Whole Exome sequencing is a technique for sequencing all the protein-coding genes in a genome (known as the exome). It consists of first selecting only the subset of DNA that encodes proteins (known as exons), and then sequencing that DNA using any high throughput DNA sequencing technology. There are 180,000 exons, which constitute about 1% of the human genome, or approximately 30 million base pairs, but mutations in these sequences are much more likely to have severe consequences than in the remaining 99%. The goal of this approach is to identify genetic variation that is responsible for both mendelian and common diseases without the high costs associated with whole-genome sequencing.
Exome selection is performed using two different approaches, amplification of exomes or capture of exomes. Whole exome sequencing can be performed with both platforms available in the lab:
ION S5TMSystem
Ion AmpliSeq Exome
The Ion AmpliSeq Exome analysis offers a simple and flexible workflow that targets ~33 Mb of coding exons, greater than 97% of coding regions as described by Consensus Coding Sequences (CCDS) annotation - in 12 primer pools for highly specific enrichment of exons within the human genome (total design coverage including padding and flanking regions is ~58 Mb).
The analysis include 1) the AmpliSeq Library construction, 2) the advantages of low DNA input, with as little as 50 ng required for each individual library, 3) with affordability and sample flexibility realized through multiplexing of up to 2 barcoded samples in a single sequencing run with >94% coverage for two samples per Ion 540TM chip. The Ion AmpliSeq Exome Kit demonstrates a high percentage of on-target bases (>90%) with excellent uniformity irrespective of sample multiplexing.
Ion TargetSeq Exome
TargetSeq exome employs hybridization based capture approach to make exome library by capturing exons identified in several data bases e.g. Gencode, RefSeq, UCSC etc, functional RNA genes, predicted MiRNA binding sites and COSMIC annotated tumor variants from human gDNA, a total of 46.2 Mb. Up to 2 exomes can be multiplexed on a Ion 540TM chip.
ILLUMINA NextSeq 500
Researchers can choose between two different Nextera Rapid Capture formats to initiate their exome sequencing studies. For the most efficient exome, the Nextera Rapid Capture Exome kit provides 37 Mb of expertly selected exonic content. The Nextera Rapid Capture Expanded Exome kit is the perfect solution when investigating coding variants and regulatory regions. It provides 62 Mb of content, including exons, 5’ and 3’ untranslated regions (UTRs), microRNA, and other non-coding regions. Researchers can supplement Nextera Rapid Capture Exome with Add-on content to interrogate additional regions of interest alongside the exome rapidly. The open-platform NextSeq 500 System can also accommodate library preparation and enrichment kits developed by third-party providers. Specifications are summarized in the following table:
