GATK_HaplotypeCaller: <NON_REF> .

Question

[iraun]

Hi there,

Could anyone explain me a little bit these two lines of my GVCF file?

Why I'm getting "NON REF" alternative allele? And why for the second line I only see END information and not the rest?

GL000192.1    546636    .    G    A,<NON_REF>    49.77    .    BaseQRankSum=1.026;ClippingRankSum=1.026;DP=4;MLEAC=1,0;MLEAF=0.500,0.00;MQ=49.02;MQ0=0;MQRankSum=-1.026;ReadPosRankSum=0.000    GT:AD:DP:GQ:PGT:PID:PL:SB    0/1:2,2,0:4:78:0|1:546636_G_A:78,0,87,84,93,177:1,1,1,1
GL000192.1    546637    .    G    <NON_REF>    .    .    END=546645    GT:DP:GQ:MIN_DP:PL    0/0:4:11:4:0,12,99

I know that NON_REF represents any possible alternative allele at this location, but if my genotype is 0/0 (homozygous for reference)... this line makes no sense for me.

Thanks in advance.

[int11ap1]

Indeed it is a very interesting question, my lady.

[singhjay]

Dear Iraun I am also facing the same issue. I am using RNA Seq Data. I am confused with the Haplotypecaller output.

After reading your post, I got some Idea but it is still not cleared. should I re run haplotype caller ? or I should exclude these "NON REF" from further analyis. I have used this output bam file for funcotator but I failed to generate a functional annotated file. It would be helpful if you can help me in this issue. Thanks and Regards

Answer 1

[iraun]

OK, I found the solution in GATK manual (https://www.broadinstitute.org/gatk/guide/article?id=4017). I'll answer my own question just in case that someone else have the same issue.

"The first thing you'll notice, hopefully, is the <NON_REF> symbolic allele listed in every record's ALT field. This provides us with a way to represent the possibility of having a non-reference allele at this site, and to indicate our confidence either way.

The second thing to look for is the END tag in the INFO field of non-variant block records. This tells you at what position the block ends."

[Istvan Albert]

Thanks for following up!

Answer 2

[Moe]

0 is reference allele. 1 is first alternative allele. 2 is second alt allele and so on

so if GT is 0/1 = heterozygous ref/alt. 0/0 = homozygous ref/ref. 1/1 = homozygous alt/alt. 0/2 = heterozygous ref/second allele. in this case you would find at least two alleles under alt column.

in your shared example, it should be:

pos: 546636 , GT: G/A pos: 546637, GT: G/G