Fu YB¹, Peterson GW², Dong Y².

G3 (Bethesda). 2016 Jan 27. pii: g3.115.025775. doi: 10.1534/g3.115.025775. [Epub ahead of print]

Abstract

Genotyping-by-sequencing (GBS) has emerged as a useful genomic approach for exploring genome-wide genetic variation. However, GBS commonly samples a genome unevenly and can generate a substantial amount of missing data. These technical features would limit the power of various GBS-based genetic and genomic analyses. Here we present software called IgCoverage for in silico evaluation of genomic coverage through GBS with an individual or a pair of restriction enzymes on one sequenced genome and report a new set of 21 restriction enzyme combinations that can be applied to enhance GBS applications. These enzyme combinations were developed through an application of IgCoverage on 22 plant, animal and fungus species with sequenced genomes, and some of them were empirically evaluated with different runs of Illumina MiSeq sequencing in 12 plant species. The in silico analysis of 22 organisms revealed up to 8 times more genome coverage for the new combinations consisted of pairing 4- or 5-cutter restriction enzymes than the commonly used enzyme combination PstI+MspI. The empirical evaluation of the new enzyme combination (HinfI+HpyCH4IV) in 12 plant species showed 1.7 to 6 times more genome coverage than PstI+MspI, and 2.3 times more genome coverage in dicots than monocots. Also, the SNP genotyping in 12 Arabidopsis and 12 rice plants revealed that HinfI+HpyCH4IV generated 7 and 1.3 times more SNPs (with 0 to16.7% missing observations) than PstI+MspI, respectively. These findings demonstrate that these novel enzyme combinations can be utilized to increase genome sampling and improve SNP genotyping in various GBS applications.

See http://www.g3journal.org/content/early/2016/01/25/g3.115.025775.long

Figure 2: Distributions of DNA fragments generated by in silico digestions with three restriction enzyme combinations on two chromosomes of Arabidopsis thaliana (A, B) and Oryza sativa (C, D). The number of DNA fragments and the average enzyme-cutting position based on a 100 kb sliding-window of a given chromosome are calculated and shown with a colored line for each enzyme combination. The corresponding horizontal linear line represents the average fragment count for an enzyme combination on the chromosome. More digestions were found for HinfI+HpyCh4IV than the other two enzyme combinations.