaffoldsfromyourassemblyinNextGenSequencing
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>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 |
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
DenovoassemblyofE.coliK12genome.2.5Mfragmentreadswereassembleddenovointo163contigsover1kbbySPAdes3.1.Scaffoldingwasperformedwithcommercialsoftwareusing3.2M8kbmatepairs.Thesinglescaffoldwascomparedtoareference genomewithMauve2.3.1.
Figure3:AssemblyofRepeat-RichMouseBACs
AssemblyFormsOne171kbScaffold
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
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ChimeraCode™Sequences
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 |
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InsertSizeFlexibility–YouChooseYourLibrarySize
TheNxSeqLongMatePairLibraryKitcanaccommodateawiderangeofinsertsizestofityourneeds. Bead-based,gel-freefragmentsizingprotocolsenablelibrariesupto8kbinsertsize,whilegel-basedsizingprotocolswillaccommodate10-20kbinsertsize.
Theresultistightsizingofyourmatepairs,enablingaccurateandcompletebioinformaticassembly.
Figure7.LongMatePairLibraries
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.
Lucigen
美国Lucigen公司主要开发各类用于基因克隆的试剂盒及相关产品，凭借其独到的产品技术，过硬的产品质量，良好的产品服务赢得了全球广大用户的信赖。
Lucigen Corporation is working to improve people’s lives by providing quality products and services to life science and healthcare professionals. Founded in Wisconsin in 1998, Lucigen offers products and services that enable life-science professionals to perform their research and testing more efficiently and effectively. The company has grown to manufacture and sell over 325 biomedical research products and services to customers worldwide, all under an ISO 13485 compliant quality system. Core competencies include enzyme evolution, protein expression, cloning, competent cells, next gen sequencing, and molecular diagnostics.
产品列表：
No. | 品牌 | 货号 | 名称 | 规格 |
1 | lucigen | 40041-2 | CloneSmart HCAmp Blunt Cloning Kit | 20 rxns |
2 | lucigen | 40300-2 | CloneSmart LCAmp Blunt Cloning Kit | 20 rxns |
3 | lucigen | RG90910K | RiboGuard™ RNase Inhibitor | 10,000 Units |
4 | lucigen | AS3107 | AmpliScribe™ T7 High Yield Transcription Kit | 50 Reactions |
5 | lucigen | ASB71110 | AmpliScribe™ T7-Flash™ Biotin-RNA Transcription Kit | 10 Reactions |
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