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The Guide to Amplicon Sequencing and Hybridization Capture

Friday, September 25, 2020 23:07

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The Guide to Amplicon Sequencing and Hybridization Capture

In recent years, significant advances in fast and efficient sequencing technology have made it possible to obtain gigabytes of genomic data from a single sequencing, which gave life to new perspectives in many fields of genomics. Next-generation sequencing (NGS) technology has become a universal and indispensable tool in molecular biology.

Next-Generation Sequencing as an Innovative Method of Reading Sequences

Next-Generation Sequencing is a non-Sanger high-speed sequencing technology. Millions of DNA fragments can be sequenced simultaneously (massively parallel sequencing), increasing sequencing efficiency. Compared to traditional sequencing techniques, the advantage of this sophisticated technology is high throughput, cost and Amplicon sequencing duration reduction, and high precision.

Next-Generation Sequencing has many applications. It can be utilized to sequence complete bacterial genomes (Whole Genome Sequencing-WGS) and sequencing of large numbers of randomly fragmented DNA sections, which are then assembled by computer (Metasequencing / Metabarcoding). Compared to traditional sequencing techniques, the advantages of this technology include high throughput, cost reduction and efficiency, duration of sequencing, and high precision.

Targeted Sequencing

Targeted sequencing methodology focuses on sequencing only specific sets of genes or genome regions. Targeted sequencing is a revolutionized, high-throughput approach that massively and efficiently reduces cost and time by analyzing data on only specific DNA regions by running samples in parallel. They can be exomes, selected genes, non-coding DNA fragments, or mitochondrial DNA. Targeted sequencing is also used to analyze regions associated with phenotypic features. This technique enables higher coverage of the analyzed sequences and the identification of rare variants or different types of mutations at reduced analysis costs.

Depending on the particular application, there are several methods of targeted sequencing available. The most utilized and prevalent techniques are hybridization capture, amplicon sequencing, and molecular inversion probes (MIPs).

Hybridization Capture Versus Amplicon Sequencing

Hybridization-based methods show significantly higher complexity levels than amplicon-based sequencing with a more generous input amount of DNA. Hybridization capture requires 1-250ng for the library preparation and 500ng for the library capture, while amplicon sequencing DNA input involves 10-100ng. In general, the hybridization capture’s process takes more steps to complete; therefore, it requires more time compared to the amplicon sequencing technique. Because of hybridization capture’s more complex and sophisticated nature, the cost of the panel and its completion vary, while amplicon sequencing is associated with a lower price per sample.

The number of targets per panel is another distinguishing feature between the hybridization-based capture and amplicon sequencing. The hybridization capture is practically unlimited by panel size, while the amplicon sequencing method applies fewer than 10,000 amplicons. The sensitivity of the hybridization capture is down to 1% without the use of UMIs. The amplicon sequencing technique’s sensitivity is down to 5%. The hybridization capture-based methods have better sequencing uniformity than the amplicon-based sequencing techniques.

Hybridization Capture and Amplicon Sequencing Applications

As stated before, the functionality of the two distinct methods of targeted sequencing has its own unique applications in various fields.

The hybridization capture technique’s best-suited applications include:

• Exome sequencing

• Gene discovery

• Genotyping

• Oncology-related applications

• Detection of rare variants

• Detection of low-frequency somatic variation of single nucleotide polymorphisms (SNPs) and insertions or deletions (indels)

Comparably, the amplicon sequencing method has found its purpose and utilization in the following:

• Genotyping by sequencing

• Detection of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing events

• Detection of disease-related variants

• Detection of germline inherited SNPs and indels.

There are several factors to be considered when selecting the appropriate targeted sequencing method, which includes the total time and cost of the process, the proper application, the expected throughput of the technique, and the target rate and uniformity.

Reference:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832303/

https://www.idtdna.com/pages/education/decoded/article/targeted-sequencing-hybridization-capture-or-amplicon-sequencing

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