Undercovered regions might lead to a missed SNP in an important region or cause an assembler to produce shorter contigs. Sequencing bias degrades genomic data applications, including genome assembly and variation discovery, which rely on genome-wide coverage. All existing sequencing technologies fall short of this ideal and exhibit various types and degrees of bias. Ideal whole-genome shotgun DNA sequencing would distribute reads uniformly across the genome and without sequence-dependent variations in quality. Development guided by these assays should result in improved genome assemblies and better coverage of biologically important loci. The assays presented in this paper provide a comprehensive view of sequencing bias, which can be used to drive laboratory improvements and to monitor production processes. Excluding locations that were similar to known bias motifs or likely due to sample-reference variations left only 0.045% of the autosomal genome with unexplained poor coverage. Analysis of Illumina data representing 120-fold coverage of a well-studied human sample reveals that 0.20% of the autosomal genome was covered at less than 10% of the genome-wide average. Our results indicate that combining data from two technologies can reduce coverage bias if the biases in the component technologies are complementary and of similar magnitude. The GC-rich regions prone to low coverage include a number of human promoters, so we therefore catalog 1,000 that were exceptionally resistant to sequencing.

Pacific Biosciences coverage levels are the least biased, followed by Illumina, although all technologies exhibit error-rate biases in high- and low-GC regions and at long homopolymer runs. As in previous work, library construction conditions significantly influence sequencing bias. We applied these methods to the Illumina, Ion Torrent, Pacific Biosciences and Complete Genomics sequencing platforms, using data from human and from a set of microbes with diverse base compositions. Accordingly, we have developed computational methods for discovering, describing and measuring bias. These biases impair scientific and medical applications. DNA sequencing technologies deviate from the ideal uniform distribution of reads.

Torrent Signal Processing First