2.2 RNA-Seq library preparation
In the last decade, there has been a considerable understanding of the
dynamic nature of organism’s genomes,13,28 this is not
limited to the modifications of DNA sequence but also to the
quantitative and qualitative measurements of RNA
sequences.13 Before sequencing a sample, a library
must be prepared for that sample.13,28,29 A library is
a collection of randomly sized DNA fragments representing the sample
input.28 Depending on the type of NGS application,
different library preparation steps are
available.28-30 Also, the sample preparation required
for RNA sample sequencing vary depending on the type of
RNA.28,30,31 For RNA-Seq, basically there are some
steps that are required which are; selection of transcript with poly(A)
tail, rRNA depletion,28,30 fragmentation followed by
cDNA synthesis, purification and amplification.30,32mRNA and lncRNA >200 nt contains a poly(A)
tail,28,29 convenient for the enrichment of poly(A) +
RNAs from total cellular RNA, which is carried out with oligo-dT
nuclease.28,29 This is followed by rRNA
depletion,32 this can be done based on
sequence-specific probes which can hybridize to rRNA then depleted with
streptavidin beads.28 RNA samples are fragmented to a
certain size range before RT,32 this is necessary due
to size limitation of most sequencing platforms.28Fragmentation of RNA can be done with alkaline solution or divalent
cations on an elevated temperature.28 Enzymes such as
RNase III can also be used to fragment RNAs but this can introduce bias
due to its preference for double-stranded RNA
sequence.28 cDNA synthesized from the RNA fragments
using RT are ligated to DNA adapters before amplification and
sequencing.28,29,32