Exome Sequencing: Current and Future Perspectives

Abstract

The completion of a reference genome sequence for humans took more than 200 scientists more than a decade in a project that cost almost $3 billion to complete (International Human Genome Sequencing Consortium 2004). During the past decade the price of genome sequencing has plummeted, much of the work has become automated, and methods have improved. These advances mean complete genomes can be sequenced quickly and affordably. Human genome sequencing is a special case and enjoys much lower cost-per-gigabase than other species because of Illumina’s HiSeq X platform (Watson 2014). Sequencing can allow for the identification of genetic variants that affect heritable phenotypes, including important disease-causing mutations and natural variation that can be exploited to improve crops and livestock. Despite the significant improvement in sequencing technology, sequencing whole genomes to a depth sufficient to find variants that affect phenotypic expression is expensive compared with targeted sequencing. This review will focus on exome sequencing, a method that targets only a subset of the genome, often the protein coding portion, significantly reducing the sequencing space and subsequently the cost. Details of the method, available platforms, uses in humans and other species, and its benefits over whole-genome sequencing (WGS) will be discussed. The exome has been defined traditionally as the sequence encompassing all exons of protein coding genes in the genome and covers between 1 and 2% of the genome, depending on species. It also may be extended to target functional nonprotein coding elements (e.g., microRNA, long intergenic noncoding RNA, etc.) as well as specific candidate loci. There are two main categories of exome capture technology: solution-based and array-based. In solution-based, whole-exome sequencing (WES), DNA samples are fragmented and biotinylated oligonucleotide probes (baits) are used to selectively hybridize to target regions in the genome. Magnetic streptavidin beads are used to bind to the biotinylated probes, the nontargeted portion of the genome is washed away, and the polymerase chain reaction (PCR) is used to amplify the sample, enriching the sample for DNA from the target region. The sample is then sequenced before proceeding to bioinformatic analysis. Array-based methods are similar except that the probes are bound to a high-density microarray. The array-based method was the first to be used in exome capture (Albert et al. 2007), but it has largely been supplanted by solution-based methods, which require less input DNA and are consequently potentially more efficient; however, studies by Asan et al. (2011) and Bodi et al. (2013) found that NimbleGen’s Sequence Capture Array performed better than the solution-based alternatives in low GC content regions; had high sensitivity and read mapping rates; and single-nucleotide polymorphism (SNP) detection from these reads was more specific to the target region. This suggests that a niche may remain for the older technology. Array-based capture has been used successfully and accurately to identify rare and common variants and identify candidate genes for monogenic diseases in small cohorts (Ng et al. 2009); however, the array-based methods are less scalable owing to the limitation of the number of probes that can be accommodated on the array and additional equipment and time required to process the microarrays. There are several differences between the available platforms, which are constantly being updated and improved. The major providers of exome capture platforms are NimbleGen, Agilent, and Illumina, and each have different designs and strengths (summarized in Table 1). The discussion of the characteristics of each platform will focus on the solution-based human kits; the performance of kits for other species has not been subjected to the same level of comparison. UTR, untranslated region; miRNA, microRNA. As described in Clark et al. (2011), NimbleGen SeqCap EZ v2.0, Agilent SureSelect Human All Exon 50Mb, Illumina TruSeq Exome Enrichment. For NimbleGen SeqCap EZ v3.0, Agilent SureSelect V5, and Illumina TruSeq and Illumina Nextera original versions. Filtering for duplicates, multiple mappers, improper pairs, and off-target reads, data from Chilamakuri et al. (2014) . UTR, untranslated region; miRNA, microRNA. As described in Clark et al. (2011), NimbleGen SeqCap EZ v2.0, Agilent SureSelect Human All Exon 50Mb, Illumina TruSeq Exome Enrichment. For NimbleGen SeqCap EZ v3.0, Agilent SureSelect V5, and Illumina TruSeq and Illumina Nextera original versions. Filtering for duplicates, multiple mappers, improper pairs, and off-target reads, data from Chilamakuri et al. (2014) . NimbleGen’s SeqCap EZ Exome Library has the greatest bait density of any of the platforms and uses short (55−105 bp), overlapping baits to cover the target region (Clark et al. 2011). This approach has been found to be an efficient method for enrichment with the least amount of sequencing needed to cover the target region and sensitively