Answers.Body: <p>You could try aligning your reads to the draft reference genome with for example <code>minimap2</code> and calling variants with <code>freebayes</code>. It appears there is a protocol for long reads: <a href="https://github.com/ekg/freebayes#re-genotyping-known-variants-and-calling-long-haplotypes" rel="nofollow noreferrer">https://github.com/ekg/freebayes#re-genotyping-known-variants-and-calling-long-haplotypes</a></p>
  
  <p>Disclaimer: I have not run <code>freebayes</code> myself, and it may be that this is a terrible idea. I would expect that calling SNVs (Single-nucleotide variants) from PacBio reads is very challenging.  However, with enough coverage, I think it could be possible.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-11-26T12:59:53.623Z
Answers.LastActivityDate: 2018-11-26T12:59:53.623Z
Answers.UserDisplayName: winni2k
Answers.Body: <p>You may want to try the longshot tool (<a href="https://github.com/pjedge/longshot" rel="nofollow noreferrer">https://github.com/pjedge/longshot</a>) developed for calling variants in diploid genomes from long read data. </p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2019-02-21T10:18:47.657Z
Answers.LastActivityDate: 2019-02-21T10:18:47.657Z
Answers.UserDisplayName: user4162
Body: <p>I'd like to call diploid SNV variants from long-reads data (~80SMRTcells PacBio). </p>
  
  <p>I have generated a draft reference genome for an indivudual from a heterozygous (~4%) species (<a href="https://github.com/marbl/canu" rel="noreferrer">Canu</a>+<a href="https://github.com/mapleforest/HaploMerger2" rel="noreferrer">Haplomerger2</a>). </p>
  
  <p>I can use this reference for some applications:</p>
  
  <ul>
  <li>RNAseq mapping;</li>
  <li>functional annotation;</li>
  <li>duplicate detection;</li>
  <li>synteny analysis</li>
  </ul>
  
  <p>but not others:</p>
  
  <ul>
  <li>analysis of compound heterozygotes; </li>
  <li>allele-specific expression; </li>
  <li>variant linkage</li>
  </ul>
  
  <p>This is mainly why I need phasing. FYI, I tried <a href="https://github.com/PacificBiosciences/FALCON/wiki/FALCON-FALCON-Unzip-%22For-Phased-Diploid-Genome-Assembly-with-Single-Molecule-Real-Time-Sequencing%22" rel="noreferrer">Falcon + FalconUnzip</a> too, but the daligner phase was just taking too much time/resources and I need to find a strategy to make it work in reasonable time (working on it).</p>
  
  <p>Meanwhile, for phasing, It was suggested to use <a href="https://github.com/vibansal/HapCUT2/blob/master/README.md" rel="noreferrer">HapCUT2</a> - from Canu's FAQ</p>
  
  <blockquote>
    <p>(1) "Avoid collapsing the genome" in Canu to get raw diploid assembly as complete &amp; separate as possible (2) HaploMerger2 to get high-quality reference and alternative haploid assemblies (3) HapCUT2 or other phasing tools to get the high-quality haplotype assembly based on the reference haploid assembly. </p>
  </blockquote>
  
  <p>However, HapCUT2 requires the input of a diploid SNV file. Which <a href="https://bioinformatics.stackexchange.com/questions/4019/converting-a-bam-file-into-vcf/4022#4022">could be done</a> if I had short-reads data, but yet haven't found a method for doing so with in my hands just long-reads data. </p>
  
  <p>Strategies I tried to get the SNVs:</p>
  
  <ul>
  <li><p><a href="https://github.com/benedictpaten/marginPhase" rel="noreferrer">marginPhase</a> : <a href="https://www.biorxiv.org/content/early/2018/04/12/293944" rel="noreferrer">method paper</a> </p>
  
  <ul>
  <li>but I had a <a href="https://github.com/benedictpaten/marginPhase/issues/12" rel="noreferrer">issue</a> in running it </li>
  </ul></li>
  <li><p><a href="https://github.com/aquaskyline/Clairvoyante" rel="noreferrer">Clairvoyante</a> : <a href="https://www.biorxiv.org/content/early/2018/04/28/310458" rel="noreferrer">method paper</a></p>
  
  <ul>
  <li>but I currently don't have access to suitable GPU machines to build the model - and I can't use the models provided by the authors because my organism is too different from human</li>
  </ul></li>
  </ul>
  
