Has anyone had any experience using SureCall software provided by Agilent to analyse Haloplex data? I have installed this but is is supposed to connect to reference genome data directly but this fails. What other workflows are commonly used to process the Haloplex data - I'd be interested to hear about these too.
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I have (sadly) had to analyse Haloplex data and the last time I did I used SureCall after spending too much time a year ago hand rolling solutions for it.
On initialisation it should as you say, download the reference data from Agilent's notoriously slow download site. If it's not doing this I would suspect you have a proxy in the way between you and the internet, and SureCall might not be aware of it. I'd look for proxy settings and talk to your IT department.
The reason I used SureCall is that my experience of trying to process Haloplex data with e.g. GATK UnifiedGenotyper has not been great, you will spend a lot of time fiddling with filters to ensure you're getting things called confidently and the GATK forums are silent on the matter of best practice for Halo data.
I also don't like the inherent variability in coverage of Haloplex data.
SureCall is basically just a wrapper for TMAP/BWA, samtools and their proprietary 'low allele frequency' caller, so BWA/samtools is a good place to start.
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Have you tried contacting Agilent? I have used SureCall and I quite like it. I haven't had any problems using it except I have to manually download the annotation (reference) data it didn't start automatically as the install guide says it will. However when I have had questions Agilent were very helpful.
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Bukowski-
Thanks for your reply. I am pretty sure the download problem is not due to a proxy server in the way. I am on a University network and as far as I know there is not proxy between me and the outside world. Thanks too for your comments about GATK and the suggestion that BWA and samtools.
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HaloPlex and SureCall
As part of the SureCall installation, SureCall downloads ~50 GB of reference genome and annotations from the Amazon cloud (not from the Agilent website). Depending on your internet speed this might take between 30 minutes and a couple of hours. [email protected] can provide further help if needed.
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An update on the installation of SureCall - It is finally working. I have to say the Agilent Tech support guys were very helpful and responsive - even though it did take quite a few hours of webex/telecon to get it working. It seems the file permissions and installation location of the aligners was the problem. Anyway it is working.
A couple of comments re the initial results. In my hands the filtering and trimming within SureCall doesn't work well. For example, with the default settings only ~1400 reads from an initial 680,000 were mapped. However if I look at the quality with FastQC and trim the reads with trimmomatic then SureCall maps ~350,000 (I know I have quite a lot of illumina adapter contamination in the library so was expecting some loss). This seems like a petty good improvement by using a different QC and trimming method.
I am still working on the variant analysis - I do see what appear to be highly significant and detrimental mutations in my tumour derived samples (so was encouraged) but I do also see apparently significant and serious mutations (stop codons etc) in my control DNA. Has any once else seen this sort of result?
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