For projects that do not require information from the non-coding regions of the human genome, whole-exome sequencing provides a great alternative. Most human whole-exome kits target a region of approximately 50 Mbases that includes all of the genes known to transfer directly to mature RNA following the removal of introns during RNA splicing (i.e. the exons).
The most common method used for hWES is custom capture of constructed whole-genome sequencing (WGS) libraries. Human genomic DNA (gDNA) is made into a shotgun library but, rather than sequencing the whole thing (as is done in whole-genome sequencing), only portions of the constructed library that contain exons are selected for sequencing. The custom capture of the exon regions is performed using hybridization capture where probes (aka “baits) of DNA oligonucleotides are designed to select only the relevant portions of the original library.
Following this crucial capture step, the final library is then sequenced to whatever coverage is needed for analysis. The most common coverages requested for hWES are 50X and 100X, but any coverage is possible. Since the region of the genome included in hWES is so much smaller than what’s used for hWGS, the total amount of sequencing required is much less. Assuming a targeted region of 50 Mbases, sequencing to 100X coverage requires a minimum of 5 Gbases of total sequencing output. Most often, this simple calculation of raw output needed is doubled to 10 Gbases of sequencing required to assure that all portions of the 50 Mbase region receive at least 100X coverage (i.e. what’s known as “on-target coverage”).
Many library preparation and custom capture kits exist for hWES. We find the most common method to be a combination of library construction using one of the Agilent SureSelect kits with sequencing performed on an Illumina sequencing platform (most often either the HiSeq 3000/4000 or the NovaSeq 6000 for large-scale projects).
Data is delivered as FASTQ.gz files through Globus Connect or a direct transfer into your Amazon Web Services (AWS) S3 account, if available.