<p>My lab has started using the <a href='http://www.halogenomics.com/haloplex/how-it-works'>Haloplex technology</a> to capture the regions of interest.</p>

<p>With this technology most of the reads have the same start/end (I've found that removing the duplicates would reduce the coverage to ~1 base). The reads are grouped in a vertical 'cluster' that can contains more than 1000 reads.</p>

<p><img src='http://i.imgur.com/nW6Le.jpg' alt='enter image description here' /></p>

<p>Have you ever used this technology ? I'm currently using <a href='http://samtools.sourceforge.net/'>samtools</a> mpileup with the  option 
<strong>"-A"</strong> ( count anomalous read pairs) and <strong>"-d 8000"</strong> ( max per-BAM depth).</p>

<p>I'm afraid there is a kind of bias with haloplex: is it safe to use <a href='http://samtools.sourceforge.net/'>samtools</a> or Gatk to call the variants ? </p>

<p>Pierre</p>

<p>Agilent will say that you should not de-duplicate Halo data. The vertical clusters are due to the placement of the restriction enzyme sites used to create the capture kits. There&#39;s very little off-target capture with Halo and the coverage can vary widely across an capture region (&#39;Manhattan skyline&#39; I think of it as).</p>

<p>You can use, and I have used samtools, GATK or VarScan to call variants from Halo data. However if you do use GATK then if you&#39;re post-filtering UnifiedGenotyper calls based on assumptions for exome or WGS data, you will probably find variants which do not pass your standard filtering test that are almost certainly genuine. Strand bias filters etc. seem to be the most prone to being tripped.</p>

<p>I&#39;ve spoke to Agilent technical support before Christmas about what they propose for best practice genotyping, and the impression I got is that it&#39;s something they&#39;re working on but don&#39;t have a definitive answer for yet. I hope you&#39;re trimming the reads in conjunction with their guidelines as well.</p>

<p>EDIT: Since I wrote this post SureCall was released:</p>

<p>http://www.genomics.agilent.com/en/NGS-Data-Analysis-Software/SureCall/?cid=AG-PT-154</p>

<p>This is capable of analysing HaloPlex data and comes directly from Agilent to support the analysis of Haloplex designs from the SureDesign wizard.</p>
