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Hi All, I'm having an issue running the chipseq pi...
# chipseq
m
Hi All, I'm having an issue running the chipseq pipeline for the first time. I've successfully run the cutandrun and rnaseq pipelines. Here's the submit script:
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#! /bin/bash
#SBATCH --job-name=nextflow-main
#SBATCH --cpus-per-task=4
#SBATCH --mem=32G
#SBATCH --gres=lscratch:200
#SBATCH --time=72:00:00

module load nextflow
export NXF_SINGULARITY_CACHEDIR=/data/$USER/nxf_singularity_cache;
export SINGULARITY_CACHEDIR=/data/$USER/.singularity;
export TMPDIR=/lscratch/$SLURM_JOB_ID
export NXF_JVM_ARGS="-Xms2g -Xmx16g"

nextflow run nf-core/chipseq -r 2.0.0 \
-profile biowulf \
--input samplesheet.csv \
--outdir output \
--gtf /data/brooksma/Index/Human/ENS/v111/Homo_sapiens.GRCh38.111.gtf.gz \
--fasta /data/brooksma/Index/Human/ENS/v111/Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa.gz \
--igenomes_ignore --genome null \
--read_length 100
Here's the error:
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Error executing process > 'NFCORE_CHIPSEQ:CHIPSEQ:FILTER_BAM_BAMTOOLS:BAM_REMOVE_ORPHANS (B4_9_H3K4me3)'

Caused by:
  Process `NFCORE_CHIPSEQ:CHIPSEQ:FILTER_BAM_BAMTOOLS:BAM_REMOVE_ORPHANS (B4_9_H3K4me3)` terminated with an error exit status (1)

Command executed:

  samtools sort -n -@ 6 -o B4_9_H3K4me3.mLb.clN.name.sorted.bam -T B4_9_H3K4me3.mLb.clN.name.sorted B4_9_H3K4me3.mLb.flT.sorted.bam
  bampe_rm_orphan.py B4_9_H3K4me3.mLb.clN.name.sorted.bam B4_9_H3K4me3.mLb.clN.bam --only_fr_pairs
  
  cat <<-END_VERSIONS > versions.yml
  "NFCORE_CHIPSEQ:CHIPSEQ:FILTER_BAM_BAMTOOLS:BAM_REMOVE_ORPHANS":
      samtools: $(echo $(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*$//')
  END_VERSIONS

Command exit status:
  1

Command output:
  (empty)

Command error:
  [E::idx_find_and_load] Could not retrieve index file for 'B4_9_H3K4me3.mLb.clN.name.sorted.bam'
  Traceback (most recent call last):
    File "/home/brooksma/.nextflow/23.10.0/assets/nf-core/chipseq/bin/bampe_rm_orphan.py", line 167, in 
      bampe_rm_orphan(BAMIn=args.BAM_INPUT_FILE, BAMOut=args.BAM_OUTPUT_FILE, onlyFRPairs=args.ONLY_FR_PAIRS)
    File "/home/brooksma/.nextflow/23.10.0/assets/nf-core/chipseq/bin/bampe_rm_orphan.py", line 82, in bampe_rm_orphan
      currRead = next(iter)
    File "pysam/libcalignmentfile.pyx", line 2209, in pysam.libcalignmentfile.IteratorRowAll.__next__
  StopIteration
However, the B4_9_H3K4me3.mLb.clN.name.sorted.bam file in the above error is empty. All the bam files are empty. Tracing back further in the pipeline, I found the trimmed fastq files from the output of NFCORE_CHIPSEQCHIPSEQFASTQC_TRIMGALORE:TRIMGALORE are all empty. The fastqc output from NFCORE_CHIPSEQCHIPSEQFASTQC_TRIMGALORE:FASTQC is fine. Here is the trimming report:
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SUMMARISING RUN PARAMETERS
==========================
Input filename: B4_9_H3K4me3_T1_1.fastq.gz
Trimming mode: paired-end
Trim Galore version: 0.6.7
Cutadapt version: 3.4
Python version: could not detect
Number of cores used for trimming: 4
Quality Phred score cutoff: 20
Quality encoding type selected: ASCII+33
Using Illumina adapter for trimming (count: 76557). Second best hit was Nextera (count: 7)
Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected)
Maximum trimming error rate: 0.1 (default)
Minimum required adapter overlap (stringency): 1 bp
Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp
Running FastQC on the data once trimming has completed
Output file will be GZIP compressed


This is cutadapt 3.4 with Python 3.9.6
Command line parameters: -j 4 -e 0.1 -q 20 -O 1 -a AGATCGGAAGAGC B4_9_H3K4me3_T1_1.fastq.gz
Processing reads on 4 cores in single-end mode ...
Finished in 134.57 s (6 µs/read; 9.29 M reads/minute).

