A modified sequence capture approach allowing standard and methylation analyses of the same enriched genomic DNA sample

Abstract

BackgroundBread wheat has a large complex genome that makes whole genome resequencing costly. Therefore, genome complexity reduction techniques such as sequence capture make re-sequencing cost effective. With a high-quality draft wheat genome now available it is possible to design capture probe sets and to use them to accurately genotype and anchor SNPs to the genome. Furthermore, in addition to genetic variation, epigenetic variation provides a source of natural variation contributing to changes in gene expression and phenotype that can be profiled at the base pair level using sequence capture coupled with bisulphite treatment. Here, we present a new 12 Mbp wheat capture probe set, that allows both the profiling of genotype and methylation from the same DNA sample. Furthermore, we present a method, based on Agilent SureSelect Methyl-Seq, that will use a single capture assay as a starting point to allow both DNA sequencing and methyl-seq.ResultsOur method uses a single capture assay that is sequentially split and used for both DNA sequencing and methyl-seq. The resultant genotype and epi-type data is highly comparable in terms of coverage and SNP/methylation site identification to that generated from separate captures for DNA sequencing and methyl-seq. Furthermore, by defining SNP frequencies in a diverse landrace from the Watkins collection we highlight the importance of having genotype data to prevent false positive methylation calls. Finally, we present the design of a new 12 Mbp wheat capture and demonstrate its successful application to re-sequence wheat.ConclusionsWe present a cost-effective method for performing both DNA sequencing and methyl-seq from a single capture reaction thus reducing reagent costs, sample preparation time and DNA requirements for these complementary analyses.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4640-y) contains supplementary material, which is available to authorized users.

Hidden variation in polyploid wheat drives local adaptation

“…Capture probes were designed (12-Mb capture targeting 36 Mb) as described in our previous work (Supplemental Fig. S1 from Olohan et al 2018).…”

“…To accurately apply MethylSeq to a diversity panel, we require bisulfite-treated and untreated sequence data for each wheat accession to identify C-T SNPs, which would otherwise be incorrectly classified as unmethylated cytosines. This was achieved using a modified sequence capture protocol (Darst et al 2010;Olohan et al 2018) that generates two libraries for sequencing from one capture; a bisulfite-treated and an untreated library for each accession. Post-sequencing, the untreated data sets were aligned to the TGAC v1 Chinese Spring reference sequence, and SNP calling was performed (Methods; Clavijo et al 2017).…”

“…The 12-Mb target sequence for this Agilent enrichment system was generated as per Supplemental Figure S1 from Olohan et al (2018). For the capture, 99,949 120-mer RNA baits were designed.…”

“…Watkins bread wheat landrace collection plus the reference variety Chinese Spring (Supplemental Table S1). DNA was extracted from seedlings, fragmented, and taken through a modified sequence capture protocol to allow genetic and methylation analysis of the same enriched genomic DNA sample by splitting the sample post-capture (Supplemental Methods; Olohan et al 2018).…”

Integrating genomic resources to present full gene and putative promoter capture probe sets for bread wheat

“…We have previously demonstrated the use of sequence capture to allow the study of both genotype and DNA methylation across targeted regions in wheat [21]. Using bisulfite treatment after sequence capture, DNA methylation analyses can be performed using the same probe sets that are implemented for genotyping [35].…”