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NGS Target Enrichment Strategies

Posted in: DNA / RNA Manipulation and Analysis

Content sponsored by Sage Science

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Next-generation sequencing (NGS) has ushered in a new era of understanding of both the inner workings and the function of the genome. NGS allows researchers to look at traits—including diseases—that are linked to differences or mutations in an individual’s genes.

Since only about 1% of the human genome constitutes genes that code for proteins, several strategies have been developed to selectively enrich these targets for sequence analysis to avoid whole-genome sequencing (WGS), which is much more expensive and does not offer consistent coverage of these regions.  Target enrichment strategies are a key component to any genomics laboratory, allowing the collection of genetic data at a fraction of the cost of WGS, and at a higher depth of coverage.

Target enrichment makes it easier to identify mutations and other changes within a single gene, across a number of genes, or across the entire exome in a single sequencing analysis.  Moreover, enrichment strategies help increase the accuracy and reliability of results.

The reduced cost of targeted NGS enables the screening of individuals for single nucleotide polymorphisms (SNPs) and small mutations such as insertions and deletions (indels). This has become important in clinical research and may become a key part of precision medicine as genomic analysis enters the clinic.

Traditional Techniques

There are two main traditional methods of targeted sequencing: hybridization capture probes and amplification by PCR.

Hybridization involves first building a sequencing library, then adding carefully designed oligo baits that hybridize to regions of interest. The hybridized library fragments are then enriched, typically using streptavidin/magnetic beads. Thousands of baits, between 55 and 125 base pairs, are used.

The second strategy uses PCR to amplify regions of interest by designing highly multiplexed primer sets.  After the PCR process, sequencing libraries are constructed from the amplicons.

In general, hybridization kits can produce more even sequencing coverage. PCR kits have a simpler workflow but can show a sequencing bias (in coverage) closer to priming sites. Manufacturers make validated kits for gene panels, whole exomes, and clinical targets.  Most offer a service to design custom panels as well, though this process can be costly and require a long lead time.

Targeted NGS is a popular and cost-effective method for screening individuals for SNPs and indels, but there are other types of mutations (structural variants) and genetic phenomena (repeat elements) that cannot be analyzed by these methods. Some of these can be somewhat resolved with WGS; the better albeit, more expensive, approach is long-read, single-molecule WGS. Pseudogenes can confound both targeted sequencing and WGS. This continues to be a challenge for clinicians, since these phenomena are associated with many diseases.

New Trends in Targeted Enrichment

Over the last several years, the genomics community has seen growing interest in gene editing with the emergence of CRISPR/Cas9 enzymology. With this approach, Cas9 endonuclease can be guided by an RNA molecule to a homologous DNA sequence on a genome and make a double-stranded cut at that site — presenting the possibility that an unwanted DNA sequence can replaced by a normal one.

NGS technologists are beginning to use this same approach for target enrichment.  Since Cas9/guide RNA complexes can make double-stranded cuts at unique sites of the genome, it is possible to enrich a gene or a genomic region with just a few guides, as opposed to using hundreds of baits or primers. This targeted approach can be used to provide information that is not collected with traditional target enrichment methods. Using short-read sequencing, additional flanking or intronic sequence can be obtained and pseudogenes can easily be resolved. With long-read methods, structural variants, repeat elements, and “dark regions” of the genome can be unraveled at a fraction of the cost of WGS.

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1 Comment

  1. Abdu Manan on July 10, 2019 at 1:17 pm

    Useful article for researchers

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