Patel et al.

High-throughputȱshort-readȱsequencingȱisȱoneȱofȱtheȱ junctions.ȱTopHatȱcanȱfindȱspliceȱjunctionsȱwithoutȱ

latestȱsequencingȱtechnologiesȱtoȱbeȱreleasedȱtoȱtheȱ aȱ referenceȱ annotation.ȱ Byȱ firstȱ mappingȱ RNA-Seqȱ

genomicsȱcommunity.ȱ

readsȱ toȱ theȱ genome,ȱ TopHatȱ identifiesȱ potentialȱ

Typically,ȱ theȱ initialȱ useȱ ofȱ short-readȱ sequencingȱ

exons,ȱsinceȱmanyȱRNA-Seqȱreadsȱwillȱcontiguouslyȱ

wasȱconfinedȱtoȱmatchingȱdataȱfromȱgenomesȱthatȱ

alignȱtoȱtheȱgenome.ȱInȱthisȱstep,ȱweȱhaveȱconsideredȱ

wereȱ nearlyȱ identicalȱ toȱ theȱ referenceȱ genome.ȱ

genomeȱ ofȱ Arabidopsis thaliana ȱ asȱ theȱ referenceȱ

Transcriptomeȱanalysisȱonȱaȱglobalȱgeneȱexpressionȱ

genomeȱforȱTophat2ȱanalysis.ȱThisȱstepȱwasȱdoneȱforȱ

levelȱisȱanȱidealȱapplicationȱofȱshort-readȱsequencing.ȱ

threeȱtimesȱforȱeachȱRNA-seqȱsampleȱandȱallȱresultsȱ

Next-generationȱ sequencingȱ hasȱ becomeȱ aȱ feasibleȱ

ofȱ TopHat2ȱ inȱ .bamȱ fileȱ formatȱ areȱ consideredȱ forȱ

methodȱ forȱ increasingȱ sequencingȱ depthȱ andȱ

furtherȱCufflinksȱanalysis.ȱ

coverageȱwhileȱreducingȱtimeȱandȱcostȱcomparedȱtoȱ

Cufflinks

theȱtraditionalȱSangerȱmethodȱ(LȱJȱCollinsȱ et al. 2008).ȱ

Cufflinksȱ assemblesȱ transcripts,ȱ estimatesȱ theirȱ

Thisȱ studyȱ showsȱ Geneȱ Expressionȱ studyȱ ofȱ threeȱ

abundances,ȱandȱtestsȱforȱdifferentialȱexpressionȱandȱ

differentȱ conditionsȱ ofȱ Arachis hypogaea ȱ L.ȱ whichȱ

regulationȱ inȱ RNA-Seqȱ samples.ȱ Itȱ acceptsȱ alignedȱ

wereȱ treatedȱ withȱ differentȱ methodȱ andȱ analysisȱ

RNA-Seqȱreadsȱandȱassemblesȱtheȱalignmentsȱintoȱaȱ

wasȱ doneȱ byȱ usingȱ variousȱ Bioinformaticsȱ toolsȱ toȱ

parsimoniousȱsetȱofȱtranscripts.ȱCufflinksȱassemblesȱ

getȱdetailȱinformationȱofȱGeneȱExpressionȱwhichȱisȱ

individualȱtranscriptsȱfromȱRNA-seqȱreadsȱthatȱhaveȱ

reportedȱinȱcurrentȱstudy.ȱ

beenȱ alignedȱ toȱ theȱ genomeȱ (Coleȱ Trapnellȱ et al.

Materials and Methods

2012).ȱInȱthisȱstep,ȱweȱhaveȱusedȱgenesȱofȱ Arabidopsis

thaliana ȱasȱreferenceȱ(.gtfȱfile)ȱannotationȱinȱCufflinks.ȱ

Sequence Retrieval

ThisȱstepȱwasȱdoneȱforȱthreeȱtimesȱforȱeachȱRNA-seqȱ

sampleȱinȱ .bamȱfilesȱ whichȱ areȱ outputȱ ofȱ TopHat2ȱ

Asȱ aȱ partȱ ofȱ research,ȱ threeȱ SRAȱ sequencesȱ

andȱallȱresultsȱofȱCufflinksȱ(.gtfȱfileȱformat)ȱwhichȱareȱ

SRR1212866,ȱ SRR1212867ȱ andȱ SRR1212868ȱ areȱ

consideredȱforȱfurtherȱCuffmergeȱanalysis.

downloadedȱfromȱBioProjectȱIDȱ243319ȱfromȱNCBIȱ

databaseȱ forȱ Geneȱ expressionȱ study.ȱ Theseȱ dataȱ Cuffmerge

filesȱsubmittedȱonȱ2-April-2014.ȱTheseȱSRAȱfilesȱareȱ

convertedȱintoȱ.fastqȱfilesȱbyȱSRAȱTOOLKITȱofȱNCBI.

CuffmergeȱusedȱtoȱmergeȱtogetherȱseveralȱCufflinksȱ

assemblies.ȱ Inȱ thisȱ step,ȱ threeȱ .gtfȱ filesȱ ofȱ eachȱ

NGS QC Toolkit

threeȱ Cufflinksȱ resultsȱ andȱ referenceȱ genesȱ fileȱ

ofȱ Arabidopsis thaliana ȱ consideredȱ asȱ referenceȱ

NGSȱ QCȱ Toolkit,ȱ itȱ isȱ anȱ applicationȱ forȱ qualityȱ

annotationȱandȱresultȱwasȱoneȱmergedȱtranscriptȱfileȱ

checkȱ andȱ filteringȱ ofȱ high-qualityȱ data.ȱ Theȱ

ofȱthreeȱtranscripts.

toolkitȱ isȱ comprisedȱ ofȱ user-friendlyȱ toolsȱ forȱ QCȱ

ofȱsequencingȱdataȱgeneratedȱusingȱRocheȱ454ȱandȱ Cuffdiff

Illuminaȱplatforms,ȱandȱadditionalȱtoolsȱtoȱaidȱQCȱ

(sequenceȱformatȱconverterȱandȱtrimmingȱtools)ȱandȱ

Cuffdiffȱ reportsȱ numerousȱ outputȱ filesȱ containingȱ

analysisȱ(statisticsȱtools)ȱ(PatelȱRK,ȱ et al. 2012).ȱNGSȱ

theȱresultsȱofȱitsȱdifferentialȱanalysisȱofȱtheȱsamples.ȱ

TOOLKITȱpackageȱisȱusedȱforȱsequenceȱfilteringȱandȱ

Geneȱ andȱ transcriptȱ expressionȱ levelȱ changesȱ areȱ

filteredȱ sequencesȱ thenȱ uploadedȱ toȱ Galaxyȱ serverȱ

reportedȱinȱsimpleȱtabularȱoutputȱfiles.ȱCuffdiffȱalsoȱ

forȱ FASTQ GROOMER ȱprocess.

reportsȱadditionalȱdifferentialȱanalysisȱresultsȱbeyondȱ

simpleȱ changesȱ inȱ geneȱ expression.ȱ Theȱ programȱ

TopHat2

canȱidentifyȱgenesȱthatȱareȱdifferentiallyȱsplicedȱorȱ

differentiallyȱregulatedȱviaȱpromoterȱswitchingȱ(Coleȱ

TopHatȱ isȱ aȱ programȱ thatȱ alignsȱ RNA-Seqȱ readsȱ

Trapnellȱ et al. 2012).ȱInȱthisȱstep,ȱoneȱmergedȱfileȱofȱ

toȱ aȱ genomeȱ inȱ orderȱ toȱ identifyȱ exon-exonȱ spliceȱ

296