<p>I am having an issue understanding some records for VCF file. We are analyzing human samples to detect somatic cancer mutations. We have a duplicate sample that was processed using Illumina Dragen Amplicon app in basespace. One of the samples had the following records (for example)</p>
<p>Sample 1</p>
<pre><code>pos, ref, alt
10, G, A  
10, GC, AA
11, C, A
</code></pre>
<p>The duplicate sample had the following:</p>
<pre><code>pos, ref, alt
10, G, A
11, C, A
</code></pre>
<p>From what I understand, in both cases positions 10 and 11 are now both CC, correct? or, is the line which is stating GC &gt; AA signifying something else?</p>
<p>Additionally, when I viewed the bam files for that position, IGV for sample number 1 showing AA against the reference, while the duplicate showing the reference bases, despite having an allele frequency of around 18% in both samples; the original and duplicate.</p>
<p>So,</p>
<ul>
<li>is there a difference in results between both samples?</li>
<li>why is IGV not showing the alternative alleles in the duplicate sample?</li>
</ul>
<p>Edit: adding the vcf lines</p>
<p>File 1</p>
<pre><code>chrX    39922208    .   G   A   .   PASS    DP=1196;MQ=249.93;FractionInformativeReads=1.000;SoftClipRatio=0.00;MNVTAG=chrX:39922208_GC-&gt;AA GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB  0/1:11.79:985,211:0.1764:482,117:503,94:1196:985,0,211,0:482,503,117,94

chrX    39922209    .   C   A   .   PASS    DP=1196;MQ=249.93;FractionInformativeReads=1.000;SoftClipRatio=0.00;MNVTAG=chrX:39922208_GC-&gt;AA GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB  0/1:11.79:985,211:0.1764:482,117:503,94:1196:985,0,211,0:482,503,117,94
</code></pre>
<p>File 2</p>
<pre><code>chrX    39922208    .   G   A   .   PASS    DP=1196;MQ=249.93;FractionInformativeReads=1.000;SoftClipRatio=0.00;MNVTAG=chrX:39922208_GC-&gt;AA GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB  0/1:11.79:985,211:0.1764:482,117:503,94:1196:985,0,211,0:482,503,117,94
chrX    39922208    .   GC  AA  .   PASS    DP=1196;MQ=249.93;FractionInformativeReads=1.000;SoftClipRatio=0.00;MNVTAG=chrX:39922208_GC-&gt;AA GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB  0/1:11.79:985,211:0.1764:482,117:503,94:1196:985,0,211,0:482,503,117,94
chrX    39922209    .   C   A   .   PASS    DP=1196;MQ=249.93;FractionInformativeReads=1.000;SoftClipRatio=0.00;MNVTAG=chrX:39922208_GC-&gt;AA GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB  0/1:11.79:985,211:0.1764:482,117:503,94:1196:985,0,211,0:482,503,117,94
</code></pre>

CC BY-SA 4.0
2024-08-08T14:02:43.153Z
What is the ploidy of your sample organism, and what is the frequency of the variants? The latter should be included in a VCF field. 

If your sample organism is haploid, then if the allele frequency (and other metrics) suggest its real, then it's as you said, GC -> AA. If its diploid or more, you could have a bunch of different outcomes in the same sample; AC on one chromosome, GA on the other, for example.
dthorbur
CC BY-SA 4.0
2024-08-08T15:00:47.073Z
My apologies, I was not clear. The samples are human, and we are detecting somatic mutations. Regarding the frequency, as I mentioned in the post, it is around 18%.
Skepto18
CC BY-SA 4.0
2024-08-12T11:52:02.173Z
What does "duplicate sample" mean? Is it the same individual sequenced two times, or is it the exact same sequencing output (fastq files) analyzed using different variant callers? Can you show us the actual VCF lines instead of this csv format? There are various pieces of relevant information included in the full line.
terdon
CC BY-SA 4.0
2024-08-12T11:53:43.770Z
Can you also please clarify your last paragraph? "*Additionally, when I viewed the bam files for that position, IGV for sample number 1 showing AA against the reference, while the duplicate showing the reference bases, despite having an allele frequency of around 18% in both samples; the original and duplicate.*" I am afraid I cannot understand anything from that, I don't know what it is supposed to mean.
terdon
CC BY-SA 4.0
2024-08-14T16:30:34.247Z
@terdon, duplicate means the same sample was sequenced twice in the same run using different index.

