Different technology features not only impact the cost and time it takes to complete your project, but it might also compromise the success of your project.
In this blog, we’ll be discussing the main factors that you should consider before choosing a NGS technology.
- Strategies for using NGS
- Depth of sequencing coverage
- Length of sequencing reads
Strategies for using NGS
Customary single gene sequencing approaches have been largely replaced by NGS. Why has this happened? Because NGS allows ultra-high throughput of RNA/DNA sequencing that is more rapid and quite less expensive, and that’s not all, it allows for very high coverage of sequences.
Some of the commonly used NGS strategies that are employed for genomic sequencing projects include:
This technique is used to read all protein-coding regions of all genes that are commonly known as exome. WES allows for the specific detection of variants in Candidate genes that may not be covered in a particular targeted sequencing approach. It can easily detect recent mutations that have not previously been associated with the disease that is being researched on.
Targeted Sequencing (Panels or Regions of Interest):
This approach is commonly used to reach a limited number of specific genomic regions that are usually well-described mutations and genes.
This approach is commonly used to entail the sequencing of the complete genome, which includes introns, regulatory regions, and even mitochondrial DNA. Whole Genome Sequencing is a very powerful approach as it allows for the identification of complex structural disparities at very high resolution, and it is often used to detect pathogenic mutations in intronic regions or novel genes.
RNA Sequencing (RNA-Seq):
This strategy is commonly used to directly sequence and enumerate the number of mRNA molecules in the complete transcriptome.
Understanding the Depth of Sequencing Coverage
The Depth of sequencing Coverage offers a sign with respect to the normal number of sequencing peruses that align to, or “cover,” every base in a sequenced test sample. This is a significant process to remember since sequencing is a process that is bound is to have errors. Hence, the higher the coverage, the higher the certainty you can have in the sequenced bases. Eminently, the degree of suggested inclusion mainly relies upon a few components, including your primary research question along with sequencing application. For example, for WGS and WES applications, the suggested inclusion is lower than different applications, such as DNA target-based sequencing or transcriptomic.
Understanding coverage and length of Sequencing Reads
Now you must be thinking about how coverage is calculated? Well, Coverage (C) is typically calculated using the Lander/Waterman equation that takes into consideration the number of reads (N), read length (L), and haploid genome length (G): C = LN / G
Read length commonly refers to the primary number of base pairs that are sequenced from a DNA/RNA fragment. The particular regions of overlap between reads are utilized to later assemble and align the reads to a reference genome that allows to reconstruct the full genomic sequence.
What can SRS methods do for you?
Millions of short DNA strands are read in parallel with high-throughput SRS technologies; SRS is the commonly used high-throughput sequencing system, while the approach is supported by a broad range of bioinformatics tools. The SRS methods usually offer high accuracy and low-cost data that is commonly used for a huge variety of applications, including variant discovery.
What can LRS technologies methods do for you?
In comparison, the high throughput LRS technologies are fully capable of generating reads that are in are hundreds of thousands of base pairs in length (averaging ~10-100kbp). Although LRS might take longer than the SRS methods and can be more expensive, it allows for better resolution of the genome since it can span multifaceted genomic features. And that’s not all; since LRS does not use PCR, the RNA/DNA stays in its native state, which allows LRS to also be used to identify base modifications such as methylation.
Determining whether to use an SRS or LRS method is not always a simple or straightforward decision, and some problems might actually require a merger of these approaches. Selecting the sequencing read length is extremely contextual and primarily depends on the sample type, application, and the desired coverage.