2010/08/26

RNA-seq から発現量が変動した遺伝子をどのように得るか

RNA-seq のデータから、統計的に有意な変動を示す遺伝子をどのように得るか。Microarray ときにも散々議論されましたが、RNA-seq でも同じく議論になっています。現在提案されている手法についてまとめてみました。

まだ全部には目を通せてません。すべて読んだ後に解説を書くかもしれませんが、網羅している自信がないので、先にリンクを列挙しておきます。主に論文とツールのソースコード、マニュアルなどです。ほかに情報をお持ちの方は教えてくださいな。

1. cufflinks と cuffdiff


http://cufflinks.cbcb.umd.edu/howitworks.html の後半に cuffdiff の説明がある。

FPKM
Trapnell C, Williams BA, Pertea G, Mortazavi AM, Kwan G, van Baren MJ, Salzberg SL, Wold B, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology doi:10.1038/nbt.1621
http://dx.doi.org/10.1038/nbt.1621


Jensen–Shannon divergence
http://en.wikipedia.org/wiki/Jensen%E2%80%93Shannon_divergence

以下の3つも知っておいたほうが理解しやすい良い。

RPKM
Ali Mortazavi, Brian A Williams, Kenneth McCue, Lorian Schaeffer and Barbara Wold Mapping and quantifying mammalian transcriptomes by RNA-Seq Nature Methods, volume 5, 621 - 628 (2008)
http://www.nature.com/nmeth/journal/v5/n7/abs/nmeth.1226.html

Hui Jiang and Wing Hung Wong, Statistical Inferences for isoform expression, Bioinformatics, 2009 25(8):1026-1032
http://bioinformatics.oxfordjournals.org/cgi/content/full/25/8/1026

2. baySeq


http://www.bioconductor.org/packages/release/bioc/html/baySeq.html
Hardcastle TJ, Kelly KA. baySeq: Empirical Bayesian Methods For Identifying Differential Expression In Sequence Count Data. BMC Bioinformatics. 2010 Aug 10;11(1):422. [Epub ahead of print] PubMed PMID: 20698981.
http://www.biomedcentral.com/1471-2105/11/422/abstract

マイクロアレイ時代に提案されていた元ネタ達
Smyth GK: Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 2004
http://www.biomedcentral.com/pubmed/16646809

Lönnstedt IM, Rimini R, Nilsson P: Empirical Bayes Microarray ANOVA and Grouping Cell Lines by Equal Expression Levels. Statistical Applications in Genetics and Molecular Biology 2005
http://dx.doi.org/10.2202/1544-6115.1125


3. DEseq


Differential expression analysis for sequence count data
Simon Anders and Wolfgang Huber
http://precedings.nature.com/documents/4282/version/2

http://www-huber.embl.de/users/anders/DESeq/
http://www.bioconductor.org/packages//release/bioc/html/DESeq.html
http://www-huber.embl.de/users/anders/DESeq/

4. EdgeR


http://www.bioconductor.org/packages/release/bioc/html/edgeR.html

Robinson, MD and Smyth GK (2007)
Moderated Statistical Tests for Assessing Differences in Tag Abundance, Bioinformatics. 23(21):2881-7.
http://www.ncbi.nlm.nih.gov/pubmed/17881408

Robinson, MD and Smyth, GK (2008)
Small Sample Estimation of Negative Binomial Dispersion, with applications to SAGE data, Biostatistics. 9(2):321-32.
http://www.ncbi.nlm.nih.gov/pubmed/17728317

EdgeR は元々 msage というパッケージだった。

古典


SAGE時代の古典的論文。

Baggerly KA, Deng L, Morris JS, Aldaz CM: Overdispersed logistic regression for SAGE: modelling multiple groups and covariates. BMC Bioinformatics 2004 , 5:144
http://www.biomedcentral.com/pubmed/15469612

Lu J, Tomfohr JK, Kepler TB: Identifying differential expression in multiple SAGE libraries: an overdispersed log-linear model approach. BMC Bioinformatics 2005 , 6:165.
http://www.biomedcentral.com/pubmed/15987513

定量化と簡単な正規化法の比較


Bullard JH, Purdom E, Hansen KD, Dudoit S. Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments. BMC Bioinformatics. 2010 Feb 18;11:94. PubMed PMID: 20167110; PMCID: PMC2838869.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838869/

正規化は
(1) total lane counts, as in RPKM
(2) per-lane counts for a "housekeeping" gene expected to be constantly expressed across biological conditions, e.g., POLR2A
(3) per-lane upper-quartile of gene counts for genes with reads in at least one lane.
の3つを比較

論文のなかに出てくる Hypergeometric model
Marioni JC, Mason CE, Mane SM, Stephens M, Gilad Y. RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res. 2008 Sep;18(9):1509-17. Epub 2008 Jun 11. PubMed PMID: 18550803; PMCID: PMC2527709.
http://www.ncbi.nlm.nih.gov/pubmed/18550803

Multiple DGE librairies comparison. (EdgeR baySeq DESeq)
http://seqanswers.com/forums/showthread.php?t=4349