For many projects, it can be overkill to do Whole Genome
Projects with a more specific focus can leverage Whole Exome
thanks to its higher coverage and lower overall cost.
WES only targets regions of the genome that translate to mature RNA after the exons have been spliced out1. This information accounts for roughly 1% of the human genome, or 50 MBases. In contrast to SNP array sequencing, which has an extremely narrow sequencing focus, exome sequencing can detect uncommon disease variants that the array won’t even be looking for. A newer technology, Low Pass Sequencing, takes a more stochastic approach and can be used as a compromise between these techniques.
Like most sequencing approaches, the WES workflow will include sample extraction, purification, library preparation and sequencing, as well as a number of downstream data manipulations. While the actual sequencing aspect remains fairly straightforward, the library preparation is what sets this technique apart procedurally.
Capturing and amplification of the exons is done using specific oligonucleotide probes or “baits” that hybridize to the regions of interest. This process is illustrated to the left2. Different library prep kits offered by Agilent, Illumina, and Roche all work towards the same goals but with different strengths and weaknesses. An analysis of earlier iterations of these offerings3 suggested that Roche required the least amount of sequencing to sensitively detect small variants, but Agilent and Illumina are able to detect a greater total number of variants with additional sequencing. These strengths and weaknesses have potentially changed, however.
Agilent’s latest iteration is the SureSelect XT V7. They boast a novel bait design algorithm that results in an end-to-end design of only 48.2Mb. A key to attaining this is only targeting genes from the most robust databases such as UCSC’s and GENCODE’s.4 Agilent’s platform makes a point of highlighting efficiency - accurately capturing everything that most projects will need, straight out of the box.
Illumina’s offering, the TruSeq Exome Kit, makes a point of highlighting its cost-efficiency and proprietary enrichment technology. TruSeq enrichment yields uniform coverage and high on-target sequencing reads5. As most WES workflows will be sequenced on Illumina platforms, using their prep chemistry affords a more seamless workflow.
The SeqCap EZ Exome kit by Roche has very similar offerings to the competitors, and perhaps slightly more. Their kit offers coverage of genes from more databases, resulting in an overall increase in genome coverage. Their kit also captures flanking regions of certain exons to provide additional context6. This higher breadth and coverage, however, will also increase the sequencing overhead.
A more recent review of these company’s offerings suggest that SeqCap and SureSelect offer excellent depth of coverage whereas Illumina shows the highest on target levels7. These pros and cons can really be seen as variances in experimental need such as read depth vs target length and number of genes being looked at. Researchers can also achieve increased flexibility by ordering a custom capture kit to focus on their specific regions of interest.
From a broader perspective, we can see that Whole Exome Sequencing services
offer a very attractive alternative to WGS if it suits your experimental design. However, it is by no means a silver bullet when searching for pathogenic variants. There are countless insights to be gained from analyzing the much larger non-coding regions of the genomes. Regulatory regions and Copy-Number Variations (CNVs), to name a few, can have tremendous impact on pathogenesis. Longer read technologies such as those offered by Pacific Biosciences
and Oxford Nanopore are needed to effectively investigate these less well-understood areas.
2Chen, Rui, Hogune Im, and Michael Snyder. "Whole-exome enrichment with the Agilent SureSelect human all exon platform." Cold Spring Harbor Protocols 2015.7 (2015): pdb-prot083659.
3Clark MJ, Chen R, Lam HY, Karczewski KJ, Euskirchen G, Butte AJ, Snyder M. 2011. Performance comparison of exome DNA sequencing technologies. Nat Biotechnol 29: 908–914.
7García-García, Gema, et al. "Assessment of the latest NGS enrichment capture methods in clinical context." Scientific reports 6 (2016): 20948.