alignment of Miseq data

mathew · 01-04-2013, 08:32 AM

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 · 01-04-2013, 09:23 AM

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 · 01-04-2013, 01:37 PM

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 · 01-07-2013, 04:32 AM

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.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
250bp paired end Miseq reads alignment	empyrean	Bioinformatics	3	12-12-2012 01:36 PM
noob questions regarding alignment of MiSeq data	Azazel	Bioinformatics	2	11-27-2012 12:35 PM
Who uses a MiSeq for 16S data?	capsicum	Metagenomics	0	11-21-2012 02:10 PM
De-Multiplexing MiSeq Data	abh	RNA Sequencing	2	11-09-2012 05:26 AM
Help with De-Multiplexing MiSeq Data	Cirno	Bioinformatics	8	08-16-2012 02:51 PM

01-04-2013, 08:32 AM	#1
mathew Member Location: australia Join Date: Jan 2011 Posts: 81	alignment of Miseq data 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 Last edited by mathew; 01-04-2013 at 08:43 AM.

01-04-2013, 01:37 PM	#3
mathew Member Location: australia Join Date: Jan 2011 Posts: 81	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.

01-04-2013, 09:23 AM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,089	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

01-07-2013, 04:32 AM	#4
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,089	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.