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).
Backtotop
Howdoesitwork?
LucigenhascreatedanewparADIgminlongspanreadtechnologyviahighlyefficientmatepairlibrarypreptechnology. GenomicDNAisshearedtothedesiredsize(2-8kbforbead-basedmethodsand10-20kbforgel-basedsizingmethods),endrepaired,A-tailedandligatedtobarcodeadaptorspriortosizeselection.TheinsertisligatedtoauniquemultiplexcouplerwithencryptedChimeraCode™sequences.SamplesarethentreatedwithexonucleasetoremoveunwantedDNA,andfinallydigestedwithaselectionofendonucleasestoproducethecorrectsizeddi-tags.BiotincaptureallowsfortheremovalofunwantedDNAfragmentspriortotheadditionofaJunctionCodeadaptorandre-circularization. LibrariesarethenPCRamplifiedandsequencedonanIlluminasequencer.
Figure4. NxSeqLongMatePairLibraryWorkflow
Backtotop
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 |
Backtotop
InsertSizeFlexibility–YouChooseYourLibrarySize
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
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.
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我分别用3ul、5ul、8ul都试过了，没有任何条带，用别人别的方法提好的DNA做，却能出现结果，不知道是自己提取的DNA浓度不够，还是根本没有提取出来。
不知道谁用过，或者有更好的全血提取NDA的试剂盒推荐呢？
本人刚刚开始做PCR，谢谢各位高手！
有试剂A和B，传说10-30分钟就可以提取出来。不知道属不属实啊？
而且1000次580块钱！如果有用过的，可否跟我传授一下心得啊？
各位战友好，因实验要求需要提取人的唾液DNA，但因标本需要在临床采集，无法做到当天就提取DNA，因此天根等漱口水提DNA的试剂盒都不满足实验需求。
之前一直在用艾德莱的唾液DNA保存运输提取试剂盒，但是这段时间下来感觉提取效率太低，而且特别不稳定，经常50%以上的标本都提不出来DNA，或者浓度特别低，完全崩溃的感觉！所以想更换试剂盒或者直接提供唾液DNA的保存液。另国外的这种试剂盒太贵了，因经费问题无法承受，所以希望战友们能够推荐一家效率高些的试剂盒，小女子不胜感激！！
