Bioinformatics 15: RNAseq

Question 1

What is RNA-Seq and what is it used for?

Answer 1

RNA-Seq uses NGS to reveal the presence and quality of RNA in a biological sample at a given moment

• provides deep insight into the complex transcriptome of the cell
• higher coverage and greater resolution than sanger, microarrays
• widely applied to investigate different types of RNA

Question 2

Principles of the RNA-Seq process?

Answer 2

1) Extract RNA
2) Convert to cDNA (Reverse Transcription)

3) Fragment, add NGS adaptors (PCR amplicons)
4) Sequence signal end fragments

5) Map reads to genome
6) Analyse transcription level at nucleotide resolution

Question 3

Advantages of RNA-Seq vs microarrays?

Answer 3

Substantially reduced cost, time and amount of RNA required

Improved

• base resolution (single base vs 100bp)
• lower background noise
• Larger dynamic range,
• allows isoform detection,
• can discover novel transcripts, not limited to known transcripts (or species)

Question 4

Transcriptional challenges with RNA-Seq?

Answer 4

Transcript bias
- fragmentation at RNA or cDNA stages introduce different biases

RNA - good representation of middle of gene, bad at 5’ and 3’ ends -> good for gene body / alternative splicing info

cDNA - bias towards 3’ end -> good for 3’ termini (transcription termination, polyadenylation )

Question 5

Bioinformatic challenges with RNA-Seq?

Answer 5

Computationally demanding mapping (100s millions of reads per experiment)

Transcript assemble complex, based on gene models, relative proportion of each isoform is inferred (probabilistic)