Answers.Body: <p>Typical SNP calling wouldn't give you good results on classical HLA genes. Ignore the VCF. Try a dedicated HLA genotyper on raw reads instead. I heard <a href="https://github.com/FRED-2/OptiType" rel="nofollow noreferrer">optitype</a> is a good one for exome or whole-genome data. I haven't used it myself, though.</p>
  
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Answers.Body: <p>The specific question and the easiest question is the absence of HLA-B*15:01 from your sample and that is not unexpected at all.</p>
  <p>The maximum frequency of this allele is 40% found in &quot;<a href="http://www.allelefrequencies.net/hla6006a.asp?page=40&amp;hla_locus=B&amp;hla_locus_type=Classical&amp;hla_allele1=15&amp;hla_allele2=&amp;hla_selection=&amp;hla_pop_selection=&amp;hla_population=&amp;hla_country=&amp;hla_dataset=&amp;hla_region=&amp;hla_ethnic=&amp;hla_study=&amp;hla_sample_size=&amp;hla_sample_size_pattern=equal&amp;hla_sample_year=&amp;hla_sample_year_pattern=equal&amp;hla_level=&amp;hla_level_pattern=equal&amp;hla_show=&amp;hla_order=order_1&amp;standard=g" rel="nofollow noreferrer">China Beijing pop 2</a>&quot; , this however is really unusually high and was published by Guang Yang et al (2009, IJI 37, 205-212).</p>
  <p>Mostly this allele is present at 5% for moderate frequency of occurrence for example Europe, such as in Germany <a href="http://www.allelefrequencies.net/hla6006a.asp?page=40&amp;hla_locus=B&amp;hla_locus_type=Classical&amp;hla_allele1=15&amp;hla_allele2=&amp;hla_selection=&amp;hla_pop_selection=&amp;hla_population=&amp;hla_country=&amp;hla_dataset=&amp;hla_region=&amp;hla_ethnic=&amp;hla_study=&amp;hla_sample_size=&amp;hla_sample_size_pattern=equal&amp;hla_sample_year=&amp;hla_sample_year_pattern=equal&amp;hla_level=&amp;hla_level_pattern=equal&amp;hla_show=&amp;hla_order=order_1&amp;standard=g" rel="nofollow noreferrer">here</a>. Moreover, in some populations the frequency is very low &lt;&lt;1%, e.g. India or Israel.</p>
  <p>So a sample genome at random the will not have the B*15:01.</p>
  <hr />
  <p>The more complicated issue is the VCF and the reference genome. This is because the basis on whether an allele is present or absent is a comparison against the reference genome which will generate the VCF. Thus understanding the HLA-B alleles of the reference genome is really important. You need a reference genome which lacks HLA-B*15:01, therefore if your VCF also lacks the allele, its missing. However, if HLA-B*15:01 is present in the reference genome your VCF will not present the allele if its present.</p>
  <p>The probability is that the allele is missing from both the reference and sample genome.</p>
  <hr />
  <p><strong>Comments</strong></p>
  <p><strong>Easy way</strong> The easiest way is search the IGV for <a href="https://www.ncbi.nlm.nih.gov/nuccore/LT618832.1" rel="nofollow noreferrer">LT618832.1</a> (HLA-B*15:01 reference), IGV has a motif finder, here <a href="https://software.broadinstitute.org/software/igv/motif_finder" rel="nofollow noreferrer">https://software.broadinstitute.org/software/igv/motif_finder</a></p>
  <p><strong>Hard way</strong>
  This is how I'd do it ..</p>
  <ul>
  <li>convert the bam to fasta then</li>
  <li>run a blast against Genbank reference LT618832.1</li>
  </ul>
  <p>I'd section the bam file into the locus of interest (e.g. chromosome 6) e.g. ... <code>samtools view ... ChrName: ... &gt; chromo6.bam</code>: you'd need to cross check how to do this last bit ... then via <code>samtools &gt;1.3</code>,</p>
  <pre><code>samtools fasta chromosome6.bam &gt; chromosome6.fasta
  </code></pre>
  <p>Finally you download a standalone version of <code>blast</code>, I assume its on <code>-c bioconda</code> ...</p>
  <pre><code>blastn -query HLAB1501.faa -db chromosome6.fa -out myhits.txt
  </code></pre>
  <p>From memory I don't think you need to <code>makeblastdb</code> for a fasta onto a fasta db.</p>
  <p>You'd need to map the SNPs in introns parsing (the coordinates are in the Genbank reference), or optically against the Genbank reference onto the <code>blast</code> output.</p>
  <p>You may want to cross check the HLA-B*15:01 against <a href="https://www.ncbi.nlm.nih.gov/nuccore/?term=HLA-B%2A15%3A01" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/nuccore/?term=HLA-B%2A15%3A01</a></p>
  <p>[but be careful about the introns]</p>
  <p><code>motif_finder</code> seems easier and accommodates potential heterozygosity.</p>
  
