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Hui Jiang and Julia Salzman have posted a new paper on the arXiv proposing a novel approach to correcting for non-uniform coverage of transcripts in RNA-Seq: “A penalized likelihood approach for robust estimation of isoform expression” (October 1, 2013).

Their paper addresses the issue of non-uniformity of read coverage across transcripts in RNA-Seq, an issue that is frustrating for the challenges it presents in analysis. The non-uniformity of read coverage in RNA-Seq was first noticed in A. Mortazavi et al., Mapping and quantifying mammalian transcriptomes, Nature Methods 5 (2008), 621–628. Figure 1 in the paper (see below) shows an example of non-uniform coverage, and the paper discusses ideas for library preparation that can reduce bias and improve uniformity.

Mortazavi_Fig1b

Figure 1b from Mortazavi et al. (2008) showing (non-uniform) coverage of Myf6.

Supp_1a_MortazaviSupplementary Figure 1a from Mortazavi et al. (2008) describing uniformity of coverage achievable with different library preparations. “Deviation from uniformity” was assessed using the Kolmogorov-Smirnov test.

The experimental approach of modifying library preparation to reduce non-uniformity has been complemented by statistical approaches to the problem. Specifically, various models have been proposed for “correcting” for experimental artefacts that induce non-uniform coverage. To understand Jiang and Salzman’s latest paper, it is helpful to review previous approaches that have been proposed. Read the rest of this entry »

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