Types and Frequencies of Sequencing Errors in Methyl-Filtered and High C0t Maize Genome Survey Sequences  

Abstract

The Maize Genome Sequencing Consortium has deposited into GenBank more than 850,000 maize (Zea mays) genome survey sequences (GSSs) generated via two gene enrichment strategies, methylation filtration and high-C 0 t (HC) fractionation. These GSSs are a valuable resource for generating genome assemblies and the discovery of single nucleotide polymorphisms and nearly identical paralogs. Based on the rate of mismatches between 183 GSSs (105 methylation filtration 1 78 HC) and 10 control genes, the rate of sequencing errors in these GSSs is 2.3 3 10 23 . As expected many of these errors were derived from insufficient vector trimming and base-calling errors. Surprisingly, however, some errors were due to cloning artifacts. These G d C to A d T transitions are restricted to HC clones; over 40% of HC clones contain at least one such artifact. Because it is not possible to distinguish the cloning artifacts from biologically relevant polymorphisms, HC sequences should be used with caution for the discovery of single nucleotide polymorphisms or paramorphisms. The average rate of sequencing errors was reduced 6-fold (to 3.6 3 10 24 ) by applying more stringent trimming parameters. This trimming resulted in the loss of only 11% of the bases (15,469/144,968). Due to redundancy among GSSs this more stringent trimming reduced coverage of promoters, exons, and introns by only 0%, 1%, and 4%, respectively. Hence, at the cost of a very modest loss of gene coverage, the quality of these maize GSSs can approach Bermuda standards, even prior to assembly.

Uneven chromosome contraction and expansion in the maize genome

“…For nonrepetitive regions, this strategy allows for high specificity to identify regions within the Zm1S and Zm9L regions that are represented among MF sequences. It should be noted that the MF libraries as well as the BAC clones were derived from the same maize inbred line, B73 (Palmer et al 2003;Whitelaw et al 2003), and the sequencing error rate of the GSS has been reported to be 2.3 ‫ן‬ 10 ‫3מ‬ or lower (Fu et al 2004). In contrast, MF sequence clones containing conserved repetitive sequence can often not be unambiguously anchored to a specific region, as many highly similar copies may be present throughout the genome.…”

Differential methylation of genes and repeats in land plants

“…Isolating and cloning the slowly annealing low-copy or high-C 0 t (HC) fraction of plant genomic DNA results in enrichment for gene sequences (Yuan et al 2003). This technology has been successfully used in maize (Whitelaw et al 2003) although, theoretically, it may select against large gene families that could be normalized in the process, and the extensive manipulation of the DNA required to construct HC libraries resulted in a high proportion of mutated sequences when applied to maize (Fu et al 2004). Furthermore, the HC maize sequences are on average 43% GC versus 50% GC for WGS (whole genome shotgun) (http://www.tigr.org/tdb/tgi/ maize/release4.0/assembly_summary.shtml), which may reflect a selection for AT-rich sequences, which reanneal more slowly than GC-rich sequences do.…”

Nearly Identical Paralogs: Implications for Maize (Zea mays L.) Genome Evolution

“…Toward this end, assemblies of B73 ESTs and geneenriched Genome Survey Sequences (GSSs) were examined for the appearance of ''polymorphic'' nucleotide positions, which we term candidate paramorphisms (CPs; Emrich et al 2004;Fu et al 2004). If a specific CP site is not due to a sequencing error or residual heterozygosity, we term this site a paramorphism (PM; Fu et al 2004). A paramorphism provides evidence of the existence of highly similar genomic loci and is strong evidence of a recent duplication without respect to the underlying duplication mechanism.…”