Technical Strategies for Targeted Sequencing and Its Present and Future

Although the emergence of a next generation sequencing (NGS) has reduced the cost of sequencing, the sequencing of the entire genome is sometimes not necessary or even counterproductive. On the contrary, it is wise to focus on a small part of it. Nowadays, by targeting enrichment strategy, people can reduce the sequencing volume of some projects, speed up the progress and reduce costs, and at the same time reduce the burden of data analysis. The exon group, for example, accounts for only 1% to 2% of the genome but 85% of the known mutations. So, compared with whole genome sequencing (WGS), exon sequencing is equivalent to a 98% reduction in sequencing. Needless to say, targeting sequencing collection is a great way to lighten your burdens. Here we look at how researchers conduct targeted enrichment and the factors they consider in their research.

Target enrichment can be achieved by two strategies: capture or amplification. That is to say, in a library of genomic DNA, you can catch the DNA you need, or you can selectively synthesize it. Many of the current technologies are mostly based on these two models, sometimes combining them.

The capture method is usually started from the preparation of NGS library, such as interrupting DNA, connecting the junction to fragment and carrying out PCR amplification. After that, the biotinylated probes complementary to the fragments of interest were hybridized with DNA and screened by streptavidin.

The complementary chain is released through the second round of PCR, and then it can be sequenced. The enrichment based on hybrid capture is carried out in the form of a chip like, but mainly in solution, "the solution is more convenient," Mike Leous, the marketing manager of the Roche sequencing section, explained. "As far as I know, no one has used solid form now."

Target enrichment can also be carried out by multiplex PCR strategy, which is called the amplification method. The DNA was digested and digested, and the probe was used to capture both ends to form a circular DNA template or to use genomic or random cut DNA templates. After that, specific primers were used to amplify the DNA region of interest and form highly enriched DNA libraries.

There are slight differences in the enrichment products of exon groups provided by different suppliers. Some products include 3 'or 5' untranslated regions, while others cover non coding RNA. The length and density of the probe and the algorithm used are slightly different, so the coverage is not the same. Before the library is prepared, it is necessary to make a good choice.

DimitriosMonos, a professor of pathology at Philadelphia children's Hospital, reminds you that if your research is cancer, you need to know where there may be inverted or missing areas, then the target area should contain non - translated DNA.

Roche NimbleGen provides a variety of DNA sets. According to Leous, the products of their exons can enrich the coding region of the human genome and cover about 4000 medical related genes. If you only want prostate cancer related genes, there is no problem. Everything is customizable.

Andrew Barry, the product marketing manager of NEB, believes that the solution type hybridization is suitable for large areas, from the Mb size to the exon group (30-60 Mb). When the scope shrinks, you will often find specific losses and begin to introduce a large number of off-target reads. The multiple PCR method is more suitable for all kinds of small focus sets. They are often faster and require less sample starting volume, but "expanding content is more difficult." Barry believes that some novel methods, such as Agilent's HaloPlex, Illumina's TruSeq Amplicon and NEB NEBNext Direct, are trying to fill the gap. Of course, there are abundant collection of solution hybrids in the market for small areas. At the same time, Ion AmpliSeq Exome RDY S5 Kit of SEM Fisher technology also uses ultra high multiple PCR to enrich exons, so that it can be sequenced on Ion S5 S5 system.

In the field of clinical research, whether to launch WES or to launch WGS has been debated in the scientific community. Some people think that finding common mutations is one thing. Finding rare mutations is another matter. For WGS, the genome coverage is 20-30 times, "you can only find mutations in the reproductive system with frequencies above 50% or above," Barry noted. "When you start studying the free DNA of liquid biopsies, you need very high coverage to find 0.1% of the mutations."

However, Janine Meienberg, a postdoctoral fellow at the rare disease foundation, said she would no longer carry out target enrichment because the target area could not be completely covered. Inadequate target enrichment and PCR in library preparation will inevitably introduce bias. The higher the GC content is, the lower the coverage of WES will be. She tends to use PCR free WGS, but the drawback is that more starting materials are needed.

Sometimes, the problem is finding sequences that can not be directly localized to the reference genome, such as translocation, structural changes or highly polymorphic regions. Monos uses his regional specific extraction (RSE) method to obtain fragments of the length exceeding 20 KB, and the 4 Mb fragments of the major histocompatibility complex (MHC) are drawn by long reading sequencing.

Another problem is that there is no reference to the genome at all, such as many important agricultural organisms. In this case, Orin McCormick, sales manager of RAPiD Genomics, says that a custom design set up to 200 thousand different regions may be a perfect substitute for WGS. "When you consider genotyping of pine trees, WGS is not an economic option."

It is a problem that enriching or not enriching. If you want to enrich, how do you choose? You can think about a few questions first: what is my target area? How many areas do I have to capture? How many starting materials do I have? How many samples do I have? At the same time, consider your funding and process, as well as various performance requirements, such as specificity, homogeneity and sensitivity. Of course, you can also see how other colleagues in similar projects do it.

Target sequence capture sequencing is currently a hot technology in genomics research. The main reason is that genome sequencing takes a lot of cost and time. Therefore, the selective (target sequence capture) depth sequencing is a wise choice for current genome research. Of course, continuous improvement of sequencing and improved bioinformatics analysis will greatly reduce cost and time. When people only sequenced some genomes, researchers could study more samples and deeper depths at the same cost. We know that the number of samples is a key indicator of the discovery of pathogenic genes, especially the more common diseases. The more sample size, the greater the possibility of locating the disease related genes. Especially for some rare mutations or partial somatic mutations, sequencing depth determines that target sequencing is an effective tool.