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Variants in pseudogene or repeat region

asakiasaki chinaMember

Hi all,

We used PCR to enrich the target amplicon, and get sequenced on HiSeq X.
In our targets, several are located in pseudogenes or repeat region.

We used bwa mem to align the reads to genome with default settings.
BQSR is neglected due to time consuming.

UG is applied to call given variants according to discussions in forum.
However, reads belong to pseudogenes or repeat region can be mapped to other regions, with XA tag in bam file.
This leads to low or even 0 mapping quality for target amplicons, and therefore, the variants are not called.

I searched the forum, and found that changing the Kmer size might fix this, but increase the false positives.

My question is,

  1. Shall I use HC instead?
  2. What can I do to get variants in these regions called, as well as those 'normal' ones.

Thanks

Tagged:

Answers

  • SheilaSheila Broad InstituteMember, Broadie admin

    @asaki
    Hi,

    Can you post some IGV screenshots of those regions? Are no variants called at all in those repeat regions? You can try lowering the mapping quality default threshold to 0 in those regions.

    -Sheila

  • asakiasaki chinaMember

    @Sheila
    Most of the uncalled variants are located in pseudogene or repeat region. After haplotype re-assembly, reads around these region were filtered due to low mapping quality.

  • SheilaSheila Broad InstituteMember, Broadie admin

    @asaki
    Hi,

    Why are there lines connecting the reads? It does not look like there are any variants in any of those reads. Are all of them mapping quality 0?

    As for variant calling, you can try reducing the mapping quality threshold to 0 in those regions. You should only do that in those regions, as it could lead to false positives in other regions.

    -Sheila

  • asakiasaki chinaMember

    @Sheila

    Lines between the reads represents pair end reads. The position I highlighted in IGV is actually a variants of homozygous REF one, which did not contain any alt alleles. I expected to see the homozygote SNP.

    How to set the mapping quality in specific regions? Any quick guide?

  • SheilaSheila Broad InstituteMember, Broadie admin

    @asaki
    Hi,

    Ah, are you asking to see hom-ref sites as well as het/hom-var sites? In that case, you can use --includeNonVariantSites.

    As for setting a lower mapping quality threshold in different regions, you can use a combination of --min_mapping_quality_score and -L.

    -Sheila

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