title: Development of strategies for SNP detection in RNA-seq data: application to lymphoblastoid cell lines and evaluation using 1000 Genomes data.
abstract: Next-generation RNA sequencing (RNA-seq) maps and analyzes transcriptomes and generates data on sequence variation in expressed genes. There are few reported studies on analysis strategies to maximize the yield of quality RNA-seq SNP data. We evaluated the performance of different SNP-calling methods following alignment to both genome and transcriptome by applying them to RNA-seq data from a HapMap lymphoblastoid cell line sample and comparing results with sequence variation data from 1000 Genomes. We determined that the best method to achieve high specificity and sensitivity, and greatest number of SNP calls, is to remove duplicate sequence reads after alignment to the genome and to call SNPs using SAMtools. The accuracy of SNP calls is dependent on sequence coverage available. In terms of specificity, 89% of RNA-seq SNPs calls were true variants where coverage is >10X. In terms of sensitivity, at >10X coverage 92% of all expected SNPs in expressed exons could be detected. Overall, the results indicate that RNA-seq SNP data are a very useful by-product of sequence-based transcriptome analysis. If RNA-seq is applied to disease tissue samples and assuming that genes carrying mutations relevant to disease biology are being expressed, a very high proportion of these mutations can be detected.
authors.affiliation: TrinSeq and Neuropsychiatric Genetics Research Group, Department of Psychiatry and Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland.
authors.forename: Emma M
authors.initials: EM
authors.lastname: Quinn
authors.forename: Paul
authors.initials: P
authors.lastname: Cormican
authors.forename: Elaine M
authors.initials: EM
authors.lastname: Kenny
authors.forename: Matthew
authors.initials: M
authors.lastname: Hill
authors.forename: Richard
authors.initials: R
authors.lastname: Anney
authors.forename: Michael
authors.initials: M
authors.lastname: Gill
authors.forename: Aiden P
authors.initials: AP
authors.lastname: Corvin
authors.forename: Derek W
authors.initials: DW
authors.lastname: Morris
mesh_terms: D002460:Cell Line; D019295:Computational Biology; D005091:Exons; D015870:Gene Expression; D023281:Genomics / Q000379:methods; D005838:Genotype; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000379:methods
chemical_list: D012313:RNA
references.citation: Nat Rev Genet. 2001 Nov;2(11):869-78
references.pmid: 11715042
references.citation: Nat Rev Genet. 2011 Feb;12(2):81-5
references.pmid: 21173775
references.citation: Genome Biol. 2010;11(5):R57
references.pmid: 20598109
references.citation: Heredity (Edinb). 2008 Jul;101(1):60-6
references.pmid: 18446183
references.citation: PLoS One. 2012;7(1):e30377
references.pmid: 22276185
references.citation: Bioinformatics. 2011 Jan 15;27(2):272-4
references.pmid: 21075741
references.citation: Nucleic Acids Res. 2009 Sep;37(16):e106
references.pmid: 19528076
references.citation: Nat Rev Genet. 2008 Apr;9(4):291-302
references.pmid: 18347592
references.citation: Nature. 2010 Apr 1;464(7289):713-20
references.pmid: 20360734
references.citation: PLoS One. 2012;7(5):e36264
references.pmid: 22574143
references.citation: Biotechniques. 2008 Jul;45(1):81-94
references.pmid: 18611170
references.citation: Nature. 2009 Sep 10;461(7261):272-6
references.pmid: 19684571
references.citation: Science. 2008 Aug 15;321(5891):956-60
references.pmid: 18599741
references.citation: Am J Med Genet B Neuropsychiatr Genet. 2010 Jun 5;153B(4):919-36
references.pmid: 20127885
references.citation: Nature. 2010 Oct 28;467(7319):1061-73
references.pmid: 20981092
references.citation: Genome Biol. 2011;12(2):R13
references.pmid: 21310039
references.citation: Am J Hum Genet. 2010 Dec 10;87(6):779-89
references.pmid: 21129726
references.citation: Genet Mol Biol. 2012 Jun;35(1 (suppl)):331-4
references.pmid: 22802718
references.citation: PLoS One. 2010 Feb 19;5(2):e9317
references.pmid: 20174472
references.citation: Mamm Genome. 2010 Dec;21(11-12):592-8
references.pmid: 21057797
references.citation: Nat Biotechnol. 2012 Feb 12;30(3):253-60
references.pmid: 22327324
references.citation: Nature. 2010 Apr 1;464(7289):773-7
references.pmid: 20220756
references.citation: Nat Genet. 2011 May;43(5):491-8
references.pmid: 21478889
references.citation: Nat Methods. 2008 Jul;5(7):613-9
references.pmid: 18516046
references.citation: Nat Rev Genet. 2006 Mar;7(3):200-10
references.pmid: 16485019
references.citation: J Neurosci Res. 2009 Jul;87(9):1953-9
references.pmid: 19224581
references.citation: PLoS One. 2009 Sep 04;4(9):e6915
references.pmid: 19730697
references.citation: Genome Res. 2008 Sep;18(9):1509-17
references.pmid: 18550803
references.citation: Nat Rev Genet. 2009 Jan;10(1):57-63
references.pmid: 19015660
references.citation: Nat Genet. 2003 Mar;33 Suppl:228-37
references.pmid: 12610532
references.citation: Science. 2007 Nov 16;318(5853):1136-40
references.pmid: 18006746
references.citation: BMC Bioinformatics. 2010 Mar 15;11:130
references.pmid: 20230626
references.citation: Nat Genet. 2007 Oct;39(10):1202-7
references.pmid: 17873877
references.citation: Nat Struct Mol Biol. 2009 Feb;16(2):130-7
references.pmid: 19136955
references.citation: Nature. 2007 Jun 7;447(7145):661-78
references.pmid: 17554300
references.citation: J Hum Genet. 2010 Jul;55(7):403-15
references.pmid: 20485443
references.citation: Hum Mol Genet. 2010 Jan 1;19(1):122-34
references.pmid: 19825846
references.citation: PLoS One. 2011;6(11):e27288
references.pmid: 22102886
references.citation: PLoS One. 2008;3(11):e3625
references.pmid: 18985160
references.citation: Bioinformatics. 2010 Jul 15;26(14):1772-6
references.pmid: 20547637
references.citation: Bioinformatics. 2009 Aug 15;25(16):2078-9
references.pmid: 19505943
references.citation: Hum Mol Genet. 2009 Nov 15;18(22):4296-303
references.pmid: 19671653
references.citation: BMC Genomics. 2011 Apr 19;12:199
references.pmid: 21504589
references.citation: Science. 2009 Sep 4;325(5945):1246-50
references.pmid: 19644074