Problem running the new version

[manarai]

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:
- CODING
- UTR
strand-strategy: SENSE
UMI-edit-distance: 1
minimum-counts-per-UMI: 0

---

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]

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]

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 ***@***.***> 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? — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <#99?email_source=notifications&email_token=AJQHVMGZPCBOM662HL5VNWLQ2XNYNA5CNFSM4J7E4HUKYY3PNVWWK3TUL52HS4DFVREXG43VMVBW63LNMVXHJKTDN5WW2ZLOORPWSZGOEHXAENA#issuecomment-569246260>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AJQHVMBTRT5O6ETNQDB4N3LQ2XNYNANCNFSM4J7E4HUA>.

[seb-mueller]

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)?