detect variants (Clark et al. 2011) and also has a high level of specificity, showing fewer off-target reads than other platforms (Clark et al. 2011; Sulonen et al. 2011). Importantly, this bait design has been found to show greater genotype sensitivity and more uniformity of coverage in difficult to sequence regions, such as areas of high GC content, than the other platforms (Asan et al. 2011; Sulonen et al. 2011; Bodi et al. 2013). Agilent’s SureSelect Human All Exon Kit is the only platform to use RNA probes, with the other platforms opting for DNA probes. The baits used are longer than those used in NimbleGen’s platform (114−126 bp) and the corresponding target sequences are adjacent to one another rather than overlapping (Clark et al. 2011). This design has been found to be good at identifying insertions and deletions (indels), because longer baits can tolerate larger mismatches (Clark et al. 2011; Bodi et al. 2013; Chilamakuri et al. 2014); it has been suggested that this also may reduce reference allele bias at heterozygous sites compared with other bait designs; however, in practice the allele bias has been similar to other platforms (Asan et al. 2011; Hedges et al. 2011). The platform has been found to produce fewer duplicate reads than NimbleGen but also fewer high-quality reads (Sulonen et al. 2011). Bodi et al. (2013) found that Agilent had a greater alignment rate and fewer PCR duplicates than NimbleGen but also had less uniform coverage. Illumina’s TruSeq Exome Enrichment Kit uses 95-bp probes that leave small gaps in the target region, with paired end reads extending outside the bait sequence during sequencing to fill the gap. This design has been found to have a high percentage of off-target enrichment (Clark et al. 2011), which reduces its target efficiency compared with the other platforms. This kit detects more single-nucleotide variants in the untranslated regions (UTRs) than the other platforms (Clark et al. 2011), although comparisons of performance with NimbleGen and Agilent’s “+UTR” kits have yet to be performed. Following filtering for duplicates, multiple mappers, improper pairs, and off-target reads, Chilamakuri et al. (2014) found that this platform retained fewer reads (54.8%) than NimbleGen (66%) or Agilent (71.7%). At high read counts (>50M), this platform outperformed Agilent’s SureSelect in downstream identification of indels (Clark et al. 2011). Illumina’s Nextera Rapid Capture Exome and Expanded Exome kits are similar to the TruSeq kit in their probe design. They differ from the other kits in that they use transposomes to fragment the genomic DNA, whereas the other platforms use ultrasonication. These kits have not been extensively compared with the other platforms, with only Chilamakuri et al. (2014) having included Nextera in a comparison study. At the time of the study there was only one Nextera kit, which was the Expanded Exome version: the kit with the larger target region of the two. The Expanded Exome kit shares a target region with the TruSeq kit, which includes UTRs and miRNAs. Chilamakuri et al. (2014) found that the Nextera kit had increased coverage of high GC content areas as the result of altered bias in the transposome technology used during fragmentation, decreasing its overall uniformity; however, recent changes to the protocol in the new versions may have improved this. They also found that out of all platforms tested, the Nextera platform retained the fewest reads after filtering for duplicates, multiple mappers, improper pairs, and off-target reads, at 40.1%. Chilamakuri et al. (2014) compared the target regions of the human kits from three providers (NimbleGen SeqCap EZ v3.0 – 64.1Mb; Agilent SureSelect V4 – 51.1Mb; and Illumina TruSeq and Illumina Nextera original version and protocol – 62.08 Mb). Despite the fact that all of the platforms are targeting the human exome, there is surprisingly little overlap between the three designs, with just 26.2 Mb covered by all three target regions. NimbleGen’s and Agilent’s coverage are more similar to one another than either are to Illumina’s; this is likely attributable to the large amount of UTRs covered in Illumina’s target region. Illumina has 22.5Mb of targets unique to their platforms, and 21.8Mb of these are UTRs. NimbleGen and Agilent have 16.1 Mb and 7 Mb of unique targets, respectively. It would be interesting to see how NimbleGen and Agilent’s “+UTR” kits compare with Illumina in single-nucleotide variant calling, particularly in UTRs. Currently, in addition to human kits, NimbleGen offers capture kits for maize, barley, wheat, soy, mouse, and pig exomes, and Agilent offers capture kits for mouse, cattle, and zebrafish exomes. Both providers also offer the opportunity to design custom kits for other species. The kits for nonhuman species use similar bait designs and protocols to the providers’ human kits. Both manufacturers offer a fle

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• Next-Generation Sequencing Technology: Current Trends and Advancements · 2023
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