  <p>Any input would be greatly appreciated!</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-11-22T16:20:05.020Z
Comments.Text: At 4% heterozygosity, falcon-unzip is probably the best hope. Trio binning would be better but you don't have the right data.
Comments.UserDisplayName: user172818
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-11-23T10:00:17.703Z
Comments.Text: @user172818 is it possible in your knowledge to run Falcon-Unzip on non-Falcon output? From [here](https://github.com/PacificBiosciences/FALCON_unzip/issues/100) it looks like it's a no.
Comments.UserDisplayName: aechchiki
Tags: <vcf><snp><variant-calling><long-reads><pacbio>
Title: calling diploid SNVs from long reads
ThreadText: # calling diploid SNVs from long reads
  
  (Asked by: username_0 on 2018-11-22)
  
  I'd like to call diploid SNV variants from long-reads data (~80SMRTcells PacBio).
  
  I have generated a draft reference genome for an indivudual from a heterozygous (~4%) species ([Canu](https://github.com/marbl/canu)+[Haplomerger2](https://github.com/mapleforest/HaploMerger2)).
  
  I can use this reference for some applications:
  
  * RNAseq mapping;
  * functional annotation;
  * duplicate detection;
  * synteny analysis
  
  but not others:
  
  * analysis of compound heterozygotes;
  * allele-specific expression;
  * variant linkage
  
  This is mainly why I need phasing. FYI, I tried [Falcon + FalconUnzip](https://github.com/PacificBiosciences/FALCON/wiki/FALCON-FALCON-Unzip-%22For-Phased-Diploid-Genome-Assembly-with-Single-Molecule-Real-Time-Sequencing%22) too, but the daligner phase was just taking too much time/resources and I need to find a strategy to make it work in reasonable time (working on it).
  
  Meanwhile, for phasing, It was suggested to use [HapCUT2](https://github.com/vibansal/HapCUT2/blob/master/README.md) - from Canu's FAQ
  
  > (1) "Avoid collapsing the genome" in Canu to get raw diploid assembly as complete & separate as possible (2) HaploMerger2 to get high-quality reference and alternative haploid assemblies (3) HapCUT2 or other phasing tools to get the high-quality haplotype assembly based on the reference haploid assembly.
  
  However, HapCUT2 requires the input of a diploid SNV file. Which [could be done](https://bioinformatics.stackexchange.com/questions/4019/converting-a-bam-file-into-vcf/4022#4022) if I had short-reads data, but yet haven't found a method for doing so with in my hands just long-reads data.
  
  Strategies I tried to get the SNVs:
  
  * [marginPhase](https://github.com/benedictpaten/marginPhase) : [method paper](https://www.biorxiv.org/content/early/2018/04/12/293944)
  
    + but I had a [issue](https://github.com/benedictpaten/marginPhase/issues/12) in running it
  * [Clairvoyante](https://github.com/aquaskyline/Clairvoyante) : [method paper](https://www.biorxiv.org/content/early/2018/04/28/310458)
  
    + but I currently don't have access to suitable GPU machines to build the model - and I can't use the models provided by the authors because my organism is too different from human
  
  Any input would be greatly appreciated!
  
  **Tags:** vcf, snp, variant-calling, long-reads, pacbio
  
  > **Comments:**
  > **username_1**: At 4% heterozygosity, falcon-unzip is probably the best hope. Trio binning would be better but you don't have the right data.  
  > **username_0**: @username_1 is it possible in your knowledge to run Falcon-Unzip on non-Falcon output? From [here](https://github.com/PacificBiosciences/FALCON_unzip/issues/100) it looks like it's a no.  
  
  ---
  
  ## 2 answers
  
  ### Answer by username_3 on 2019-02-21. Score: 2 | Accepted answer
  
  You may want to try the longshot tool (<https://github.com/pjedge/longshot>) developed for calling variants in diploid genomes from long read data.
  
  
  ---
  
  ### Answer by username_2 on 2018-11-26. Score: 2
  
  You could try aligning your reads to the draft reference genome with for example `minimap2` and calling variants with `freebayes`. It appears there is a protocol for long reads: <https://github.com/ekg/freebayes#re-genotyping-known-variants-and-calling-long-haplotypes>
  
  Disclaimer: I have not run `freebayes` myself, and it may be that this is a terrible idea. I would expect that calling SNVs (Single-nucleotide variants) from PacBio reads is very challenging. However, with enough coverage, I think it could be possible.
  
  
  ---
  
  