How to extract uniquely aligned paired end reads obtained from freebayes using diffrent parmeter combinations of samtools?
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3.8 years ago

I have 10x reads and I need uniquely aligned reads for SNP calling. I am using samtools to extract uniquely aligned reads but I am not satisfied by the results. It would be great if someone can share the exact way of filtering these reads. I tried many combinations and checked many sources online but could not found any satisfactory answer. Here are the parameters that I already tried.

     step:01 #Initial stats after alignment using freebayes:
        172387804 + 0 in total (QC-passed reads + QC-failed reads)
        9715270 + 0 secondary
        0 + 0 supplementary
        22559490 + 0 duplicates
        165148332 + 0 mapped (95.80% : N/A)
        162672534 + 0 paired in sequencing
        81336267 + 0 read1
        81336267 + 0 read2
        109869698 + 0 properly paired (67.54% : N/A)
        152047167 + 0 with itself and mate mapped
        3385895 + 0 singletons (2.08% : N/A)
        25207784 + 0 with mate mapped to a different chr
        20769368 + 0 with mate mapped to a different chr (mapQ>=5)

 #step:02: Picard tools was used to mark and remove duplicates
     146765957 + 0 in total (QC-passed reads + QC-failed reads)
     9715270 + 0 secondary
     0 + 0 supplementary
     0 + 0 duplicates
     139526485 + 0 mapped (95.07% : N/A)
     137050687 + 0 paired in sequencing
     68390482 + 0 read1
     68660205 + 0 read2
     91962549 + 0 properly paired (67.10% : N/A)
     128054904 + 0 with itself and mate mapped
     1756311 + 0 singletons (1.28% : N/A)
     21673676 + 0 with mate mapped to a different chr
     18001625 + 0 with mate mapped to a different chr (mapQ>=5)

       # To get uniquely aligned reads "parameters1:-h -q 20 -F 256 test.bam"
        107311384 + 0 in total (QC-passed reads + QC-failed reads)
        0 + 0 secondary
        0 + 0 supplementary
        0 + 0 duplicates
        107311384 + 0 mapped (100.00% : N/A)
        107311384 + 0 paired in sequencing
        53437167 + 0 read1
        53874217 + 0 read2
        80455227 + 0 properly paired (74.97% : N/A)
        105892555 + 0 with itself and mate mapped
        1418829 + 0 singletons (1.32% : N/A)
        15343533 + 0 with mate mapped to a different chr
        15343533 + 0 with mate mapped to a different chr (mapQ>=5)

        ##2nd try:parameters2: -q 20 -f 0x02
        82937879 + 0 in total (QC-passed reads + QC-failed reads)
        2482652 + 0 secondary
        0 + 0 supplementary
        0 + 0 duplicates
        82937879 + 0 mapped (100.00% : N/A)
        80455227 + 0 paired in sequencing
        40153047 + 0 read1
        40302180 + 0 read2
        80455227 + 0 properly paired (100.00% : N/A)
        80455226 + 0 with itself and mate mapped
        1 + 0 singletons (0.00% : N/A)
        0 + 0 with mate mapped to a different chr
        0 + 0 with mate mapped to a different chr (mapQ>=5)

        #3rd try:
        -f 0x02 -bq 1 (properly paired end reads)
        93036316 + 0 in total (QC-passed reads + QC-failed reads)
        2817262 + 0 secondary
        0 + 0 supplementary
        0 + 0 duplicates
        93036316 + 0 mapped (100.00% : N/A)
        90219054 + 0 paired in sequencing
        45085486 + 0 read1
        45133568 + 0 read2
        90219054 + 0 properly paired (100.00% : N/A)
        90219033 + 0 with itself and mate mapped
        21 + 0 singletons (0.00% : N/A)
        0 + 0 with mate mapped to a different chr
        0 + 0 with mate mapped to a different chr (mapQ>=5)

        #4th: -F 3852
        105892555 + 0 in total (QC-passed reads + QC-failed reads)
        0 + 0 secondary
        0 + 0 supplementary
        0 + 0 duplicates
        105892555 + 0 mapped (100.00% : N/A)
        105892555 + 0 paired in sequencing
        52606133 + 0 read1
        53286422 + 0 read2
        80455226 + 0 properly paired (75.98% : N/A)
        105892555 + 0 with itself and mate mapped
        0 + 0 singletons (0.00% : N/A)
        15343533 + 0 with mate mapped to a different chr
        15343533 + 0 with mate mapped to a different chr (mapQ>=5)

        #5th: -q 20 -F 268
        105892555 + 0 in total (QC-passed reads + QC-failed reads)
        0 + 0 secondary
        0 + 0 supplementary
        0 + 0 duplicates
        105892555 + 0 mapped (100.00% : N/A)
        105892555 + 0 paired in sequencing
        52606133 + 0 read1
        53286422 + 0 read2
        80455226 + 0 properly paired (75.98% : N/A)
        105892555 + 0 with itself and mate mapped
        0 + 0 singletons (0.00% : N/A)
        15343533 + 0 with mate mapped to a different chr
        15343533 + 0 with mate mapped to a different chr (mapQ>=5)

        #6th:-q 20 -F 0x100
        107311384 + 0 in total (QC-passed reads + QC-failed reads)
        0 + 0 secondary
        0 + 0 supplementary
        0 + 0 duplicates
        107311384 + 0 mapped (100.00% : N/A)
        107311384 + 0 paired in sequencing
        53437167 + 0 read1
        53874217 + 0 read2
        80455227 + 0 properly paired (74.97% : N/A)
        105892555 + 0 with itself and mate mapped
        1418829 + 0 singletons (1.32% : N/A)
        15343533 + 0 with mate mapped to a different chr
        15343533 + 0 with mate mapped to a different chr (mapQ>=5)

Anyone suggest me the right parameter out of these tries.
I am loosing lot of reads. Is there any better way or parameter to solve this issue?
Thanks
NGS read filtering Samtools freebayes • 787 views
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