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Old 01-14-2016, 02:40 PM   #1
Nikvailo
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Default Possible Cause for False Homozygous Reads (Heterozygous in Real)

While we sequencing CFTR with long-range PCR + Nextera XT + MiSeq 2x250bp, we detected homozygous variant in sample. (upper part of image)

However, when we sequenced the same sample with short-range PCR with same conditions, the variant seemed heterozygous. (lower part of image)

What could be the reason for that?

Is it possible that our long-range primers bind and amplify only one copy of CFTR?

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Old 01-14-2016, 07:05 PM   #2
Brian Bushnell
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Certainly, if your SNP of interest is linked to another mutation that happens to be at your primer site, you'll get a major bias. Perhaps you should examine both of your long-amplicon primer sites with Sanger.
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Old 01-15-2016, 09:30 AM   #3
ECO
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I also have run into a situation where Illumina's adapter trimming was too promiscuous, and was actually trimming regions of the genome!

Ha! I just found my old example and it was CFTR also (albeit exon 10):

http://seqanswers.com/forums/showthread.php?t=23265

How did you do adapter trimming? Are you able to visualize soft-clipped reads?
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Old 01-15-2016, 09:58 AM   #4
HESmith
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Another possibility is that the exon 3 sequence is duplicated in your genome, and the short-range (but not long-range) PCR primers are amplifying both copies.
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Old 01-15-2016, 10:27 AM   #5
Richard Finney
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Are other exons in your target?
What's the coverage of the other exons relative to exon 3 ?
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Old 01-16-2016, 10:08 PM   #6
Nikvailo
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Thank you for all answers. And sorry for my late reply. We were quite busy.

Quote:
Originally Posted by Brian Bushnell View Post
Certainly, if your SNP of interest is linked to another mutation that happens to be at your primer site, you'll get a major bias. Perhaps you should examine both of your long-amplicon primer sites with Sanger.
Thank you Brian. It is quite possible. We will design primers to check that if there is any variant in the primer binding site.

Quote:
Originally Posted by ECO
I also have run into a situation where Illumina's adapter trimming was too promiscuous, and was actually trimming regions of the genome!
Ha! I just found my old example and it was CFTR also (albeit exon 10):
http://seqanswers.com/forums/showthread.php?t=23265
How did you do adapter trimming? Are you able to visualize soft-clipped reads?
We are actually using MiSeq built-in adapter trimming option. I understand you example. I is very strange that, that trimmer can also remove genomic regions. But I couldn't figure out that how such bug results with my problem?

Quote:
Originally Posted by HESmith
IAnother possibility is that the exon 3 sequence is duplicated in your genome, and the short-range (but not long-range) PCR primers are amplifying both copies.
So you say that one of duplicated exon has variant but other has not? Right?

Quote:
Originally Posted by Richard Finney
Are other exons in your target?
What's the coverage of the other exons relative to exon 3 ?
It changes. Because of that the PCR efficiency of each amplicon is different the coverage is different in each of exonic regions.
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