Does This Looks Like An Antisense Rna?

Question

[biotech]

Hi community,

I would like to know your opinions about the image attached. enter link description here

We have sequenced a bacterial transcriptome in two conditions to compare differential expression (Ion Torrent with strand specific libraries). The feature were is centered the IGV snapshot has a logFC = 10, that is, this gene is more expressed in sample 69. Do you thinks this could be caused by library preparation artefacts or on the contrary looks promising enough to verify via RT-qPCR?

For this gene, logCPM = 10. Please let me know if you need some additional information.

Thanks for your help. Best, Bernardo

[Devon Ryan]

Do only these 2 genes show this or is this a more general and broader pattern? That's probably the easier way to eye-ball artifact.

[biotech]

I don't have this pattern for all DEG, but here is a similar one:

The opposite situation, logFC = -4.9 and logCPM = 11.06282357 This time sample 69 has a mix of sense and antisense reads.

https://drive.google.com/file/d/0B8-ZAuZe8jldWEg3VEIwRmVrcEE/edit?usp=sharing

[Devon Ryan]

You have some seriously ginormous fold-changes! Since these sorts of opposite-strand patterns seem to be more the exception than the rule, that's definitely something I would test further.

[David Westergaard]

Are you sure you mean _log_FC, and not just FC? A logFC of 10 correspond to a 1024 increase, and it most likely the highest logFC I have ever seen! Even in some of the microarray drug response experiments I've analyzied, a logFC of 5 was very rare.

[biotech]

Yes, I'm sure is logFC, but what I would like to know is your opinion about the possibility of being this an antisense regulation. I only have logFC > 5 for ten genes, though.

[biotech]

logFC results were retrieved using edgeR, they are log2 fold changes

[David Langenberger]

I would recommend you to check some reads that create your potential antisense RNA. Go to the BAM file and check, if these reads are mapped uniquely to this region.

Answer 1

[Michael]

Whether this is real or an artifact cannot be decided based on looking at a coverage plot or the reads alone. Every conclusion would be mere speculation, but the observation might warrant an experimental validation by sequencing the genomic region first to exclude genomic variation, then RT-PCR to validate the transcript.

There are many possible reasons to exclude:

• transposable element (check what this region is by blasting against NR/NT)
• repeat region, or duplicated region, check by blasting against the rest of the genome
• genomic re-arrangements (different strains, mutations, etc.)
• problem with the library prep or sequencing
• contamination with another bacterium, strain