I would check what format your files were in to start with and that you had converted them sanger fastq before running BWA. I've had this problem before and it was because of the format which caused all the quality scores to be too low meaning all bases were called as Ns (I think they had already been converted to fastq sanger and I didn't realise and converted them again). Nicola On 26/08/2011 00:50, Alison Gardner wrote:

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Hello,

I am aligning Illumina paired end sequence using BWA, then run SAM-to-BAM resulting in a 1.1 Gb file.

But when I then generate pileup on this file column 4 (consensus bases) are all N's and the file is now 8.4 Gb.

Can someone tell me what might be going on?

Any help much appreciated.

Thanks

Alison Gardner

Neurogenetics Research Program, SA Pathology (at Women's and Children's Hospital), Level 9, Clarence Rieger Bldg., 72 King William Road, North Adelaide, S.A., Australia, 5006

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