Question

Next-generation sequencing technology has revolutionized nearly every biological field of study. A variety of techniques are used to create a genomic library prior to sequencing. However, cost is still a factor when deciding on which method to use for a particular research question.

(A) Briefly describe the technique of restriction-site associated DNA sequencing (RADseq). What is the difference between single-end and paired-end sequencing?

(B) Suppose you were interested in examining the population genomic structure of NYC rats throughout the five boroughs. Would it be better to use RADseq or shotgun sequencing? Explain your reasoning.

Answer 1

(A) Restriction enzyme digestion and Next-generation sequencing of regions adjacent to restriction sites are used in Restriction-site associated DNA sequencing (RADSeq). The fragments from one individual are ligated to a modified Illumina adapter containing a unique identifying sequence.
The fragments from many samples can be pooled together and sequenced on a single flow cell lane. The resulting reads can be identified and separated by identifying the unique barcodes at the start of each sequencing read. By sequencing a lot of samples of interest in just one lane this way, and comparing the tags to the individuals, many biologically relevant SNPs and genetic loci can be identified in a single experiment. It is a very economical way of identifying SNPs even when the reference genome is not defined.
The steps of RadSeq follows like this:
obtain very high-molecular-weight DNA,
digest with one or more restriction endonucleases,
ligate adapters to the DNA fragments for sequencing,
select specific-sized fragments (some RadSeq protocols),
incorporate barcodes into adaptors to identify individual samples in library preparation and sequence.
Most methods use Illumina sequencing to produce reads of 50–300 bp.
Sequencing can be single-end or paired-end.
In single-end reading, the sequencer reads a fragment from only one end to the other, generating the sequence of base pairs. In paired-end reading it starts at one read, finishes this direction at the specified read length, and then starts another round of reading from the opposite end of the fragment.

(B) (RADSeq) allows analysis of the same subsets across the genome for hundreds to thousands of individuals, providing hundreds or thousands of genetic markers. It is cost-effective, allowing sampling of the genome without having to sequence the entire genome. RadSeq does not require prior genetic information so is useful for evolutionary and ecological studies of nonmodel species.
Thus, It is a very economical way of identifying SNPs even when the reference genome is not defined, whereas shotgun sequencing is not economical, since it has to analyse entire genome sequence.
Hence it would be better to use RadSeq than shotgun sequencing.