MiSeq Analysis Steps to Examine Recombination

Question

[Sara S.]

Hello,

I have paired end MiSeq data (2x250) from several evolved bacteriophages. I'm looking to align them to 2 different reference genomes (Lambda and Phi80) in order to see which genes in the evolved phages are from which ancestral phage- looking at the recombination between the 2 phages. I've figured out how to QC and trim the .fastq files and used Bowtie2 (in Galaxy) to align them to each reference genome separately. I'm not sure what I should be doing next though. I think I need to process the BAM alignment files now? And look at the summary stats, but I'm not sure what constitutes a good alignment/how to tell how much mapped correctly. I think I also need to remove duplicates, correct? But after this I'm not sure what I should do in order to visualize or determine what aligned to each of the 2 reference genomes. Any suggestions would be greatly appreciated! Thank you.

Answer 1

[Asaf]

I think that you can use tophat-fusion to find your recombination events. You should construct a new genome composed of the two phages' genomes and look for fusions between them.

I think you should remove PCR duplicates unless you have a reason to believe that the start and end points of two independent fragments could be equal with high probability (fragmentation bias, no fragmentation at all etc., since it's a whole genome I can't see why it should apply but you know the protocol)