failed in discover one single mutation

[lmanchon]

--Hi,

i don't know why this record is discarded: chr21 43104346 . G T 273 Q10 SAMPLE=SAD190004_S5;TYPE=SNV;DP=636;END=43104346;VD=289;AF=0.4544;BIAS=2:2;REFBIAS=170:177;VARBIAS=143:146;PMEAN=39.1;PSTD=1;QUAL=33.5;QSTD=1;SBF=0.93657;ODDRATIO=1.01975260801729;MQ=0;SN=35.125;HIAF=0.4554;ADJAF=0.0079;SHIFT3=0;MSI=2;MSILEN=2;NM=1.1;HICNT=281;HICOV=617;LSEQ=TCGGTTTATTGTGCAACCGA;RSEQ=AGCACCTGTCTCCATGACGA;DUPRATE=0;SPLITREAD=0;SPANPAIR=0 GT:DP:VD:AD:AF:RD:ALD 0/1:636:289:347,289:0.4544:170,177:143,146

which parameter i need to adjust ?

thank you --

[PolinaBevad]

Hi @lmanchon , these is MQ=0, that means that mapping quality of this variant is zero - so the reads supporting the variant should have MAPQ=0 as well and seems are not mapped correctly to the reference. Better to check with genome browser, but I believe it was filtered out correctly (FILTER field is Q10 for this case).

[lmanchon]

seems to be well mapped with BWA-MEM, see IGV panel here:

[PolinaBevad]

Maybe those are secondary alignments? Do you have XA tags in these reads? Just show any read supporting the variant. We filter secondary alignments, but probably they were included in tags instead of flags (-F 0x504).

[lmanchon]

this the corresponding sam lines: samtools view mybam.bam chr21:43104255-43104428 | grep "43104346" M02792:126:000000000-K9RHL:1:1114:12346:24983 163 chr21 43104253 0 147M = 43104346 240 TTAACTGTCTTTGAAAAGAACATGAAGTTTTTATAATTTACATGAAAAAAAGGCAAACAAACCTGGCTAAACGTCGGTTTATTGTGCAACCGAGAGCACCTGTCTCCATGACGACATGCTCCAATTTTGAAATAAAATGAACAGTTG BEDHBFFDGFEEDEGGHHFIDECKFJGEFGFEBEBJEFEBDEEKGJJJJHGHDIEJJEEJJDEIKIFGBJJDBEHBHDFFBEEKFGFFJDEBFHFHGEFHHKEHHHHEELFDBFFEDLFHIHEJEFFFKFJKFBJJJDKGKFEGEFD XA:Z:chr21,+6495931,147M,0; ZA:Z:CGCT ZB:Z:CCGT BC:Z:5 MD:Z:147 RG:Z:SAD190004_S5 NM:i:0 AS:i:147 XS:i:147 QX:Z:CCB ABC RX:Z:CGC-CCG xc:i:1 zd:Z: xm:i:1 M02792:126:000000000-K9RHL:1:1114:12346:24983 83 chr21 43104346 0 147M = 43104253 -240 GAGCACCTGTCTCCATGACGACATGCTCCAATTTTGAAATAAAATGAACAGTTGACTCTGTAAGGGAAAATGAGAGCTGATTATTTTGCTGGGAAGATATCAAACACATGGAATATGTCAGCAGCATGACATACACTATCAAATTAC HHFLEHGEFFFGIKFEGDEHEKEEFGGIKGEIJJEHEFEBFFGEEHFFKHEJEHEGGGEDBGHHHHFGGEFHHGIFHFHEJCEJJJEFHFIHHFIHEBEHKGFFLEKEEHHGEBEEEHLHGLHFLEEHEDDCDCDHCDEHDECHCCD XA:Z:chr21,-6496024,147M,0; ZA:Z:CGCT ZB:Z:CCGT BC:Z:5 MD:Z:147 RG:Z:SAD190004_S5 NM:i:0 AS:i:147 XS:i:147 QX:Z:CCB ABC RX:Z:CGC-CCG xc:i:1 zd:Z: xm:i:1

[PolinaBevad]

Those are secondary alignments: there is XA:Z tag that shows where was another location in genome where the read can be mapped. And this one will have MAPQ 0. Usually these should be also put to SAM flags (i.e. 163/83 for those reads) but for some reason it wasn't - probably BWA setup. But anyway - this variant is likely to be on another position in genome, so it was filtered out here.

[lmanchon]

okay thank you for your help.

[PolinaBevad]

Glad to help! :)