Flexible and scalable genotyping-by-sequencing strategies for population studies

Abstract

BackgroundMany areas critical to agricultural production and research, such as the breeding and trait mapping in plants and livestock, require robust and scalable genotyping platforms. Genotyping-by-sequencing (GBS) is a one such method highly suited to non-human organisms. In the GBS protocol, genomic DNA is fractionated via restriction digest, then reduced representation is achieved through size selection. Since many restriction sites are conserved across a species, the sequenced portion of the genome is highly consistent within a population. This makes the GBS protocol highly suited for experiments that require surveying large numbers of markers within a population, such as those involving genetic mapping, breeding, and population genomics. We have modified the GBS technology in a number of ways. Custom, enzyme specific adaptors have been replaced with standard Illumina adaptors compatible with blunt-end restriction enzymes. Multiplexing is achieved through a dual barcoding system, and bead-based library preparation protocols allows for in-solution size selection and eliminates the need for columns and gels.ResultsA panel of eight restriction enzymes was selected for testing on B73 maize and Nipponbare rice genomic DNA. Quality of the data was demonstrated by identifying that the vast majority of reads from each enzyme aligned to restriction sites predicted in silico. The link between enzyme parameters and experimental outcome was demonstrated by showing that the sequenced portion of the genome was adaptable by selecting enzymes based on motif length, complexity, and methylation sensitivity. The utility of the new GBS protocol was demonstrated by correctly mapping several in a maize F2 population resulting from a B73 × Country Gentleman test cross.ConclusionsThis technology is readily adaptable to different genomes, highly amenable to multiplexing and compatible with over forty commercially available restriction enzymes. These advancements represent a major improvement in genotyping technology by providing a highly flexible and scalable GBS that is readily implemented for studies on genome-wide variation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-979) contains supplementary material, which is available to authorized users.

GBS-based single dosage markers for linkage and QTL mapping allow gene mining for yield-related traits in sugarcane

“…This great alignment rate may be related to the greater amount of scaffolds for this reference compared with the other three references and to the fact that the Pst I enzyme used for library formation is sensitive to DNA methylation [102]; thus, more polymorphic sites are expected from the methyl-filtered genome. Additionally, GBS-based markers had more markers mapped to parent SP80-3280 than to parent RB835486 (Table 4).…”

“…This result can be explained by some factors: a) the possible presence of more Pst I enzyme restriction sites in cultivar SP80-3280 than in RB835486, leading to more polymorphic sites in the first cultivar, as shown for barley cultivars by Liu et al [54]; b) also could be due to more similarity between the genome of SP80-3280 with the reference; or c) due to different methylation effects between the two cultivars. Furthermore, inconsistencies in the number of sites sequenced per sample [102] and in the number of reads per site [103, 104], in addition to the filtering steps applied to the GBS libraries to obtain the markers, can influence the observed result. Other factors that can influence these results are the quality and quantity of the biological replicates used for GBS-based marker calling.…”

Double‐digest RADseq loci using standard Illumina indexes improve deep and shallow phylogenetic resolution of Lophodermium, a widespread fungal endophyte of pine needles

“…Clustering thresholds between 55 and 70% sequence similarity are considered to reconstruct the most accurate phylogenetic topologies for recent species divergences (e.g., <60 million years) but is highly recommended to be tested at various clustering values (Rubin, Ree, & Moreau, 2012). As our reads are longer than typical ddRAD reads sequenced on the HiSeq, we tested clustering at 85% threshold (DaCosta & Sorenson, 2014; Heffelfinger et al., 2014) allowing 15 Ns as well as at a lower 70% threshold allowing 30 Ns. Hereafter, the final datasets using these two criteria are referred to as 85/15N and 70/30N matrices, respectively.…”

Harnessing the power of RADseq for ecological and evolutionary genomics

“…Inconsistency in size selection could produce variation among libraries for methods that use size selection to reduce the set of loci. The consistency of different size selection techniques (automated or manual gel extraction vs. bead-based selection) has not been rigorously quantified, but magnetic beads are likely much less consistent 56 . Methods that target every cut site (original RAD, 2bRAD) are generally expected to be more consistent across libraries (but see Sources of Error).…”

“…Variations on the above techniques include using methylation-sensitive enzymes 70 ; adding more restriction enzymes to existing protocols to further reduce the set of loci 67,71 ; adding a second digestion to eliminate adaptor dimers 18 ; adapting RADseq techniques to other sequencing platforms such as Ion Torrent 71–73 ; and other minor technical modifications 56,74 .…”