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Body: <p>I'm trying to find the HLA-B*15:01 variant in my DNA results, prompted from this research paper: <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142661/#:%7E:text=HLA%2DB*15%3A01%20is%20strongly%20associated%20with%20asymptomatic,mechanism%20underlying%20early%20viral%20clearance" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142661/#:~:text=HLA%2DB*15%3A01%20is%20strongly%20associated%20with%20asymptomatic,mechanism%20underlying%20early%20viral%20clearance</a>.</p>
  <p>I have DNA test results from Dante Labs that contain the following files</p>
  <pre><code>&quot;*.bam&quot;
  &quot;*.bam.bai&quot;
  &quot;*.cnv.vcf.gz&quot;
  &quot;*.cnv.vcf.gz.tbi&quot;
  &quot;*.filtered.indel.vcf.gz&quot;
  &quot;*.filtered.indel.vcf.gz.tbi&quot;
  &quot;*.filtered.snp.vcf.gz&quot;
  &quot;*.filtered.snp.vcf.gz.tbi&quot;
  &quot;*1_SA_L001_R1_001.fastq.gz&quot;
  &quot;*SA_L001_R2_001.fastq.gz&quot;
  </code></pre>
  <p>I tried loading this on the Genome Explorer on Sequencing.com, but it couldn't find HLA-B at all, only HLA-A and HLA-C.</p>
  <p>Then I tried loading the VCF files in IGV. I was able to filter to Chromosome 6 and HLA-B, which resolved to position</p>
  <pre><code>chr6:31,319,652-31,326,956
  </code></pre>
  <p>The CNV VCF says no variables found for chr6:31,319,652-31,326,956
  The filtered Indel VCF did produce some variants for this position, but I have no clue how to search deeper specifically for HLA-B*15:01. Here is a screenshot: <a href="https://i.sstatic.net/eCnFM.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/eCnFM.png" alt="enter image description here" /></a></p>
  <p>The filtered SNP VCF produced <em>alot</em> more variants for this position, but again, I don't know how to go deeper than this.</p>
  <p>I'm not really sure what to do from here?</p>
  <p>I don't have data on what the reference was, so that is unknown to me. I will reach out to Dante Labs about that, is there some sort of standard that is supposed to be used, or is it different for every genome provider?</p>
  <p>How do I actually perform the check in IGV assuming the reference did not have it and the VCF did. I also assume this VCF does not have this allele, but I frankly am lost into how to confirm it to the &quot;last mile&quot; through the VCF so to speak, or even just go straight to BAM</p>
  
Tags: <phylogenetics><phylogeny><genome><gene><igv>
Title: Searching for HLA-B in DNA results
ThreadText: # Searching for HLA-B in DNA results
  
  (Asked by: username_0 on 2022-09-25)
  
  I'm trying to find the HLA-B\*15:01 variant in my DNA results, prompted from this research paper: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142661/#:~:text=HLA%2DB\*15%3A01%20is%20strongly%20associated%20with%20asymptomatic,mechanism%20underlying%20early%20viral%20clearance](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142661/#:%7E:text=HLA%2DB*15%3A01%20is%20strongly%20associated%20with%20asymptomatic,mechanism%20underlying%20early%20viral%20clearance).
  
  I have DNA test results from Dante Labs that contain the following files
  
  ```
  "*.bam"
  "*.bam.bai"
  "*.cnv.vcf.gz"
  "*.cnv.vcf.gz.tbi"
  "*.filtered.indel.vcf.gz"
  "*.filtered.indel.vcf.gz.tbi"
  "*.filtered.snp.vcf.gz"
  "*.filtered.snp.vcf.gz.tbi"
  "*1_SA_L001_R1_001.fastq.gz"
  "*SA_L001_R2_001.fastq.gz"
  
  ```
  
  I tried loading this on the Genome Explorer on Sequencing.com, but it couldn't find HLA-B at all, only HLA-A and HLA-C.
  
  Then I tried loading the VCF files in IGV. I was able to filter to Chromosome 6 and HLA-B, which resolved to position
  
  ```
  chr6:31,319,652-31,326,956
  
  ```
  
  The CNV VCF says no variables found for chr6:31,319,652-31,326,956
  The filtered Indel VCF did produce some variants for this position, but I have no clue how to search deeper specifically for HLA-B\*15:01. Here is a screenshot: [![enter image description here](https://i.sstatic.net/eCnFM.png)](https://i.sstatic.net/eCnFM.png)
  
  The filtered SNP VCF produced *alot* more variants for this position, but again, I don't know how to go deeper than this.
  
