STAR crashing due to "FATAL ERROR: Read1 and Read2 are not consistent"

Question

[satshil.r]

Hello,

I'm trying to use STAR as my aligner, but I get this error:

EXITING because of FATAL ERROR: Read1 and Read2 are not consistent, reached the end of the one before the other one
SOLUTION: Check you your input files: they may be corrupted

I checked the logs, and it's my last mate pair, L004, which is causing this issue.

I did a wc -l on the L004 reads, but they both have the same number of lines. I'm not sure why this error is occurring. Can someone point me in the right direction? This is happening for 8 of my samples. All 8 samples, L004 is behaving the same way.

Thank you

[Ashutosh Pandey]

Use a trimmer like Trimmomatic that takes care of the orphan reads. If you don't want to repeat the trimming analysis check out this post Combining The Paired Reads From Illumina Run. I would still suggest you to start with the original fastq files and process it through Trimmomatic.

[satshil.r]

I've already trimmed the data and removed all bad-quality reads as well.

Answer 1

[michael.ante]

Hi satshil,

If the line numbers are identical, the ordering might be different. Compare the read-ids line-by line (e.g. use awk to extract the IDs into two files and compare them with diff).

It also might be the case, that the reads from lane 4 are corrupt, check them with e.g the FastQValidator.

You can also use FastQC to have a look at the R1 and R2 reads from the different lanes and compare them to those from lane 4.

Cheers,
Michael

[satshil.r]

Just checked all 4 lanes through FastQValidator. Awesome tool, thank you.

Fortunately L004 is not corrupt. both reads return

fastQValidator --file L004_R2.fq
Finished processing L004_R2.fq with 25780124 lines containing 6445031 sequences.
There were a total of 0 errors.
Returning: 0 : FASTQ_SUCCESS

Same number of lines, same number of sequences... Anything else might be a reason?

I also extracted the IDs to two files and ran diff, and it found no differences between the two files, both are identical.

Answer 2

[satshil.r]

I just ran L004 by it's self, and it ran perfectly. Ran it again with all 4 reads in one and it crashed with the same error.

Guess I'll just do them individual and merge the sam files together after then... Such a weird error.

[michael.ante]

Have you tried to merge the Fastq-files? In case of junction-detection it might be better to have more reads.
You should be aware of the same input order.