tophat error "does not have a matching mate in the same order"

[bob-loblaw]

Hey guys I'm getting this error with Tophat and I can't seem to figure out why, I'm using paired end reads from Illumina HiSeq. Any tips? I figured it might just be one read thats the problem so I removed it but then I got another one.


[2012-11-21 16:45:33] Beginning TopHat run (v2.0.3)
-----------------------------------------------
[2012-11-21 16:45:33] Checking for Bowtie
Bowtie version: 2.0.0.6
[2012-11-21 16:45:33] Checking for Samtools
Samtools version: 0.1.18.0
[2012-11-21 16:45:33] Checking for Bowtie index files
[2012-11-21 16:45:33] Checking for reference FASTA file
Warning: Could not find FASTA file hg19.fa
[2012-11-21 16:45:33] Reconstituting reference FASTA file from Bowtie index
Executing: /usr/local/bowtie2/bowtie2-inspect hg19 > ./tophat_out/tmp/hg19.fa
[2012-11-21 16:48:12] Generating SAM header for hg19
format: fastq
quality scale: phred64 (reads generated with GA pipeline version >= 1.3)
[2012-11-21 16:49:04] Preparing reads
[FAILED]
Error running 'prep_reads'
Error: read #1 (HWI-ST993:228:C0RWJACXX:6:2316:17166:97507) does not have a matching mate in the same order (HWI-ST993:228:C0RWJACXX:6:2316:12084:97378 found instead)

[lollysticky]

Just removing that one read does not solve your problem
the order of the paired reads is not the same.

When you have two files representing your reads, many alignment programs expect each 'mate' to be found in the exact same order in each file. The error itself says so... As it already fails on the very first read, I expect all your mates to be out of order.


Just write a small script that screens both your files at the same time to detect possible mate-errors. Perhaps think about making new mate files.