  I'm not really sure what to do from here?
  
  I don't have data on what the reference was, so that is unknown to me. I will reach out to Dante Labs about that, is there some sort of standard that is supposed to be used, or is it different for every genome provider?
  
  How do I actually perform the check in IGV assuming the reference did not have it and the VCF did. I also assume this VCF does not have this allele, but I frankly am lost into how to confirm it to the "last mile" through the VCF so to speak, or even just go straight to BAM
  
  **Tags:** phylogenetics, phylogeny, genome, gene, igv
  
  
  ---
  
  ## 2 answers
  
  ### Answer by username_1 on 2022-09-27. Score: 2
  
  Typical SNP calling wouldn't give you good results on classical HLA genes. Ignore the VCF. Try a dedicated HLA genotyper on raw reads instead. I heard [optitype](https://github.com/FRED-2/OptiType) is a good one for exome or whole-genome data. I haven't used it myself, though.
  
  
  ---
  
  ### Answer by username_2 on 2022-09-25. Score: 1
  
  The specific question and the easiest question is the absence of HLA-B\*15:01 from your sample and that is not unexpected at all.
  
  The maximum frequency of this allele is 40% found in "[China Beijing pop 2](http://www.allelefrequencies.net/hla6006a.asp?page=40&hla_locus=B&hla_locus_type=Classical&hla_allele1=15&hla_allele2=&hla_selection=&hla_pop_selection=&hla_population=&hla_country=&hla_dataset=&hla_region=&hla_ethnic=&hla_study=&hla_sample_size=&hla_sample_size_pattern=equal&hla_sample_year=&hla_sample_year_pattern=equal&hla_level=&hla_level_pattern=equal&hla_show=&hla_order=order_1&standard=g)" , this however is really unusually high and was published by Guang Yang et al (2009, IJI 37, 205-212).
  
  Mostly this allele is present at 5% for moderate frequency of occurrence for example Europe, such as in Germany [here](http://www.allelefrequencies.net/hla6006a.asp?page=40&hla_locus=B&hla_locus_type=Classical&hla_allele1=15&hla_allele2=&hla_selection=&hla_pop_selection=&hla_population=&hla_country=&hla_dataset=&hla_region=&hla_ethnic=&hla_study=&hla_sample_size=&hla_sample_size_pattern=equal&hla_sample_year=&hla_sample_year_pattern=equal&hla_level=&hla_level_pattern=equal&hla_show=&hla_order=order_1&standard=g). Moreover, in some populations the frequency is very low <<1%, e.g. India or Israel.
  
  So a sample genome at random the will not have the B\*15:01.
  
  ---
  
  The more complicated issue is the VCF and the reference genome. This is because the basis on whether an allele is present or absent is a comparison against the reference genome which will generate the VCF. Thus understanding the HLA-B alleles of the reference genome is really important. You need a reference genome which lacks HLA-B\*15:01, therefore if your VCF also lacks the allele, its missing. However, if HLA-B\*15:01 is present in the reference genome your VCF will not present the allele if its present.
  
  The probability is that the allele is missing from both the reference and sample genome.
  
  ---
  
  **Comments**
  
  **Easy way** The easiest way is search the IGV for [LT618832.1](https://www.ncbi.nlm.nih.gov/nuccore/LT618832.1) (HLA-B\*15:01 reference), IGV has a motif finder, here <https://software.broadinstitute.org/software/igv/motif_finder>
  
  **Hard way**
  This is how I'd do it ..
  
  * convert the bam to fasta then
  * run a blast against Genbank reference LT618832.1
  
  I'd section the bam file into the locus of interest (e.g. chromosome 6) e.g. ... `samtools view ... ChrName: ... > chromo6.bam`: you'd need to cross check how to do this last bit ... then via `samtools >1.3`,
  
  ```
  samtools fasta chromosome6.bam > chromosome6.fasta
  
  ```
  
  Finally you download a standalone version of `blast`, I assume its on `-c bioconda` ...
  
  ```
  blastn -query HLAB1501.faa -db chromosome6.fa -out myhits.txt
  
  ```
  
  From memory I don't think you need to `makeblastdb` for a fasta onto a fasta db.
  
  You'd need to map the SNPs in introns parsing (the coordinates are in the Genbank reference), or optically against the Genbank reference onto the `blast` output.
  
  You may want to cross check the HLA-B\*15:01 against <https://www.ncbi.nlm.nih.gov/nuccore/?term=HLA-B%2A15%3A01>
  
  [but be careful about the introns]
  
  `motif_finder` seems easier and accommodates potential heterozygosity.
  
  
  ---
  
  