Question

“Next generation RNA sequencing (RNA-Seq) has transformed our ability to explore the bacterial transcriptome and is leading to new RNA discoveries”. Discuss.

Answer 1

Main goal of RNA SEQUENCING :

Goal of RNA-Seq is :

• identify alternative splicing events
• test if they differ between conditions
• Long-read sequencing captures the full transcript thereby minimizes many of issues in estimating isoform abundance, like ambiguous read mapping.

(read means sequence of a cluster that is obtained after the end of the sequencing process)

GENERAL ASPECTS OF RNA-Seq

Major considerations include :

(1) how to capture RNA molecules of interest;

(2) how to convert RNA to double-stranded cDNAs with defined size ranges;

(3) how to place adapter sequences on the cDNA ends for amplification and sequencing.

These are discussed in the following sections.

• Selection of Poly(A) + Transcripts

• rRNA Depletion

• Fragmentation

• Adapters and Directionality

• Amplification and Molecular Labels