alignment of Miseq data

[mathew]

To identify presence and absence of a bacterial in clinical samples as well as sequence variation among various states of diseases if any; we recently generated 2X250bp miseq data. I used BWA it is not working. I was wondering what should be the best way to align to reference genome or any other suggestion how best I can achieve seq variations

Thanks

[GenoMax]

Can you be more specific about the "BWA not working" bit?

Did you have inserts of sufficient length so the reads remained in the "insert" part? With 250 bp it is possible to have overlap between the two reads. See this thread for software that can help you check the overlap: http://seqanswers.com/forums/showthread.php?t=23482

[mathew]

miseq alignments

The genome is ~5K and we used Nextera DNA libraries and then seq with 2X250 bp. It appears that first I need to find out overlap two mates and then align?
BWA I used default option with my custom genome.

[GenoMax]

Since you are working with a small genome you may need to create BWA indexes with "is" option (rather than the "bwasw") as indicated in the bwa help: http://bio-bwa.sourceforge.net/bwa.shtml.

If you use a program like FLASH (to collapse the R1/R2 reads into a single sequence) you could also use BLAT to do the sequence search.