Dealing with the GC-content bias in second-generation DNA sequence data

Terry Speed
University of California, Berkeley (UC Berkeley)

GC-content bias describes the dependence between fragment count (read coverage) and GC content found in high-throughput sequencing assays, particularly the Illumina Genome Analyzer technology. For analyses that
focus on measuring fragment abundance within a genome, this bias can dominate the signal of interest. There is no consensus as to the source or shape of the bias; current methods to remove it do not assume a knowledge
of the curve shape or scale. In this work we analyze regularities in the GC-bias patterns, and find a compact description for this curve family. It is the GC content of the full DNA fragment, not only the sequenced read,
that influences fragment counts. This GC effect is unimodal: both GC rich fragments and AT rich fragments are under-represented in the sequencing results. Moreover, the size of the fragment may interact with the shape
and peak of the GC curve. Based on these findings, we propose a new method to calculate expected coverage. This single-bp GC correction and accommodates library, strand, and fragment lengths information, as well as
non-uniform bin sizes. We show that it outperforms current approaches in copy-number estimation tasks. These GC-modeling considerations can inform other high-throughput sequencing analyses, such as ChIP-seq and RNA-seq,
and illuminate possible causes for the GC-content bias.

Presentation (PDF File)

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