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Old 07-23-2018, 04:13 PM   #1
NM1000
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Default Rockhopper Analysis (IGV Viewer)

Hello Everyone,

I would greatly appreciate if anyone can help me out with analyzing "Rockhopper IGV Viewer Results". We are trying to figure out what does the dip or downward spike (indicated by red arrow in the attached image) mean. Could it possibly mean that no reads were found during sequencing? Is it safe to correlate it to mRNA degradation or mRNA stability?

RNA-Seq was performed on Illumina Next Seq 500, and the RNA samples were extracted from gram-positive bacteria.

Thank you for your time and effort.
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Old 10-05-2018, 09:27 AM   #2
falconpunch
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Quote:
Originally Posted by NM1000 View Post
Hello Everyone,

I would greatly appreciate if anyone can help me out with analyzing "Rockhopper IGV Viewer Results". We are trying to figure out what does the dip or downward spike (indicated by red arrow in the attached image) mean. Could it possibly mean that no reads were found during sequencing? Is it safe to correlate it to mRNA degradation or mRNA stability?

RNA-Seq was performed on Illumina Next Seq 500, and the RNA samples were extracted from gram-positive bacteria.

Thank you for your time and effort.
I haven't used Rockhopper yet, but I have done similar analysis of bacterial mRNA with HiSeq 2000 reads in Geneius (expensive software).

I assume the blue area represents read depth by genome location? The "dips" could be caused by a number of things, in rough order of decreasing likelihood here:
1) variable mRNA degradation in the bacterium (as you said)
2) variable RNA degradation during isolation (i.e. maybe it got too warm or picked up an RNase)
3) variable amplification in RT or PCR steps
4) variable sequencing in the NextSeq, possibly due to base repetition within the sequence or extreme GC content or hairpins or something

#1 seems most logical to me. You usually don't get a stretch that long from bacteria with high, consistent reads unless it's a particularly long and highly expressed gene.

There are some other things that would shed much more light on it, though:
--Do you know predicted genes, operons, intrinsic terminators, etc. in this area? If drops happen right after a predicted intrinsic terminator, for example, then there's your answer.
--Related to that, is this is "supposed to be" one long transcript (as I think the solid blue suggests, if it were switching strands the opposite strands would be red? I don't know the software well enough)
--What sort of read depth is this? I don't see any readout of the y-axis, for all I know the "high" parts really go much higher and the "dips" go all the way down to 5-10 reads, or maybe not
--What was your read configuration? 150bp? 300bp? Single or paired-end? This could help a lot too.

Hope some of that rambling helps!
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