Analysis of genome-scale count data in Bioconductor

High read number is relevant for RNA-Seq because our ability toreliably detect and measure rare, yet physiologically relevant, RNAspecies (those with abundances of 1–10 RNAs per cell) depends onthe number of independent pieces of evidence (sequence reads)obtained for transcripts from each gene. This constraint influencedour sequencing strategy, choice of instrument and choice of the25-bp read length.The sensitivity of RNA-Seq will be a function of both molar concentrationMortazaviand transcriptetlength.al. 2008We thereforeNaturequantifiedMethodstranscriptlevels in reads per kilobase of exon model per million mapped reads(RPKM) (Fig. 1a,c). The RPKM measure of read density reflectsthe molar concentration of a transcript in the starting sample bynormalizing for RNA length and for the total read number in themeasurement. This facilitates transparent comparison of transcriptlevels both within and between samples.Wang et al. 2008 Nature Reviews GeneticsBut, this is not the full story. Examination of a well-characterized locusData from a 21-million-read transcriptome measurement of adultmouse skeletal muscle (Fig. 1b,c) illustrate some key characteristicsof our results. Myf6 (also known as Mrf4) is a much-studied29and plong (transc(~0.5transca dyntion. Sand qsequedetecbase (valueimpacthe coRPKMin conlute trthe baand thabout