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Old 03-18-2019, 09:59 AM   #1
pitch_
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Default Strange coverage histogram of museum samples

Hi everyone,

I had sequenced ancient museum samples on Illumina Hiseq 4000 platform. After running through typical pre-processing steps to generate a bam file (eg. cutadapt, bwa mem, mark duplicate, indel realignment). I then input the bam files into qualimap and obtained very strange coverage histograms (50x) (attached). I obtained coverage histograms with a similar pattern even after rescaling the bam file using mapdamage 2.0.

I am wondering what is the reason for this pattern in coverage?

Thanks in advance!
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File Type: pdf Histo.pdf (727.0 KB, 18 views)
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Old 03-18-2019, 07:33 PM   #2
luc
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Just a guess: paired-end reads of short fragments; thus they are mostly overlapping; resulting in counts as multiples of two?
More info would help.
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Old 03-18-2019, 08:13 PM   #3
pitch_
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Quote:
Originally Posted by luc View Post
Just a guess: paired-end reads of short fragments; thus they are mostly overlapping; resulting in counts as multiples of two?
More info would help.
Hi Luc, that does sound like a plausible reason. What other info would help in confirming it?
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Old 03-19-2019, 12:05 PM   #4
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They are paired-end? Which read length?
You should be able to determine the insert sizes from the alignments (bam files).
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Old 03-20-2019, 07:55 AM   #5
pitch_
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Quote:
Originally Posted by luc View Post
They are paired-end? Which read length?
You should be able to determine the insert sizes from the alignments (bam files).
They are paired-end reads with a read length of 150bp sequenced on illumina Hiseq4000 platform.

The insert size determined by qualimap for the 2 samples are
1st: 4194.82
2nd: 33,264.66
I am unsure how else to determine the insert size..
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Old 03-20-2019, 09:23 AM   #6
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I think luc is correct. You can inspect the bam file to see if the paired reads map to the same location. You can also run bbmerge and see if the two reads overlap and what percent of the library does that.
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Old 03-24-2019, 11:50 PM   #7
pitch_
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Hi all,

After checking the fragment size and mapping, I found that both of you were right, the coverage pattern is due to overlap of read 1 and read 2.

Thanks for the help!
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