Genome-wide in situ exon capture for selective resequencing

Abstract

Increasingly powerful sequencing technologies are ushering in an era of personal genome sequences and raising the possibility of using such information to guide medical decisions. Genome resequencing also promises to accelerate the identification of disease-associated mutations. Roughly 98% of the human genome is composed of repeats and intergenic or non-protein-coding sequences. Thus, it is crucial to focus resequencing on high-value genomic regions. Protein-coding exons represent one such type of high-value target. We have developed a method of using flexible, high-density microarrays to capture any desired fraction of the human genome, in this case corresponding to more than 200,000 protein-coding exons. Depending on the precise protocol, up to 55-85% of the captured fragments are associated with targeted regions and up to 98% of intended exons can be recovered. This methodology provides an adaptable route toward rapid and efficient resequencing of any sizeable, non-repeat portion of the human genome.

Capturing variation in Lens (Fabaceae): Development and utility of an exome capture array for lentil

“…Despite the decreasing cost and the advancement of whole genome sequencing methods, data storage and computation time are still issues for organisms with large and highly repetitive genomes. Exome capture is a cost‐effective sequencing method that generates reduced representation libraries by targeting the protein‐coding region of a genome (Hodges et al., 2007). The method starts with total genomic DNA sheared into fragments, and target‐specific probes hybridize with the specific regions of interest.…”

Review of massively parallel DNA sequencing technologies

“…Unwanted DNA is then washed off, and the captured material eluted and prepared for sequencing. The capture capacity ranges from a few Mb up to the entire exome (Hodges et al 2007;Porreca et al 2007) using two general methodologies: conventional solid state arrays (on-array capture) and paramagnetic beads (in-solution capture) (Albert et al 2007;Chou et al 2010;Gnirke et al 2009). A number of custom hybridisation platforms are available including Agilent, Roche Nimblegen and Illumina.…”

“…The method of choice is dependent on application; in particular target size, type of target and sample number, required performance, ease of use and costs (Albert et al 2007;Mamanova et al 2010). Ideally the targeting method should allow enrichment of multiple different loci independent of their size, sequence composition or spatial distribution, and should be amenable to automation so that it can match the sequencing capacity.…”

“…Factors affecting the quality score include signal intensity, background noise in the reaction itself or generated by the instrument and crosstalk between clusters. Errors can include overcalls and undercalls (insertions or deletions of bases from the sequence) as well as miscalls (incorrect base assigned) (Albert et al 2007;Brockman et al 2008). Different sequencing technologies are prone to different systematic errors, which influence their utility for different applications; for example, accurately sequencing homopolymeric regions can be difficult using pyrosequencing due to intermediate fluorescence signal intensities resulting from the incorporation of n identical nucleotides.…”

On the future of genetic risk assessment

“…Since the first description of NGS systems (Bentley et al 2008;Margulies et al 2005) and the development of methods for genome partitioning, including genome-wide exon capture and droplet PCR-based DNA enrichment (Gnirke et al 2009;Hodges et al 2007;Hu et al 2009a, b;Mondal et al 2011), numerous groups have successfully combined these methods to identify the molecular causes of monogenic disorders, and whole exome sequencing has been established as a potent and affordable strategy to identify diseasecausing mutations in the 1 % of the human genome that codes for protein (reviewed by Gilissen et al 2011). It is becoming clear, however, that this approach is not successful in a significant proportion of families with monogenic disorders (e.g.…”