=== Summary ===

Total reads processed:              20,825,534
Reads with adapters:                 8,978,111 (43.1%)
Reads written (passing filters):    20,825,534 (100.0%)

Total basepairs processed: 2,103,378,934 bp
Quality-trimmed:               3,217,395 bp (0.2%)
Total written (filtered):  2,033,313,363 bp (96.7%)
Yet, the B4_9_H3K4me3_T1_1_val_1.fq.gz file is empty. Any help would be greatly appreciated!
l
Hi, I'm running chipseq on some samples and have encountered the exact same issue. same error message and all. I've checked the files and no index appears. Was wondering what is the solution for this
Hi, so I was able to solve the issue by using an older reference (initially was using GRCm39 to mm10 and got through the BAM_REMOVE_ORPHANS portion. but now i'm having errors with computeMatix in the chipseq pipeline.
Caused by: Process 
NFCORE_CHIPSEQ:CHIPSEQ:DEEPTOOLS_COMPUTEMATRIX (Pol_J_T_REP1)
terminated with an error exit status (1) Command executed: computeMatrix \ scale-regions --regionBodyLength 1000 --beforeRegionStartLength 3000 --afterRegionStartLength 3000 --missingDataAsZero --skipZeros --smartLabels \ --regionsFileName genes.bed \ --scoreFileName Pol_J_T_REP1.mLb.clN.bigWig \ --outFileName Pol_J_T_REP1.mLb.clN.computeMatrix.mat.gz \ --outFileNameMatrix Pol_J_T_REP1.mLb.clN.computeMatrix.vals.mat.tab \ --numberOfProcessors 32 cat <-END_VERSIONS versions.yml "NFCORE_CHIPSEQCHIPSEQDEEPTOOLS_COMPUTEMATRIX": deeptools: $(computeMatrix --version | sed -e "s/computeMatrix //g") END_VERSIONS Command exit status: 1 Command output: (empty) Command error: multiprocessing.pool.RemoteTraceback: """ Traceback (most recent call last): File "/home/ubuntu/mount_1/work/conda/env-a3c287b5939a5abb-a99740b96a460a4d021aea67a9fa0fe9/lib/python3.12/multiprocessing/pool.py", line 125, in worker result = (True, func(*args, **kwds)) ^^^^^^^^^^^^^^^^^^^ File "/home/ubuntu/mount_1/work/conda/env-a3c287b5939a5abb-a99740b96a460a4d021aea67a9fa0fe9/lib/python3.12/multiprocessing/pool.py", line 48, in mapstar return list(map(*args)) ^^^^^^^^^^^^^^^^ File "/home/ubuntu/mount_1/work/conda/env-a3c287b5939a5abb-a99740b96a460a4d021aea67a9fa0fe9/lib/python3.12/site-packages/deeptools/heatmapper.py", line 174, in compute_s> return heatmapper.compute_sub_matrix_worker(*args) ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ File "/home/ubuntu/mount_1/work/conda/env-a3c287b5939a5abb-a99740b96a460a4d021aea67a9fa0fe9/lib/python3.12/site-packages/deeptools/heatmapper.py", line 379, in compute_s> sub_matrix[:] = np.NAN ^^^^^^ File "/home/ubuntu/mount_1/work/conda/env-a3c287b5939a5abb-a99740b96a460a4d021aea67a9fa0fe9/lib/python3.12/site-packages/numpy/__init__.py", line 414, in getattr raise AttributeError("module {!r} has no attribute " AttributeError: module 'numpy' has no attribute 'NAN' """ The above exception was the direct cause of the following exception: Traceback (most recent call last): File "/home/ubuntu/mount_1/work/conda/env-a3c287b5939a5abb-a99740b96a460a4d021aea67a9fa0fe9/bin/computeMatrix", line 10, in module sys.exit(main()) ^^^^^^ File "/home/ubuntu/mount_1/work/conda/env-a3c287b5939a5abb-a99740b96a460a4d021aea67a9fa0fe9/lib/python3.12/site-packages/deeptools/computeMatrix.py", line 398, in main hm.computeMatrix(scores_file_list, args.regionsFileName, parameters, blackListFileName=args.blackListFileName, verbose=args.verbose, allArgs=args) File "/home/ubuntu/mount_1/work/conda/env-a3c287b5939a5abb-a99740b96a460a4d021aea67a9fa0fe9/lib/python3.12/site-packages/deeptools/heatmapper.py", line 252, in computeMa> res, labels = mapReduce.mapReduce([score_file_list, parameters], ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ File "/home/ubuntu/mount_1/work/conda/env-a3c287b5939a5abb-a99740b96a460a4d021aea67a9fa0fe9/lib/python3.12/site-packages/deeptools/mapReduce.py", line 142, in mapReduce res = pool.map_async(func, TASKS).get(9999999) ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ File "/home/ubuntu/mount_1/work/conda/env-a3c287b5939a5abb-a99740b96a460a4d021aea67a9fa0fe9/lib/python3.12/multiprocessing/pool.py", line 774, in get raise self._value AttributeError: module 'numpy' has no attribute 'NAN' Work dir: /home/ubuntu/mount_1/work/a7/48ad9ca118f1de19a81a73c728037c
I think it may have to do with numpy but what needs to be changed to get computematrix to work
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