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Figure 2

Nature Methods - 5, 621 - 628 (2008)
Published online: 30 May 2008; | doi:10.1038/nmeth.1226

Mapping and quantifying mammalian transcriptomes by RNA-Seq

Ali Mortazavi, Brian A Williams, Kenneth McCue, Lorian Schaeffer & Barbara Wold

Fig 2 full size
Figure 2. Reproducibility, linearity and sensitivity.
(a) Comparison of two brain technical replicate RNA-Seq determinations for all mouse gene models (from the UCSC genome database), measured in reads per kilobase of exon per million mapped sequence reads (RPKM), which is a normalized measure of exonic read density; R 2 = 0.96. (b) Distribution of uniquely mappable reads onto gene parts in the liver sample. Although 93% of the reads fall onto exons or the RNAFAR-enriched regions (see Fig. 3 and text), another 4% of the reads falls onto introns and 3% in intergenic regions. (c) Six in vitro–synthesized reference transcripts of lengths 0.3–10 kb were added to the liver RNA sample (1.2 times 104 to 1.2 times 109 transcripts per sample; R 2 > 0.99). (d) Robustness of RPKM measurement as a function of RPKM expression level and depth of sequencing. Subsets of the entire liver dataset (with 41 million mapped unique + splice + multireads) were used to calculate the expression level of genes in four different expression classes to their final expression level. Although the measured expression level of the 211 most highly expressed genes (black and cyan) was effectively unchanged after 8 million mappable reads, the measured expression levels of the other two classes (purple and red) converged more slowly. The fraction of genes for which the measured expression level was within plusminus5% of the final value is reported. 3 RPKM corresponds to approximately one transcript per cell in liver. The corresponding number of spliced reads in each subset is shown on the top x axis.

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