Error in rule AberrantSplicing_pipeline_Counting_03_filter_expression_FraseR_R

[canankolakoglu]

Hello,

Firstly I would like to thank you for this amazing tool.
I've dealt with aberrant splicing analysis using DROP workflow for 203 samples. During the analysis the execution was corrupted, and then rerun with snakemake aberrantSplicing --cores 80 --rerun-incomplete. After counting split and non-split reads with subread and calculating the FRASER metrics, the execution halted with an error in rule AberrantSplicing_pipeline_Counting_03_filter_expression_FraseR_R.
I've sampled this cohort for 10 samples and started over an analysis again. I ended up with the same error.
Could you please help me find where I am doing wrong?
Please find the attached sneakemake.log file and sample_annotation file.

Thanks.

sample_annotation.csv
2023-01-24T180827.985403.snakemake.log

[vyepez88]

Hi, can you also share your config file?

[canankolakoglu]

Hi again,

Thank you for your quick response.
You can see the content of config file below;

projectTitle: "DROP: Detection of RNA Outliers Pipeline"
root: /mnt/NAS_DEPO_1/1501/RNA-Seq/170722_RNAseq/OUTPUTS/splicing/fraser_drop_R7567/Output
htmlOutputPath: /mnt/NAS_DEPO_1/1501/RNA-Seq/170722_RNAseq/OUTPUTS/splicing/fraser_drop_R7567/Output/html
indexWithFolderName: true

hpoFile: null
root: Output
sampleAnnotation: /mnt/NAS_DEPO_1/1501/RNA-Seq/170722_RNAseq/OUTPUTS/splicing/fraser_drop_R7567/sample_annotation.csv
geneAnnotation:
v38: /mnt/NAS_DEPO_1/REF_GENOMES/GRCh38_gencode_v38/gencode.v38.primary_assembly.annotation.gtf
genomeAssembly: hg38
genome:
gencode: /mnt/NAS_DEPO_1/REF_GENOMES/GRCh38_gencode_v38/GRCh38.primary_assembly.genome.fa

exportCounts:
geneAnnotations:
- v38
excludeGroups:

aberrantExpression:
run: false
groups:
- group1
- group2
fpkmCutoff: 1
implementation: autoencoder
padjCutoff: 1
zScoreCutoff: 0
maxTestedDimensionProportion: 3
dassie:
tssWindow: 500
pasWindow: 1000

aberrantSplicing:
run: true
groups:
- fraser
recount: false
longRead: false
keepNonStandardChrs: true
filter: true
minExpressionInOneSample: 20
minDeltaPsi: 0.05
implementation: PCA
padjCutoff: 0
zScoreCutoff: 0
deltaPsiCutoff: 0.05
maxTestedDimensionProportion: 6

mae:
run: false
groups:
- group1
- group2
- group3
gatkIgnoreHeaderCheck: true
padjCutoff: .05
allelicRatioCutoff: 0.8
addAF: false
maxAF: .001
maxVarFreqCohort: .04
# VCF-BAM matching
qcVcf: Data/qc_vcf_1000G.vcf.gz
qcGroups:
- mae

rnaVariantCalling:
run: false
groups:
- batch_0
highQualityVCFs:
dbSNP:
repeat_mask:
createSingleVCF: true
addAF: true
maxAF: .001
maxVarFreqCohort: .1
hcArgs:
minAlt: 3

tools:
gatkCmd: gatk
bcftoolsCmd: bcftools
samtoolsCmd: samtools

[vyepez88]

I think I found the error in the STRAND column. It is currently TRUE/FALSE, but it shouldn't. From the DROP paper:

"STRAND: either yes, no, or reverse: ‘no’ means that the sequencing was not strand specific; ‘yes’ that it was strand specific, and the first read in the pair is on the same strand as the feature and the second read on the opposite strand; and ‘reverse’ that the sequencing is strand specific and the first read in the pair is on the opposite strand to the feature and the second read on the same strand."

[canankolakoglu]

Hello again,

I rearranged the sample_annotation file according to your point and started a new analysis. It ended up with the same error again;

Error in rule AberrantSplicing_pipeline_Counting_03_filter_expression_FraseR_R:
    jobid: 3
    output: Output/processed_data/aberrant_splicing/datasets/savedObjects/fraser/fds-object.RDS, Output/processed_data/aberrant_splicing/datasets/savedObjects/fraser/filter.done
    log: /mnt/NAS_DEPO_1/1501/RNA-Seq/170722_RNAseq/OUTPUTS/splicing/fraser_drop_R7567_new/.drop/tmp/AS/fraser/03_filter.Rds (check log file(s) for error message)

RuleException:
CalledProcessErrorin line 89 of /mnt/NAS_DEPO_1/tmp/tmp8owpzaov:
Command 'set -euo pipefail;  Rscript --vanilla /mnt/NAS_DEPO_1/1501/RNA-Seq/170722_RNAseq/OUTPUTS/splicing/fraser_drop_R7567_new/.snakemake/scripts/tmp_rolako4.03_filter_expression_FraseR.R' returned non-zero exit status 1.
  File "/mnt/NAS_DEPO_1/tmp/tmp8owpzaov", line 89, in __rule_AberrantSplicing_pipeline_Counting_03_filter_expression_FraseR_R
  File "/home/canan/miniconda3/envs/drop_env/lib/python3.10/concurrent/futures/thread.py", line 58, in run
Shutting down, this might take some time.
Exiting because a job execution failed. Look above for error message
Complete log: .snakemake/log/2023-01-27T104100.607576.snakemake.log
 

Analysis files are attached.

sample_annotation.csv

2023-01-27T104100.607576.snakemake.log