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Old 03-31-2017, 08:13 AM   #1
sitapriyamoorthi
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Default RNAseq alignment -misaligned on the genome!

Hello ,

My colleague did the mapping of publicly available human RNAseq data. However when I view this alignment on IGV the reads map next to the genes and not above it? Why would this be happening? Any suggestions?
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Old 03-31-2017, 08:27 AM   #2
GenoMax
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Can you clarify what do you mean by that? Perhaps attach a screenshot to explain.
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Old 03-31-2017, 11:09 AM   #3
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You're looking at the wrong genome build/annotation in IGV almost certainly.
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Old 03-31-2017, 12:28 PM   #4
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I agree with Bukowski, if you're visualizing the data against hg19, try switching to GRCh38 - or vice-versa. Your collaborator aligned the data against a genome build that's different from the one you're using on IGV.

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Old 03-31-2017, 12:37 PM   #5
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Classic mistake, but you'll make this one only once
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Old 03-31-2017, 01:38 PM   #6
sitapriyamoorthi
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Default Nope correct genome build

Nope we are looking at it with the same genome build that he used to align same problem.
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Old 03-31-2017, 04:23 PM   #7
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Curious... can you provide a screenshot?
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Old 04-01-2017, 10:41 AM   #8
sitapriyamoorthi
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Default Here are the screen shots

So this is the screen shot for ACTB gene and as you can see the reads show up ~1kb downstrem
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Old 04-04-2017, 12:25 PM   #9
r.rosati
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I have half an hypothesis. See if you agree.
By the (blurry) coordinates it seems like you're using hg19, is this correct? If so, then ACTB is within the window chr7:5,566,700-5,570,300. I'll use these numbers.

First thing, if your IGV is set to show mismatches in color, then you will agree with me that the build used for alignment and the build you're using on IGV don't match, because the nucleotides are all in color = mismatched. On the other side, if you've set IGV to show all nucleotides in color regardless of match, then disregard this comment.

You do see some reads about 1kb downstream, but you will agree with me that the exon structure totally doesn't match that of ACTB. You see a long exon, then three short ones, then one last long exon. This doesn't match ACTB.

However, 22kb downstream you have the FSCN1 gene, whose exon structure really seems to match the transcriptome reads. I'm not affirming those reads totally absolutely belong to FSCN1, but...

Untitled.jpg

If this is the case, and you say that you see these reads 1kb downstream to ACTB, then maybe your reads are actually shifted 60kb upstream.
And if so, then you should see transcriptome reads matching the ACTB exon structure within about the region: chr7:5,500,000-5,518,000.

The hole in this hypothesis is: I don't know which genome build would have ACTB shifted 60kb upstream compared to hg19. It's not GRCh38 nor hg18. Could it be an earlier version of hg19?

In the end, I would talk to your collaborator and be 100% sure about the genome build he's used to align the data. Although the mapping doesn't match hg19 the data is likely not wrong, but it's best to realign it to GRCh38 or hg19.

Last edited by r.rosati; 04-04-2017 at 01:19 PM.
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Old 04-13-2017, 11:33 AM   #10
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...Any news?
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Old 04-14-2017, 05:03 AM   #11
sitapriyamoorthi
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Dear All,
I thought I had replied to this thread but I hadn't. R.Rosati, thank you for you painstaking response. I asked my collaborator again to check the genome build he had used. Being a yeast person he did not realize how different genome annotations were version to version, infact he had used GRCh38 and not hg 19. Problem resolved! Thank you all for your prompt responses it helped me query the problem with my collaborator more confidently!
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