https://github.com/ekg/freebayes

Calling variants: from fastq to VCF

You've sequenced some samples. You have a reference genome or assembled set of contigs, and you'd like to determine reference-relative variants in your samples. You can use freebayes to detect the variants, following these steps:

• Align your reads to a suitable reference (e.g. with bwa or MOSAIK)
• Ensure your alignments have read groups attached so their sample may be identified by freebayes. Aligners allow you to do this, but you can also use bamaddrg to do so post-alignment.
• Sort the alignments (e.g. bamtools sort).
• Mark duplicates, for instance with samtools rmdup (if PCR was used in the preparation of your sequencing library)
• Run freebayes on all your alignment data simultaneously, generating a VCF. The default settings should work for most use cases, but if your samples are not diploid, set the --ploidy and adjust the --min-alternate-fraction suitably.
• Filter the output e.g. using reported QUAL and/or depth (DP) or observation count (AO).
• Interpret your results.
• (possibly, Iterate the variant detection process in response to insight gained from your interpretation)

More steps in the above link.

http://www.htslib.org/workflow/