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nf-core/rnaseq: Troubleshooting

Input files not found

If only no file, only one input file , or only read one and not read two is picked up then something is wrong with your input file declaration

  1. The path must be enclosed in quotes (' or ")
  2. The path must have at least one * wildcard character. This is even if you are only running one paired end sample.
  3. When using the pipeline with paired end data, the path must use {1,2} or {R1,R2} notation to specify read pairs.
  4. If you are running Single end data make sure to specify --singleEnd

If the pipeline can't find your files then you will get the following error

ERROR ~ Cannot find any reads matching: *{1,2}.fastq.gz

Note that if your sample name is "messy" then you have to be very particular with your glob specification. A file name like L1-1-D-2h_S1_L002_R1_001.fastq.gz can be difficult enough for a human to read. Specifying *{1,2}*.gz wont work give you what you want Whilst *{R1,R2}*.gz will.

Some samples are missing

If the pipeline ran without crashing and some samples are missing then there are two possible explanations:

  1. You made a mistake when declaring the input files - in that case see above.
  2. Your sample didn't reach 5 % read alignment.

Samples that receive less than 5% alignment are skipped for further analysis. You will thus see STAR/Hisat2 output for such samples but nothing more. This limit was set for two reasons, one it doesn't really make any sense to waste CPU hours running on data that bad. Secondly, and most importantly we found that some of the downstream processes would occasionally crash if we allowed it.

Data organization

The pipeline can't take a list of multiple input files - it takes a glob expression. If your input files are scattered in different paths then we recommend that you generate a directory with symlinked files. If running in paired end mode please make sure that your files are sensibly named so that they can be properly paired. See the previous point.

Extra resources and getting help

If you still have an issue with running the pipeline then feel free to contact us. Have a look at the pipeline website to find out how.

If you have problems that are related to Nextflow and not our pipeline then check out the Nextflow gitter channel or the google group.