Haloplex and SureCall

[steveCJ]

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.

[Bukowski]

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.

[AStretton]

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.

[steveCJ]

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.

[steveCJ]

AStretton -
Thanks for the reply. The install guide does sort to allude to manual download when you try to run the program but that does nothing either. I have contracted Agilent (via their website) but I've not had a reply as yet.

[AStretton]

Have you tried contacting your sales rep? Whilst they might not know the answer the should put you in contact with someone who can help. When you run SureCall have you gone in to the admin tab and annotation download and tried from there?

[lnakhwa]

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.

[steveCJ]

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?

[lnakhwa]

Thanks for the update. Am glad that you were able to get it working and that the tech support team was helpful. Regarding variant analysis - Agilent Tech support should be able to figure out what is going on with the variant analysis as well.