Bowtie2 mapping error

Hi all,

I have 4 ATAC-seq pair-end datasets would like to align with same reference genome (hg38 Canonical) using Bowtie2 under the same condition. Of the 4 datasets, 1 is uploaded to the galaxy library for the purpose of teaching so I have that to be my control dataset. the other 3 are published datasets.

Before mapping, I processed all dataset for pre-trimmed fastqc, cutadapt/ trimmomatic and post-trimmed fastqc and it all went well. However, 2 testing datasets failed to map in Bowtie2 (both gives this error message 'samtools sort: failed to read header from “-” '. the control dataset and the other testing dataset is fine to map.

I couldn’t figure out why but here’s my guess

  1. I realized in the failed datasets, the size of starting materials (ID_1 or ID_2 fastq.gz) are either 1.4GB or 3.6GB, whereas the other 2 datasets fastq.gz are only either 100MB or 15MB. is the size matter ?
  2. while compared the Bowtie2 command lines between failed and passed datasets, this command line “/mnt/pulsar/files/staging/3529357/inputs/dataset_8950718.dat” appeared in the failed set and “/mnt/user-data-4/008/909/dataset_8909088.dat” appeared in passed dataset

Not sure if they are related.

Does any know how to fix it ?


Dear @Frank,
(1) No, the size is irrelevant. Bowtie2 can handle large files.
(2) No, what you have posted are just the different file locations and names, probably.

Your error point more to a malformatted fastq file or wrong setup of Bowtie2.

(A) Your R1 and R2 files are out of sync. Meaning you have applied cutadapt/trimmomatic only to one mate of the read pair.
(B) Check the setting once again of Bowtie2. It might be the case that Bowtie2 tries to map, but the resulting file is empty.

If, you cannot fine the error than maybe share your history.


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Hi @Frank,

do you still have problem with Bowtie2 on Galaxy Australia? There was an issue with a particular destination (one of the pulsar servers) few days ago, but it is resolved now.

You can report errors like this to the Galaxy Australia support team as following: click at any output of a failed job, click at View or report this error icon, the one looking like ladybird bug, add any description in the middle window (optional) and hit Report. Do not delete outputs of the failed job when you report the error.

Kind regards,

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