周二培训材料功能注释及blast2go的使用trinity lecture

vs -firstmethod:firstalignallthereadstoareferencegenomeandthenmergesequenceswithoverlap tools:Scripture, umsensitivity,butdependoncorrectread-to-referencealignment,thereferencegenomeisavailableassembly-firsttools:?:donotrequireanyread-referencealignments,importantwhenthegenomicsequenceisnotavailable,isgapped,highlyfragmentedorsubstantiallyaltered,asincancercellsTrinity,developedattheBroadInstituteandtheHebrewUniversityofJerusalem(耶路撒冷希伯莱大学),representsanovelmethodfortheefficientandrobustdenovoreconstrucionoftranscriptomesfromRNA-seqTrinitycombinesthreeindependentsoftwaremodules:Inchworm,Chrysalis,andButterfly,appliedsequentiallytoprecesslargevolumesofRNA-seqreads.psBroad,一个附属于MIT和harvard的，在生物医学和基1.分 reads，构建k-mer字典“25-mersworkverywellforbothhighlyandlowlyexpresseddeBruijn字典--seqType (JellyfishMemory)(~1GRAMper1Mpairsof2.从k-mer字典中移除error-containingk-1.k-mersthathaveidenticalk-1prefixes,differingonlyattheirterminalnumleotide,andremovingthosek-mersthatare<5%abundantascomparedtothemosthighlyabundantk-merofthe2.error-containingkmerswillbegreatlyenrichedwithinthelow-abundancekmercountsandcanbeexcludedwithminimalloss.--min_kmer_cov3.选择seedk-4.Seedk-mer延伸 kmer,每使用一个kmer，将其从kmer字典中移除thesequenceyieldedfromthebidirectionalseedk-merextensionisreportedasadrafttranscriptcontig5.重复seedselectionbidirectionalk-merextension直到k-mer字典耗尽6.Withanaveragek-mercoverageofLengthatleast (2*(k-InchwormdoesaverygoodjobatreconstructingtranscriptsfromRNA-seqdata,butsinceitleveragesonlyuniquek-mersforcontigconstruction,itcanonlyreportthepartsofalternativelysplicedisoformsthatareunique.SubsequentTrinitystepsreconstructthefull-lengthalternativelysplicedtranscripts.由于config的构造方法，使得各个config之间不可能共享k，因此这些Inchwormconfigs不能很好的表征各种可变剪切形式和同 1.groupscontigsintoconnectedcommonk-1basesperfectReadsupport:therearenreadsthatspanacrossapotentialjunction(welds)andextendperfectmatchesby(k-1)/2basesoneachside; (default:2.buildadeBruijngraphforeachK-1mer Kmer 3.readsthereadsarethenmappedtocomponentsbyselectingthecomponentthatsharesthemostk-1-merswiththeread.Chrysalisalsocountsallk-mers(inassignedreads)andstoresthemas'edgeweight'(fordeBruijnconstructedinlaststep)toindicatetheirsupportinthereadset.ComponentswithlessthanaminimumnumberofnodesareButterflyresolvesalternativelysplicedandparalogousgraph1.mergingconsecutivenodesinlinearpathsinthedeBruijngraphtoformnodesthatrepresentlongersequences2.pruningedgesthatrepresentminordeviations(supportedbycomparativelyfewreads),whichlikelycorrespondtosequencingpathtracesthepathsthatreadsandpairsofreadstake withinthegraphandreportsthemostprobabletranscriptsasafastafile.InchwormassemblestheRNA-seqdataintouniquesequencesoftranscripts,oftengeneratingfull-lengthtranscriptsforadominantisoform,butthenreportsjusttheuniqueportionsofalternativelysplicedtranscripts.ChrysalisclusterstheInchwormcontigsintoclustersandconstructscompletedeBruijngraphsforeachcluster.Eachclusterrepresentsthefulltranscriptonalcomplexityforagivengene(orsetsofgenesthatsharesequencesincommon).Chrysalisthenpartitionsthefullreadsetamongthesedisjointgraphs.Butterflythenprocessestheindividualgraphsinparallel,tracingthepathsthatreadsandpairsofreadstakewithinthegraph, yreportingfull-lengthtranscriptsforalternativelysplicedisoforms,andteasingaparttranscriptsthatcorrespondstoparalogousgenes.--Ifdataarederivedfromagene-densecompactgenome,suchasfromfungalgenomes,wheretranscriptsmayoftenoverlapinUTRregions.Trinitywillexaminetheconsistencyofreadpairingsandfragmenttranscriptsatpositionsthathavelittleread-pairing--SS_lib_type(Strand-specificlibraryPairedRF:firstread(/1)offragmentpairissequencedasanti-sense(reverse(R)),andsecondread(/2)isinthesensestrand(forward(F));typicalofthedUTP/UDGsequencingmethod.FR:firstread(/1)offragmentpairissequencedassense(forward),andsecondread(/2)isintheantisensestrand(reverse)Unpaired(single)F:thesinglereadisinthesense(forward)R:thesinglereadisintheantisense(reverse)>comp2_c0_seq1len=2364path=[0:0-587588:588-10761146:1077-2363]comp2:sequenceisderivedfromChrysaliscomponent#2c0:sequencealsocorrespondstoButterflysub-component#0(duringgraphcompactionandpruning,somecomponentsarepartitionedintodisconnectedponents).seq1:sequencecountfromchrysaliscomponent2,butterflyponentzero.Ifthisponentyieldsmultiplesequences,thesewillhavedifferentseqnumbers.len:lengthofthetranscript“Thegeneralideaistocombineallyourrna-seqdataandgenerateoneassembly.Then,toalignthereadsfromthedifferentsamplessepara totheTrinityassemblies,computingabundanceestimatesbas