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samplesheet with 3 columns #251

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rhodesch opened this issue Jul 17, 2023 · 1 comment
Closed

samplesheet with 3 columns #251

rhodesch opened this issue Jul 17, 2023 · 1 comment
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bug Something isn't working

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@rhodesch
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rhodesch commented Jul 17, 2023 •
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Description of the bug

The INPUT_CHECK:SAMPLESHEET_CHECK appears to be failing when samplesheet has 3 columns on an HPC when -profile singularity is specified. When I test on my local machine with -profile docker or on an HPC with -profile test,singularity, it reads the samplesheet.csv properly with only 3 required columns (name, fast1, fastq2) - see "testing" sections below.

Command used and terminal output

nextflow run nf-core/scrnaseq -r 2.3.2 \ # -r 2.3.2 or -r dev raise identical errors
   -profile singularity \
   --input data/samplesheet.csv \
   --fasta data/genome.fa \
   --gtf data/genes.gtf \
   --protocol 10XV3 \
   --aligner cellranger \
   --cellranger_index data/refdata-gex-GRCh38-2020-A \
   --outdir ~/work

samplesheet 1

sample,fastq_1,fastq_2
test_gex,test_gex_S1_L001_R1_001.fastq.gz,test_gex_S1_L001_R2_001.fastq.gz

error for samplesheet 1

[49/2e17b8] process > NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet.csv) [100%] 1 of 1, failed: 1 ✘
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC                             -
[46/ccff72] process > NFCORE_SCRNASEQ:SCRNASEQ:GTF_GENE_FILTER (genome.fa)                     [100%] 1 of 1 ✔
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_COUNT               -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_H5AD                      -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:CONCAT_H5AD                      -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_SEURAT                    -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:CUSTOM_DUMPSOFTWAREVERSIONS                     -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MULTIQC                                         [  0%] 0 of 1
Execution cancelled -- Finishing pending tasks before exit

ERROR ~ Error executing process > 'NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet.csv)'

Command output:
  ERROR: Please check samplesheet -> Invalid number of columns (minimum = 3)!
  Line: ''

Command error:
  WARNING: Skipping mount /cm/local/apps/singularitypro/3.9/var/singularity/mnt/session/etc/resolv.conf [files]: /etc/resolv.conf doesn't exist in container
  ERROR: Please check samplesheet -> Invalid number of columns (minimum = 3)!
  Line: ''

samplesheet 2:

sample,fastq_1,fastq_2,expected_cells,seq_center
test_gex,/expanse/lustre/projects/test_gex_S1_L001_R1_001.fastq.gz,/expanse/lustre/projects/test_gex_S1_L001_R2_001.fastq.gz,,test_center

error for samplesheet 2:

ERROR ~ Error executing process > 'NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet.csv)'

Command output:
  ERROR: Please check samplesheet -> Expected cells must be an integer
  Line: 'test_gex,/expanse/lustre/projects/test_gex_S1_L001_R1_001.fastq.gz,/expanse/lustre/projects/test_gex_S1_L001_R2_001.fastq.gz,,test_center

Command error:
  WARNING: Skipping mount /cm/local/apps/singularitypro/3.9/var/singularity/mnt/session/etc/resolv.conf [files]: /etc/resolv.conf doesn't exist in container
  ERROR: Please check samplesheet -> Expected cells must be an integer
  Line: 'test_gex,/expanse/lustre/projects/test_gex_S1_L001_R1_001.fastq.gz,/expanse/lustre/projects/test_gex_S1_L001_R2_001.fastq.gz,,test_center

