Hi All,

I am working with a viral sequence. Upon investigating the freebayes output vcf file I found that there are two positions where variants are reverse complement of the reference.

    Reference	527	.	CCCGGGCGTCGGGCGAC	GTCGCCCGACGCCCGGG	0	.	AB=0;ABP=0;AC=0;AF=0;AN=1;AO=1837;CIGAR=2X2M2X2M1X2M2X2M2X;DP=8277;DPB=9767.53;DPRA=0;EPP=3415.19;EPPR=9687.22;GTI=0;LEN=17;MEANALT=29;MQM=40.3887;MQMR=39.4471;NS=1;NUMALT=1;ODDS=65195.1;PAIRED=0.997278;PAIREDR=0.968095;PAO=0;PQA=0;PQR=137846;PRO=4415;QA=0;QR=230712;RO=6394;RPL=63;RPP=3463.56;RPPR=11044.7;RPR=1774;RUN=1;SAF=1831;SAP=3940.06;SAR=6;SRF=6101;SRP=11459.1;SRR=293;TYPE=complex	GT:DP:DPR:RO:QR:AO:QA:GL	0:8277:8277,1837:6394:230712:1837:0:0,-30803.9

and

    Reference	5586	.	GTCGCCCGACGCCCGGG	CCCGGGCGTCGGGCGAC	1.19962e-12	.	AB=0;ABP=0;AC=0;AF=0;AN=1;AO=1439;CIGAR=2X2M2X2M1X2M2X2M2X;DP=6715;DPB=8094.82;DPRA=0;EPP=2732.86;EPPR=7945.83;GTI=0;LEN=17;MEANALT=27;MQM=39.3489;MQMR=38.6015;NS=1;NUMALT=1;ODDS=54869.5;PAIRED=0.994441;PAIREDR=0.963359;PAO=0;PQA=0;PQR=125375;PRO=3860;QA=0;QR=188745;RO=5240;RPL=1401;RPP=2806.41;RPPR=9203.98;RPR=38;RUN=1;SAF=9;SAP=3050.08;SAR=1430;SRF=271;SRP=9149.39;SRR=4969;TYPE=complex	GT:DP:DPR:RO:QR:AO:QA:GL	0:6715:6715,1439:5240:188745:1439:0:0,-25889.3

When I visualized the alignment in IGV the variant was present at the specified location. This is visualization with IGV.

![enter image description here][1]

How can I know for sure that,

 1. Is this variant a sequencing or mapping artifact or an inversion
    variant?
 2. Is visualization in IGV a correct method of validating a
    variant?

  [1]: https://s6.postimg.cc/6jwcjicg1/position527.png
Hello,

> Is this variant a sequencing or mapping artifact or an inversion
> variant?

this is very likely some kind of artifact. Have a look at the 6th column of your vcf. This is a quality value for the variant site. Your value is 0 or something very close to it. Normally this value should be at least something around 20. Depending on the read depth this value is much higher.

Also freebayes gives you the genotype `0` which means `REF`

> Is visualization in IGV a correct method of validating a variant?

For a quick check this is absolutely fine. In this case you can also see that the bases which doesn't match the ref are faded. This normally means they have bad quality values.

For a real validation, each variant that has an impact on your goal, has to be confirmed by another method like sanger sequencing.

fin swimmer