Reliable variant calling

[Mudal]

Hello!

I'm new to PacBio and NGS sequence analysis, so I hope someone here can point me in the right direction.
I have 1 SMRT cell worth of sequencing data (just a pilot for now).

The sample is a mix of libraries (amplicons) of about 800 bp. Each of these libraries is derived from one and the same gene, but they've been through a mutagenic process and should carry some diversity = 1-10 SNPs per molecule, relative to the reference.
We have a non-mutagenized wild-type library in the sample as well.

All of these libraries are barcoded. The amplicon structure is:
Barcode - Amplicon - Universal_primer.

Even though barcoding was not exactly done according to PacBio barcoding recommendations, I feel resolving barcodes will not be a huge problem. However, calling variants might be difficult. Does anyone here have experience with reliably detecting SNPs in samples with similar characteristics?

[phenotype]

Hi Mudal,

Here is a good place to start:

https://github.com/PacificBiosciences/Bioinformatics-Training/wiki/Barcoding

I think you might be able to use SMRT Analysis to do this. If not, the tutorial also walks you through command-line steps. Which ever you choose, you'll still need to download and install SMRT Analysis http://www.pacbiodevnet.com on some sort of linux server (https://github.com/PacificBioscience...-Analysis%3F):

[IonTom]

Maybe try the deepSNV library:

http://bioconductor.org/packages/devel/bioc/html/deepSNV.html

[Mudal]

Maybe try the deepSNV library:
http://bioconductor.org/packages/dev...l/deepSNV.html

Thanks. I aligned my reads to the reference using BLASR from the SMRTAnalysis software. From there, it was easy to demultiplex based on barcodes and get separate BAM files.
I ended up using deepSNV and freebayes for variant calling.