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