Mapping Accuracy of Short Reads from Massively Parallel Sequencing and the Implications for Quantitative Expression Profiling 英文参考文献.docVIP

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Mapping Accuracy of Short Reads from Massively Parallel Sequencing and the Implications for Quantitative Expression Profiling 英文参考文献.doc

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Mapping Accuracy of Short Reads from Massively Parallel Sequencing and the Implications for Quantitative Expression Profiling 英文参考文献

MappingAccuracyofShortReadsfromMassivelyParallel SequencingandtheImplicationsforQuantitative ExpressionProfiling NicolaPalmieri,ChristianSchlo¨tterer* Institutfu¨rPopulationsgenetik,Veterina¨rmedizinische Universita¨t Wien,Vienna,Austria Abstract Background: Massively parallel sequencing offers an enormous potential for expression profiling, in particular for interspecificcomparisons.Currently,differentplatformsformassivelyparallelsequencingareavailable,whichdifferinread lengthandsequencingcosts.The454-technologyoffersthehighestreadlength.Theothersequencingtechnologiesare morecosteffective,ontheexpenseofshorterreads.Reliableexpressionprofilingbymassivelyparallelsequencingdepends cruciallyontheaccuracytowhichthereadscouldbemappedtothecorrespondinggenes. Methodology/Principal Findings: We performed an in silico analysis to evaluate whether incorrect mapping of the sequencereadsresultsinabiasedexpressionpattern.Acomparisonofsixavailablemappingsoftwaretoolsindicateda considerable heterogeneity in mapping speed and accuracy. Independently of the software used to map the reads, we foundthatforcompactgenomesbothshort(35bp,50bp)andlongsequencereads(100bp)resultinanalmostunbiased expressionpattern.Incontrast,forspecieswithalargergenomecontainingmoregenefamiliesandrepetitiveDNA,shorter reads(35–50bp)producedaconsiderablebiasingeneexpression.Inhumans,about10%ofthegeneshadfewerthan50% ofthesequencereadscorrectlymapped.Sequencepolymorphismupto9%hadalmostnoeffectonthemappingaccuracy of100bpreads.For35bpreadsupto3%sequencedivergencedidnotaffectthemappingaccuracystrongly.Theeffectof indelsonthemappingefficiencystronglydependsonthemappingsoftware. Conclusions/Significance: In complex genomes, expression profiling by massively parallel sequencing could introduce a considerable biasduetoincorrectlymapped sequencereadsiftheread lengthisshort. Nevertheless,thisbiascouldbe accounted for if the genomic sequence is known. Furthermore, sequence polymorphisms and indels also affect th