Tophat2 error when mapping a sample file to a reference

Question

[debitboro]

Hi Everyone,

Can somebody help me about the following post, I explain;

I'm doing some analysis with my RNA-Seq data (PE from Illumina, 88 files and each file is almost 5 Go). Since I have big files, and any task like mapping to the hg reference or something like this take a lot of time, I have the idea to work with small files instead of the original files, and I just used seqtk to pick randomly a sub-sample of 10000 reads with the same seed from each original file, and thus I have my new small data (convenient way to test my scripts). So, I want to map my first PE reads (R1 and R2) with tophat2, and I got the following error:

...
...
Preparing reads
[FAILED]
Error running 'prep_reads'
Error: qual length (156) differs from seq length (101) for fastq record !
...
...

thanks

[GenoMax]

Was this data trimmed? It sounds like the trimming did not work right and something is out of order or that file may be corrupt.

[debitboro]

Thank you for your response,

Yeah, before doing the sampling with seqtk I used skewer for quality trimming and adapter clipping. I think the problem is skewer, because I tried with only the sampling with seqtk and it is OK.

[debitboro]

Hi all,

As specified in the above post, for the test of my scripts I want to apply that scripts to a small data instead of using a huge data because the mapping take a lot of time, so I used seqtk to pick randomly a given number of reads (for example 10000 reads) from each original file, and thus I construct my new small dataset. However, when I fastqc the obtaining files I got an error which means that my new files are corrupted or something like this. So, I decide to search another method for sampling (picking a number of reads from the original files). Do you have any idea ?

[GenoMax]

First of all seqtk sample should have worked without problems. What command did you run? As given in the example did you used an identical seed to keep pairing of the reads intact?
If you do want to use a different program then you can use reformat.sh from BBMap as described here.