Description of the bug
Hi
I have hit an error trying to run one of the test datasets that merges multiple fastq files per sample (samplesheet.multi_lanes.csv).
I have to say first that when running with the testing dataset that includes only the chr17.fa the pipeline runs great. When i run it with my local version of the hg38 igenomes fasta however it hits an error at the GroupReadsByUmi step. The file should be accessible by downloading the hg38 version in this link - here.
I download the sample sheet from the test-dataset repo and I run the pipeline using:
nextflow run nf-core/fastquorum -r 1.0.0 -params-file params.yaml -w work.nobackup -resume
And the params.yaml file is:
input: ./samplesheet.multi_lanes.csv
outdir: ./results.nobackup
duplex_seq: yes
fasta: <<path to>>/iGenomes/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/genome.fa
call_min_reads: 6 3
filter_min_reads: 3 3
The error is in the GroupReadsByUmi step, it claims that a given read is not sorted correctly (SRR6109255.382418).
Command error:
INFO: Environment variable SINGULARITYENV_TMP is set, but APPTAINERENV_TMP is preferred
INFO: Environment variable SINGULARITYENV_TMPDIR is set, but APPTAINERENV_TMPDIR is preferred
INFO: Environment variable SINGULARITYENV_NXF_TASK_WORKDIR is set, but APPTAINERENV_NXF_TASK_WORKDIR is preferred
INFO: Environment variable SINGULARITYENV_NXF_DEBUG is set, but APPTAINERENV_NXF_DEBUG is preferred
[2024/05/27 03:00:20 | FgBioMain | Info] Executing GroupReadsByUmi from fgbio version 2.0.2 as <<USER>>@<<machine>> on JRE 11.0.9.1-internal+0-adhoc..src with snappy, IntelInflater, and IntelDeflater
[2024/05/27 03:00:20 | GroupReadsByUmi | Info] Filtering the input.
[2024/05/27 03:00:20 | GroupReadsByUmi | Info] Assigning reads to UMIs and outputting.
[2024/05/27 03:00:20 | FgBioMain | Fatal] ########################################################################################
[2024/05/27 03:00:20 | FgBioMain | Fatal] Execution failed!
[2024/05/27 03:00:20 | FgBioMain | Fatal] Template SRR6109255.382418 has only one read, paired-reads required for paired strategy.
[2024/05/27 03:00:20 | FgBioMain | Fatal] ########################################################################################
[2024/05/27 03:00:20 | FgBioMain | Info] GroupReadsByUmi failed. Elapsed time: 0.04 minutes.
When going into the directory I can see that the problematic read (the first read in the file) is actually properly paired but and not in order?
> samtools view SRR6109255_three_lanes.bam | cut -f 1,2,3,4 | head
SRR6109255.382418 99 chr1 10466962
SRR6109255.143407 163 chr1 1913705
SRR6109255.143407 83 chr1 1913705
SRR6109255.441042 99 chr1 18777854
SRR6109255.382418 147 chr1 10467504
SRR6109255.441042 147 chr1 18777853
SRR6109255.58832 163 chr1 26281528
SRR6109255.58832 83 chr1 26281528
When I track down the merged bam one level more I reach the MERGE_BAM process. The individual bams seem to be all sorted correctly but the merged one (see above) may not be?
> samtools view 1/SRR6109255_three_lanes.mapped.bam | cut -f 1,2,3,4 | head
SRR6109255.382418 99 chr1 10466962
SRR6109255.382418 147 chr1 10467504
SRR6109255.145917 99 chr1 28242786
SRR6109255.145917 147 chr1 28243430
> samtools view 2/SRR6109255_three_lanes.mapped.bam | cut -f 1,2,3,4 | head
SRR6109255.143407 163 chr1 1913705
SRR6109255.143407 83 chr1 1913705
SRR6109255.764263 113 chr1 30571796
SRR6109255.764263 177 chr1 197441624
> samtools view 3/SRR6109255_three_lanes.mapped.bam | cut -f 1,2,3,4 | head
SRR6109255.441042 99 chr1 18777854
SRR6109255.441042 147 chr1 18777853
SRR6109255.58832 163 chr1 26281528
SRR6109255.58832 83 chr1 26281528
I am not sure if I am doing smth wrong.
Command used and terminal output
nextflow run nf-core/fastquorum -r 1.0.0 -params-file params.yaml -w work.nobackup -resume
(information in the description)
Relevant files
No response
System information
HPC, running on SGE
Ubuntu Linux
N E X T F L O W
version 24.04.1 build 5913
created 20-05-2024 09:48 UTC (02:48 PDT)
cite doi:10.1038/nbt.3820
http://nextflow.io
nf-core/fastquorum = 1.0.0
Description of the bug
Hi
I have hit an error trying to run one of the test datasets that merges multiple fastq files per sample (
samplesheet.multi_lanes.csv).I have to say first that when running with the testing dataset that includes only the
chr17.fathe pipeline runs great. When i run it with my local version of thehg38igenomes fasta however it hits an error at the GroupReadsByUmi step. The file should be accessible by downloading the hg38 version in this link - here.I download the sample sheet from the test-dataset repo and I run the pipeline using:
And the
params.yamlfile is:The error is in the
GroupReadsByUmistep, it claims that a given read is not sorted correctly (SRR6109255.382418).When going into the directory I can see that the problematic read (the first read in the file) is actually properly paired but and not in order?
When I track down the merged bam one level more I reach the
MERGE_BAMprocess. The individual bams seem to be all sorted correctly but the merged one (see above) may not be?I am not sure if I am doing smth wrong.
Command used and terminal output
Relevant files
No response
System information
HPC, running on SGE
Ubuntu Linux
N E X T F L O W
version 24.04.1 build 5913
created 20-05-2024 09:48 UTC (02:48 PDT)
cite doi:10.1038/nbt.3820
http://nextflow.io
nf-core/fastquorum = 1.0.0