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Hoohm / dropSeqPipe

A SingleCell RNASeq pre-processing snakemake workflow
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Problem running the new version #99

Open manarai opened 4 years ago

manarai commented 4 years ago

Hi,

This used to work for me with the older version of dropseqpipe but I updated it and am having so issues 30% half way through. Not sure if I am even setting this up correctly. I am trying to run a mix sample of human and mouse from Nadia. Any help would be great!

Thanks for this great package.

[Wed Dec 25 21:57:12 JST 2019] org.broadinstitute.dropseqrna.barnyard.DigitalExpression done. Elapsed time: 0.00 minutes. Runtime.totalMemory()=2058354688 [Wed Dec 25 21:57:12 2019] Error in rule extract_umi_expression_species: jobid: 52 output: /home/user/Nadia_projects/Nadia_analysis/5_mix_mouse_human_miseq/results/samples/N702_S2_L001/homo_sapiens/umi/expression.txt, /home/user/Nadia_projects/Nadia_analysis/5_mix_mouse_human_miseq/results/samples/N702_S2_L001/homo_sapiens/umi/expression.long conda-env: /home/user/Nadia_projects/Nadia_analysis/dropSeqPipe_2/.snakemake/conda/e90f4a5b shell: export _JAVA_OPTIONS=-Djava.io.tmpdir=/home/user/Nadia_projects/Nadia_analysis/dropSeqPipe/tmpdir && DigitalExpression -m 20g I=/home/user/Nadia_projects/Nadia_analysis/5_mix_mouse_human_miseq/results/samples/N702_S2_L001/homo_sapiens/unfiltered.bam O=/home/user/Nadia_projects/Nadia_analysis/5_mix_mouse_human_miseq/results/samples/N702_S2_L001/homo_sapiens/umi/expression.txt EDIT_DISTANCE=1 OUTPUT_LONG_FORMAT=/home/user/Nadia_projects/Nadia_analysis/5_mix_mouse_human_miseq/results/samples/N702_S2_L001/homo_sapiens/umi/expression.long STRAND_STRATEGY=SENSE OUTPUT_READS_INSTEAD=false LOCUS_FUNCTION_LIST={CODING,UTR} MIN_BC_READ_THRESHOLD=0 CELL_BC_FILE=/home/user/Nadia_projects/Nadia_analysis/5_mix_mouse_human_miseq/results/samples/N702_S2_L001/homo_sapiens/barcodes.csv (exited with non-zero exit code)

Shutting down, this might take some time. Exiting because a job execution failed. Look above for error message Complete log: /home/user/Nadia_projects/Nadia_analysis/dropSeqPipe_2/.snakemake/log/2019-12-25T215241.015436.snakemake.log

config.yaml

CONTACT: email: ' person: '' LOCAL: temp-directory: /home/user/Nadia_projects/Nadia_analysis/dropSeqPipe/tmpdir memory: 20g raw_data: /home/tuserNadia_projects/Nadia_analysis/5_mix_mouse_human_miseq/fastq_files/ results: /home/user/Nadia_projects/Nadia_analysis/5_mix_mouse_human_miseq/results META: species: mus_musculus: build: 38 release: 94 homo_sapiens: build: 38 release: 91 ratio: 0.2 reference-directory: /home/user/Nadia_projects/Nadia_analysis/reference gtf_biotypes: /home/user/Nadia_projects/Nadia_analysis/5_mix_mouse_human_miseq/gtf_biotypes.yaml

FILTER: barcode-whitelist: '' 5-prime-smart-adapter: AAAAAAAAAAA cell-barcode: start: 1 end: 12 UMI-barcode: start: 13 end: 25 cutadapt: adapters-file: /home/user/Nadia_projects/Nadia_analysis/5_mix_mouse_human_miseq/NexteraPE-PE.fa R1: quality-filter: 20 maximum-Ns: 1 extra-params: '' R2: quality-filter: 20 minimum-adapters-overlap: 6 minimum-length: 15 extra-params: '' MAPPING: STAR: genomeChrBinNbits: 18 outFilterMismatchNmax: 10 outFilterMismatchNoverLmax: 0.3 outFilterMismatchNoverReadLmax: 1 outFilterMatchNmin: 0 outFilterMatchNminOverLread: 0.66 outFilterScoreMinOverLread: 0.66 EXTRACTION: LOCUS:

samples.csv contains 2 mixed samples from human and mouse Nadia scRNAseq

samples,expected_cells,read_length,batch N701_S1_L001,1000,100,batch1 N702_S2_L001,1000,100,batch2

Hoohm commented 4 years ago

We have made quite a few changes for the new version and our test pipeline for mixed species is not that robust so it's possible that some bugs made it through the cracks.

I would not be surprised if your bam files are empty and the extraction can't work since there is nothing to extract.

What does the species plot look like?

manarai commented 4 years ago

Hi,

The pipeline failed before making the plot. As you guessed the Bam files were empty. Do you still have the older version that I could download until the new version becomes stable?

Thanks

On Dec 27, 2019, at 19:59, Patrick Roelli notifications@github.com wrote:

We have made quite a few changes for the new version and our test pipeline for mixed species is not that robust so it's possible that some bugs made it through the cracks.

I would not be surprised if your bam files are empty and the extraction can't work since there is nothing to extract.

What does the species plot look like?

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seb-mueller commented 4 years ago

Version 0.5 should be most stable in fact. In mixed species, it can happen that one species is in fact missing in one dataset which makes the pipeline fail. Could you try running with one species only to see if it works? Are both homo_sapiens/unfiltered.bam and mus_musculus/unfiltered.bam empty? If it fails for single species, could you attach the log file for trouble shooting (including how you run the pipeline)?