Why Hybridization Capture?
Crop plant genomes typically have hundreds or more distinct NLR regions, each of which are several Kb in length. These regions can be independent, or found in clusters close to other NLR regions (Jupe et al 2013). As a large, complex gene family with relatively high sequence similarity between members, this creates challenges for resolving these regions with certain next-generation sequencing (NGS) methods, such as low-pass whole genome sequencing or amplicon-based studies. The technique of hybridization-based capture, on the other hand, provides an ideal balance of robust target recovery in terms of both breadth and depth.
In hybridization capture, NGS libraries are denatured and allowed to hybridize to customized biotinylated probes which are complimentary to regions of interest as shown in Figure 1. Affordable oligo synthesis technologies from vendors such as Arbor Biosciences, can generate custom probe sets containing tens of thousands of unique probes, allowing a single RenSeq experiment to comprise the full sequence diversity of all known R genes in a given genome. This approach delivers recovery of both known NLR regions as well as novel ones, due to the nature of hybridization-based capture to enrich molecules that have sequence divergence up to 20% from a given probe. Such R genes may have been missing during annotation of a given genome sequence, or missing altogether from the reference genome. Thus hybridization capture can function both as a tool for discovery of novel R gene genomic content, as well as a tool to help improve the annotation of NLR regions in a genome (Jupe et al 2013).
The ability to fully sequence one or more complete NLR genes, typically 3.2 kb in length, improves the fine resolution of genomic regions relevant to R gene studies. An advantage of hybridization capture with myBaits® Custom probe libraries is they are fully compatible with any sequencing platform, meaning that the same probe set can be used to enrich DNA samples prepared for Illumina® short-read sequencing or long-read sequencing platforms such as Oxford Nanopore® or PacBio®. Arbor Biosciences is happy to discuss your RenSeq project and help design a custom panel or create a full service project performed by scientists on our myReads® NGS services team.
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