Sure, whole genome sequencing (WGS) sounds fancy—and why not get as much information as possible by sequencing the entire genome? But it’s not always the best option. For many purposes, WGS can be too expensive and too slow, and it yields unwieldy amounts of data (much of which is unnecessary to the task at hand). The alternative for researchers is whole exome sequencing (WES). And no, it’s not a “lite version” of WGS.
WES is the sequencing of the exome—all of the coding regions, or exons, of the genome. Like WGS, WES starts by creating a DNA library from a sample. The methods diverge at this point, because in WES, the DNA-library fragments are then enriched for exons. Next-generation sequencing (NGS) of the resulting exon-enriched DNA library yields a sequenced exome. WES is increasingly valuable to disease researchers, because most disease-causing mutations are located in the exome. Here are some examples of advances in WES and applications in disease research.
Library prep influences WES success
Far from a routine process, library preparation can greatly influence whether exome sequencing is successful.
“Library diversity and recovery play a huge role in how much coverage you actually see in certain regions of the exome,” says Brian Dugan, global strategic marketing manager for NGS at QIAGEN. QIAGEN’s QIAseq FX kit prepares DNA libraries using enzymatic fragmentation rather than mechanical shearing, enabling researchers to customize the fragment size using only a standard thermocycler. Although tagmentation-based methods can incur sequence bias that leads to less diverse libraries, “the QIAseq FX kit displays little bias, similar to mechanical shearing methods,” says Dugan. For difficult samples, such as formalin-fixed paraffin-embedded (FFPE) tissue or liquid biopsies, QIAGEN’s QIAseq Ultralow Input Library Kit makes it easier to generate libraries from the low-input amounts typical of these sample types.
Biocompare’s Library Preparation Kit Search Tool
Find, compare and review library
preparation kits from different suppliers Search
Swift Biosciences, which focuses on DNA-library preparation for NGS, offer two technologies that are especially useful for whole exome sequencing. The Accel-NGS® 2S Kit repairs DNA fragments on both the 3’ and the 5’ end and uses sequential ligation of their adapter molecules. “This results in a much higher conversion rate of DNA molecules into library molecules,” says Tim Harkins, president and CEO of Swift Biosciences. “And that higher conversion rate then allows for very deep sequencing.” This is advantageous when constructing a library from samples like FFPE tissue, which can contain damaged DNA. The conversion rate is even higher using circulating, cell-free DNA (cfDNA) from liquid biopsies, because cfDNA does not require size selection, which can result in DNA loss.
Swift Biosciences’ molecular identifiers (MIDs) technology (available as Accel-NGS® 2S MID Indexing Kits) is based on a 9-base-pair tag on one of the DNA adapters. MIDs provide greater discrimination by combining sequencing start points with uniquely identifiable DNA molecules. “The probability that you would have the exact same MID with the exact same start point is very low,” says Harkins. MIDs are helpful in finding PCR duplicates and sequencing errors and in detecting low-frequency alleles.
Candia Brown, VP of global marketing at Swift Biosciences, notes that the company’s technology is well suited to translational researchers who want to compare patient samples over time. Past samples of the initial tumor (stored as FFPE tissue) can be compared with fresh tissue from the present, and then with samples over time in longitudinal studies. Such studies may monitor patients by analyzing cfDNA from liquid biopsies taken regularly to assess the efficacy of cancer therapies, for example. The ability to assess FFPE, fresh and cfDNA samples “gives researchers many more options for longitudinal, prognostic studies,” says Brown. “They can monitor variations that might affect therapeutic efficacy or driver mutations that affect tumor growth.”
Exome enrichment for disease research
The exome-enrichment step of WES enables disease researchers to focus on genes of interest.
Agilent Technologies recently partnered with researchers from Emory University and The Children’s Hospital of Philadelphia to develop the most current exome, the SureSelect Clinical Research Exome V2. The collaboration resulted in curated disease-associated targets and created “an exome that delivers 10% more reads at 20X coverage in these specific regions, as well as comprehensive coverage of the rest of the exome,” says Corinna Nunn, associate director for NGS at Agilent.
Another target-enrichment tool is Agilent’s SureSelect Human All Exon V6, aimed at regions of the exome critical to clinical and translational researchers. “SureSelect exomes have been instrumental in identifying the causative variants in more than 50 Mendelian diseases,” says Nunn. Another tool from Agilent, the OneSeq Target Enrichment System, detects genome-wide copy number changes, single nucleotide polymorphisms, insertions/deletions and loss of heterozygosity—without the complications that WGS entails. “The current cost and turnaround time of deep-coverage WGS prevent it from being used routinely in clinical labs,” says Nunn.
Though most exon-enrichment methods are hybridization-based, Thermo Fisher Scientific offers an amplicon-based method that is especially fast and user-friendly. The Ion AmpliSeq™ Exome RDY S5 Kit is designed to produce an exome library in less than six hours. “Using a simple workflow, our customers can go from sample to answer in just two days using small amounts of input material,” says Arvind Kothandaraman, senior product manager in Clinical NGS at Thermo Fisher Scientific. The kit includes a 96-well plate preloaded with dried-down reagents that are ready to use.
In addition, Thermo Fisher Scientific’s Ion AmpliSeq™ exome panels leverage the ultrahigh multiplex PCR approach of Ion AmpliSeq™ technology. “Ion AmpliSeq™ exome products are primarily used for inherited disease research when the disease etiology is unknown, and the phenotype is highly complex,” says Kothandaraman. Researchers are also combining whole exome sequencing with other genomic tools, such as microarrays. “Some of our customers use chromosomal microarray analysis and whole exome sequencing for autism spectrum disorder research,” says Kothandaraman.
Expanding the power of WES
Some clinical researchers want to broaden their scope and look at noncoding regions of the genome, in addition to exons—but without resorting to WGS. Agilent Technologies and 10x Genomics are collaborating to provide tools for this. They combine Agilent’s SureSelect technology with 10x Genomics’ Linked-Read technology to let researchers look at regions of the genome that are otherwise difficult to map, detect structural variation and phase entire genes to resolve compound heterozygotes.
In 10x Genomics’ Chromium™ System, molecular barcodes attach to DNA fragments, refining the alignment process during analysis and creating linked reads that phase across exons. This provides greater long-range information, enabling researchers to detect larger events, such as structural variants, that are challenging with standard exomes.
“In general, balanced events like inversions and translocations are really difficult to call with just short-range sequencing, but the information from molecular barcoding now gives us the power to identify those events,” says Deanna Church, senior director of applications at 10x Genomics. Another advantage of molecular barcoding is the ability to construct individual haplotypes. “Haplotype reconstruction actually gives you improved power for calling events that are typically really difficult to call with a standard exome,” shares Church.
Looking forward, Church is interested in applying 10x Genomics’ technology to cancer exomes, using neoantigen prediction to enable immunotherapy or vaccines. “Understanding what the potential neoepitope landscape of the tumor looks like is really important,” she says. “And having a fully phased exome could help to predict epitopes that are close to exon boundaries.” It’s another reminder that WES isn’t just “WGS lite,” but rather a quickly evolving technique with diverse applications that are only just beginning.
Image: Shutterstock Images