GATK IndelRealigner error

[shocker8786]

Hello,

I'm attempting to use GATK 2.3-9-ge5ebf34 to re-align some RNA-seq datasets. Below is the code I'm using.

java -Xms16g -jar ~/bin/GenomeAnalysisTK-2.3-9-ge5ebf34/GenomeAnalysisTK.jar -T IndelRealigner -R ../../../transcripts/reference_wMT.fa -I fat_no_dup.bam -targetIntervals fat_no_dup.intervals -o fat_nd_realigned.bam

I'm receiving 2 errors that occur at many locations in the output. The first is below:

IndelRealigner - When first element of the alt consensus is M, the second one must be I or D. Actual: 85M208M2D15M.

I see that the issue is there are 2 M fields when it is only expecting 1. But I'm unsure of what this means, or what I can do to fix it. The bam file was aligned using tophat and allowing only 1 alignment per read. Any thoughts?

The second error is:

IndelRealigner - Not attempting realignment in interval 12:26475533-26475760 because there are too many reads.

I know the issue here is there are too many reads at that interval, so I have to increase the -maxReadsForRealignment setting. Does anyone know of a way to determine what the highest coverage is for one of these regions, so I can set the max reads above that level, instead of picking a random number and trying again? Thanks!

[dpryan]

In the first instance, the CIGAR string makes no sense. It should be 293M2D15M. For the second warning (these aren't errors), that's just an informative message to tell you that the coverage in that region is too high. There's no reason to play with -maxReadforRealignment too much, since those regions should just be ignored anyway.