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.
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便宜点的就是国产的tiangen，效果会差一点点 如果是大样本的全血 用这个也凑合
普通的手提或者试剂盒提取的土壤基因组DNA由于常常残留有PCR的强烈抑制物如腐殖酸、棕黄酸等杂质造成实验失败，此外，由于采用了剧烈的玻璃珠击打来破裂菌体，常常造成DNA剪切和降解。本公司经过长期研发开发出了具有自主知识产权的土壤基因组DNA，通过专利配方的腐殖酸和棕黄酸去除试剂配合特殊处理的纯化柱，可以最大程度的去除这些杂质，同时加上多次柱漂洗，确保得到的DNA具有极高纯度，此外独特的抽提和裂解体系可以迅速裂解细胞（壁）和灭活细胞内核酸酶，不需要借助玻璃珠破壁，有效保证了基因组DNA的完整性。
产品特点：
1.本公司独有的专利配方和纯化柱能有效去除腐殖酸等杂质。
2.不需要借助玻璃珠破壁，有效保证了基因组DNA的完整性，长度可达30kb -50kb，可直接用于PCR，Southern-blot和各种酶切反应。
3.兼容性强，适用于各种不同的土壤包括淤泥等提取困难的土壤。
4.多步骤去除各种杂质和抑制物，保证了极高纯度，OD260/OD280典型的比值达1.7～1.9。
5.不需要使用有毒的苯酚等试剂，也不需要乙醇沉淀等步骤。
6.快速，简捷，单个样品操作一般可在60分钟内完成。
你找下，杭州昊鑫生物
2、电泳问题：如果你核酸定量浓度不低的话就考虑电泳问题,电泳的时候加上1kb的marker,如果marker跑不出来说明你凝胶有问题,一般只要marker跑出来了,DNA浓度又比较高,而没有条带这样的现象很少见,DNA提取比较简单而且相当稳定,本人当时做甲基化提取的DNA有一次拿出来电泳忘了放回冰箱了,结果大夏天的在外面放了一天,心怀忐忑的进行了一次电泳,结果条带依然给力,一点都没有降解.
根据模板的类型分为DNA类PCR试剂盒和RNA类PCR试剂盒
PCR mix厂家：普洛麦格公司,QIAGEN公司,罗氏公司,TAKARA公司.
成品PCR试剂盒厂家：深圳匹基,上海之江,中山达安,上海科华,上海复星.
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！