Due to space limitation I will post the lines in the original question.
Skepto18
<vcf><variant-calling><variants><igv>
Understanding VCF file records
# Understanding VCF file records

(Asked by: username_0 on 2024-08-08)

I am having an issue understanding some records for VCF file. We are analyzing human samples to detect somatic cancer mutations. We have a duplicate sample that was processed using Illumina Dragen Amplicon app in basespace. One of the samples had the following records (for example)
Sample 1
pos, ref, alt
10, G, A  
10, GC, AA
11, C, A

The duplicate sample had the following:
pos, ref, alt
10, G, A
11, C, A

From what I understand, in both cases positions 10 and 11 are now both CC, correct? or, is the line which is stating GC > AA signifying something else?
Additionally, when I viewed the bam files for that position, IGV for sample number 1 showing AA against the reference, while the duplicate showing the reference bases, despite having an allele frequency of around 18% in both samples; the original and duplicate.
So,

is there a difference in results between both samples?
why is IGV not showing the alternative alleles in the duplicate sample?

Edit: adding the vcf lines
File 1
chrX    39922208    .   G   A   .   PASS    DP=1196;MQ=249.93;FractionInformativeReads=1.000;SoftClipRatio=0.00;MNVTAG=chrX:39922208_GC->AA GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB  0/1:11.79:985,211:0.1764:482,117:503,94:1196:985,0,211,0:482,503,117,94

chrX    39922209    .   C   A   .   PASS    DP=1196;MQ=249.93;FractionInformativeReads=1.000;SoftClipRatio=0.00;MNVTAG=chrX:39922208_GC->AA GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB  0/1:11.79:985,211:0.1764:482,117:503,94:1196:985,0,211,0:482,503,117,94

File 2
chrX    39922208    .   G   A   .   PASS    DP=1196;MQ=249.93;FractionInformativeReads=1.000;SoftClipRatio=0.00;MNVTAG=chrX:39922208_GC->AA GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB  0/1:11.79:985,211:0.1764:482,117:503,94:1196:985,0,211,0:482,503,117,94
chrX    39922208    .   GC  AA  .   PASS    DP=1196;MQ=249.93;FractionInformativeReads=1.000;SoftClipRatio=0.00;MNVTAG=chrX:39922208_GC->AA GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB  0/1:11.79:985,211:0.1764:482,117:503,94:1196:985,0,211,0:482,503,117,94
chrX    39922209    .   C   A   .   PASS    DP=1196;MQ=249.93;FractionInformativeReads=1.000;SoftClipRatio=0.00;MNVTAG=chrX:39922208_GC->AA GT:SQ:AD:AF:F1R2:F2R1:DP:SB:MB  0/1:11.79:985,211:0.1764:482,117:503,94:1196:985,0,211,0:482,503,117,94

**Tags:** vcf, variant-calling, variants, igv
> **Comments:**
> **username_1**: What is the ploidy of your sample organism, and what is the frequency of the variants? The latter should be included in a VCF field.   If your sample organism is haploid, then if the allele frequency (and other metrics) suggest its real, then it's as you said, GC -> AA. If its diploid or more, you could have a bunch of different outcomes in the same sample; AC on one chromosome, GA on the other, for example.  
> **username_0**: My apologies, I was not clear. The samples are human, and we are detecting somatic mutations. Regarding the frequency, as I mentioned in the post, it is around 18%.  
> **username_2**: What does "duplicate sample" mean? Is it the same individual sequenced two times, or is it the exact same sequencing output (fastq files) analyzed using different variant callers? Can you show us the actual VCF lines instead of this csv format? There are various pieces of relevant information included in the full line.  
> **username_2**: Can you also please clarify your last paragraph? "*Additionally, when I viewed the bam files for that position, IGV for sample number 1 showing AA against the reference, while the duplicate showing the reference bases, despite having an allele frequency of around 18% in both samples; the original and duplicate.*" I am afraid I cannot understand anything from that, I don't know what it is supposed to mean.  
> **username_0**: @terdon, duplicate means the same sample was sequenced twice in the same run using different index.  Due to space limitation I will post the lines in the original question.  

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