Using freebayes for variant calling, separate file for reference genome and expected mutations

Question

[sglass]

Hello,

I am attempting to use freebayes for variant calling. The sequencing data I am going to use freebayes on consists of a yeast sample containing a yeast artificial chromosome. I am only interested in the yeast artificial chromosome, and have little interest in the original yeast chromosomes. The yeast artificial chromosome consist of a bacterial chromosome with portions of a plasmid installed. From previous analysis I know that pieces of the plasmid have been installed into the genome.

My primary objective is to have a sequence for my yeast artificial chromosome, from this I want to be able to know where the genes from the plasmid have inserted into the bacterial chromosome and how the bacterial chromosome differs from my reference.

So far I have aligned my samples to my bacterial chromosome using bwa package. I have also aligned to my yeast chromosomes to check the statistics for how much of my sequencing data has been aligned. Between these two alignments I have 88.24% of the sequence mapped.

Should I include the reference allele options and provide the reference sequence for my plasmid?

Are there additional options I should specify to get a better output to achieve my end goal.

[Pierre Lindenbaum]

see Exome Sequencing: Masking The Non-Genic Sequences ?

The main consequence of removing information is increasing the risk of false alignments