testing with -profile test

nextflow run nf-core/scrnaseq -r dev -profile test,singularity --outdir ~/work

Reads test samplesheet successfully

executor >  local (2)
executor >  local (2)
[13/66700c] process > NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet-2-0.csv) [100%] 1 of 1 ✔
executor >  local (3)
[13/66700c] process > NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet-2-0.csv)      [100%] 1 of 1 ✔
executor >  local (3)
[13/66700c] process > NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet-2-0.csv)      [100%] 1 of 1 ✔
executor >  local (3)
[13/66700c] process > NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet-2-0.csv)      [100%] 1 of 1 ✔
executor >  local (4)
[13/66700c] process > NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet-2-0.csv)      [100%] 1 of 1 ✔
executor >  local (5)
[13/66700c] process > NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet-2-0.csv)      [100%] 1 of 1 ✔
executor >  local (5)
[13/66700c] process > NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet-2-0.csv)      [100%] 1 of 1 ✔
executor >  local (5)
[13/66700c] process > NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet-2-0.csv)      [100%] 1 of 1 ✔
executor >  local (5)
executor >  local (6)
executor >  local (7)
executor >  local (7)
executor >  local (7)
executor >  local (8)
executor >  local (8)
executor >  local (9)
executor >  local (9)
executor >  local (9)
executor >  local (9)
executor >  local (9)
executor >  local (9)
executor >  local (9)
[13/66700c] process > NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet-2-0.csv)      [100%] 1 of 1 ✔
[94/0ef6ec] process > NFCORE_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC (Sample_Y)                           [100%] 2 of 2 ✔
[cb/544432] process > NFCORE_SCRNASEQ:SCRNASEQ:GTF_GENE_FILTER (GRCm38.p6.genome.chr19.fa)              [100%] 1 of 1 ✔
[c1/412784] process > NFCORE_SCRNASEQ:SCRNASEQ:STARSOLO:STAR_GENOMEGENERATE (GRCm38.p6.genome.chr19.fa) [100%] 1 of 1 ✔
[8d/e4cce9] process > NFCORE_SCRNASEQ:SCRNASEQ:STARSOLO:STAR_ALIGN (Sample_Y)                           [100%] 2 of 2 ✔
[1c/db87f2] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_H5AD (Sample_X)                    [100%] 1 of 1
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:CONCAT_H5AD                               -
[26/f8c154] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_SEURAT (Sample_X)                  [100%] 1 of 1, failed: 1
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:CUSTOM_DUMPSOFTWAREVERSIONS                              -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MULTIQC                                                  [  0%] 0 of 1
Pulling Singularity image docker://quay.io/nf-core/seurat:4.3.0 [cache /expanse/lustre/projects/jan109/crhodes4/quay.io-nf-core-seurat-4.3.0.img]
Pulling Singularity image https://depot.galaxyproject.org/singularity/scanpy:1.7.2--pyhdfd78af_0 [cache /expanse/lustre/projects/jan109/crhodes4/depot.galaxyproject.org-singularity-scanpy-1.7.2--pyhdfd78af_0.img]
Execution cancelled -- Finishing pending tasks before exit


ERROR ~ Error executing process > 'NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_SEURAT (Sample_X)'

Caused by:
  Process `NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_SEURAT (Sample_X)` terminated with an error exit status (1)

Command executed:

  mtx_to_seurat.R \
      *.Solo.out/Gene*/filtered/matrix.mtx.gz \
      *.Solo.out/Gene*/filtered/barcodes.tsv.gz \
      *.Solo.out/Gene*/filtered/features.tsv.gz \
      Sample_X/Sample_X_matrix.rds \
      star

Command exit status:
  1

Command output:
  (empty)

Command error:
  Error: package or namespace load failed for ‘Seurat’ in loadNamespace(j <- i[[1L]], c(lib.loc, .libPaths()), versionCheck = vI[[j]]):
   there is no package called ‘future’
  Execution halted

testing on local machine with -profile docker

nextflow run nf-core/scrnaseq -r 2.3.2 -params-file nf-params.json -profile docker

sample,fastq_1,fastq_2
test_gex,test_gex_S1_L001_R1_001.fastq.gz,test_gex_S1_L001_R2_001.fastq.gz

executor >  local (3)
[30/fcc82f] process > NFCORE_SCRNASEQ:SCRNASEQ:INPUT_CHECK:SAMPLESHEET_CHECK (samplesheet.csv) [100%] 1 of 1 ✔
[68/94d3c9] process > NFCORE_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC (test_gex)                  [  0%] 0 of 1
[e9/076cdf] process > NFCORE_SCRNASEQ:SCRNASEQ:GTF_GENE_FILTER (genome.fa)                     [100%] 1 of 1 ✔
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_COUNT               [  0%] 0 of 1
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_H5AD                      -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:CONCAT_H5AD                      -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_SEURAT                    -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:CUSTOM_DUMPSOFTWAREVERSIONS                     -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MULTIQC                                         -

Relevant files

Can't seem to recreate log currently. When running nextflow command above to generate log file, for -r dev getting this:

Project `nf-core/scrnaseq` contains uncommitted changes -- Cannot switch to revision: dev

Happy to provide log files or anything else if needed.

System information

Nextflow version: 23.04.1
Hardware: HPC
Executor: local
Container engine: Singularity
OS: Rocky Linux 8.7 (Green Obsidian) / ID_LIKE="rhel centos fedora"
Version of nf-core/scrnaseq: 2.3.2 and dev
@rhodesch rhodesch added the bug Something isn't working label Jul 17, 2023
@rhodesch
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Updating for future reference. The following worked: nextflow run nf-core/scrnaseq -r dev as long as there is no empty trailing line at the end of samplesheet.csv. The error was due to the final empty line.

Works:

sample,fastq_1,fastq_2
test_gex,test_gex_S1_L001_R1_001.fastq.gz,test_gex_S1_L001_R2_001.fastq.gz

Error:

sample,fastq_1,fastq_2
test_gex,test_gex_S1_L001_R1_001.fastq.gz,test_gex_S1_L001_R2_001.fastq.gz

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