Review: –
We all as biotechnologist know the importance of polymorphisms in DNA. These polymorphisms help the scientists to identify regions of similarity in various genomes and are generally referred to as molecular markers. NGS (next generation sequencing) has enhanced our abilities to trace these molecular markers.
Like most other molecular markers, the NGS also depend on restriction enzymes. Different parts of the genome can be targeted using different restriction enzymes. Various types of markers produced by NGS include the following:
RNA-seq
This involves sequencing of transcriptomes. Through RNA-seq, functional SNPs, generally related to the disease can be detected. However, the accuracy of this method to find out the molecular marker is suspected because there is no reference genome available. But the advantage of this method is its cost-effective nature, specificity to the particular species/population. Restriction enzymes.
Sequence capture
This method is a highly accurate method because reference genome is available. It involves targeting the regions of interest directly. Various sequence capture methods like Sure Select, Nimblegen and Raindance are known.
Reduced representation sequencing
This method involves RRLs and CRoPS to sequence a particular region of the genome and not the entire genome. This is done because whole genome sequencing is costly. An SNP map of the whole genome is prepared first of all using automated DNA sequencing method. Restriction enzymes are employed in the entire protocol for cutting small sections of DNA which are to be sequenced. Through this method, polymorphism from multiple individuals can be found out.
RAD-seq
This method sequences regions surrounding the restriction sites of almost all restriction endonucleases. Even if the length of restriction fragments is very large, then also sequencing is done. This method has been used to create linkage maps, the creation of SNP and investigation of phylogeography.
Advantages of NGS
The tediousness of the entire task is reduced because less amount of genome/DNA is sequenced.
The methods used in NGS are cost-effective. When the amount of sequenced DNA is shortened, so the cost of the overall procedure is also shortened.
Each marker analyzed receives high coverage in these limited resources.
Studies on a large population can be performed effectively.
Studies on recombinant populations can be performed effectively.
Factors which affect NGS
Availability of reference genome enhances the specificity of the process.
Knowledge about a number of markers required provides a well-defined approach for the entire work.
Knowledge about the expected degree of polymorphism in a particular genome that is being studied.
Choice of restriction enzyme.
DNA sample preparation