epi2me-labs / wf-transcriptomes

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Data output files and report don't match - e.g. # of total reads aren´t assigned to the same samples #19

Closed SamanthaStewart closed 10 months ago

SamanthaStewart commented 1 year ago

What happened?

When running the pipeline after multiplexing my RNA samples via the SQK-PCB109 protocol, the transcriptomes report in the end shows inconsistently assigned e.g. total read values compared to the single output files.

When comparing the Output files "..._read_aln_stats.tsv" I get the same values but they are assigned differently.

e.g. sample 12_X shows 271880 total reads (Report) And the Output file sample 12_X shows 384585 total reads

12_G shows 265334 total reads (Report) And the Output file sample 12_G shows 222981 total reads

The Outputfile of sample D_X of the output tsv file suits the numbers of the total reads of G_G of the Report.

It looks like the report isn’t sorting the sample ID correctly. The numbers are found 1:1. They’re just not assigned to the same samples.

Which assignment can be trusted now? The report or the Outputs of e.g., "..._read_aln_stats.tsv"?

I already checked my sample and condition sheets. I ran the pipeline when sorted by barcodes, sorted by sampleIDs, and made sure that both sheets were sorted the same way. The troubleshooting did not change the results. The inconsistencies still occurred. issue

What´s the problem here? How can I solve it?

Operating System

Windows 10

Workflow Execution

EPI2ME Labs desktop application

Workflow Execution - EPI2ME Labs Versions

version 22.04.5

Workflow Execution - CLI Execution Profile

None

Workflow Version

transcriptomes v0.1.8

Relevant log output

not sure which log output would be relevant here...

N E X T F L O W ~ version 22.04.5
Launching `/mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs/workflows/epi2me-labs/wf-transcriptomes/main.nf` [distracted_tesla] DSL2 - revision: 50fc30ae91

WARN: Found unexpected parameters:
* --wf.agent: epi2melabs/4.1.1
* --version: false
- Ignore this warning: params.schema_ignore_params = "wf.agent,version" 

|||||||||| _____ ____ ___ ____ __ __ _____ _ _
|||||||||| | ____| _ \_ _|___ \| \/ | ____| | | __ _| |__ ___
||||| | _| | |_) | | __) | |\/| | _| _____| |/ _` | '_ \/ __|
||||| | |___| __/| | / __/| | | | |__|_____| | (_| | |_) \__ \
|||||||||| |_____|_| |___|_____|_| |_|_____| |_|\__,_|_.__/|___/
|||||||||| wf-transcriptomes v0.1.8
--------------------------------------------------------------------------------
Core Nextflow options
runName : distracted_tesla
containerEngine : docker
launchDir : /mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs/instances/wf-transcriptomes_f8ffb00a-7e22-49ec-b292-3ea56c4ad79e
workDir : /mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs/instances/wf-transcriptomes_f8ffb00a-7e22-49ec-b292-3ea56c4ad79e/work
projectDir : /mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs/workflows/epi2me-labs/wf-transcriptomes
userName : stewart
profile : standard
configFiles : /mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs/workflows/epi2me-labs/wf-transcriptomes/nextflow.config

Input Options
fastq : /mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/08032023_12DG_all_purifications_on_one_Flow_cell/fastq_pass_08032023_all_purification_on_one_flow_cell
transcriptome_source: reference-guided
ref_genome : /mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/Vadim and Adithi/gencode/GRCh38.p13.genome.fa
ref_transcriptome : /mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/Vadim and Adithi/gencode/gencode.v42.transcripts_corrected.fa
ref_annotation : /mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/Vadim and Adithi/gencode/gencode.v42.chr_patch_hapl_scaff.annotation.gtf

Output Options
out_dir : /mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs/instances/wf-transcriptomes_f8ffb00a-7e22-49ec-b292-3ea56c4ad79e/output

Sample Options
sample_sheet : /mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/08032023_12DG_all_purifications_on_one_Flow_cell/sample_sheet_purificationtestallononeflowcell_changed_v1_with_adithi.txt

Options for reference-based workflow
plot_gffcmp_stats : true

Gene Fusion Detection Options
jaffal_dir : /mnt/c/home/epi2melabs/JAFFA

Differential Expression Options
condition_sheet : /mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/08032023_12DG_all_purifications_on_one_Flow_cell/condition_sheet_purificationtestallononeflowcell_changed_with_adithi.txt

Advanced Options
bundle_min_reads : 50000

!! Only displaying parameters that differ from the pipeline defaults !!
--------------------------------------------------------------------------------
If you use epi2me-labs/wf-transcriptomes for your analysis please cite:

* The nf-core framework
https://doi.org/10.1038/s41587-020-0439-x

--------------------------------------------------------------------------------
This is epi2me-labs/wf-transcriptomes v0.1.8.
--------------------------------------------------------------------------------
Reference Transcriptome provided will be used for differential expression.
Checking fastq input.
Barcoded directories detected.
Checking sample sheet.
Doing reference based transcript analysis
[c7/26100e] Submitted process > pipeline:getVersions
[fd/533582] Submitted process > checkSampleSheet
[c9/6acb92] Submitted process > pipeline:getParams
[a1/ee8609] Submitted process > pipeline:build_minimap_index
[fd/b7edd8] Submitted process > pipeline:summariseConcatReads (5)
[73/042a89] Submitted process > pipeline:summariseConcatReads (11)
[45/7c8f24] Submitted process > pipeline:summariseConcatReads (2)
[f3/a4a93c] Submitted process > pipeline:summariseConcatReads (3)
[67/5bd75b] Submitted process > pipeline:summariseConcatReads (4)
[42/96e4e8] Submitted process > pipeline:summariseConcatReads (9)
[11/579c98] Submitted process > pipeline:summariseConcatReads (8)
[b8/836f12] Submitted process > pipeline:summariseConcatReads (7)
[fa/798edb] Submitted process > pipeline:summariseConcatReads (6)
[77/0b7f67] Submitted process > pipeline:summariseConcatReads (10)
[bb/46c4f7] Submitted process > pipeline:summariseConcatReads (12)
[c5/42fe2b] Submitted process > pipeline:summariseConcatReads (1)
[65/7432ac] Submitted process > pipeline:preprocess_reads (1)
[7c/e8273f] Submitted process > pipeline:preprocess_reads (2)
[84/5d2d2f] Submitted process > pipeline:preprocess_reads (3)
[1d/abd68a] Submitted process > pipeline:preprocess_reads (4)
[96/35da54] Submitted process > pipeline:preprocess_reads (5)
[56/1ad77d] Submitted process > pipeline:preprocess_reads (6)
[0d/8c3b67] Submitted process > pipeline:preprocess_reads (7)
[d5/9a37ab] Submitted process > pipeline:preprocess_reads (8)
[8a/d26241] Submitted process > pipeline:preprocess_reads (9)
[2a/b47b9f] Submitted process > pipeline:preprocess_reads (10)
[76/6cd6b5] Submitted process > pipeline:preprocess_reads (11)
[1d/1beff9] Submitted process > pipeline:preprocess_reads (12)
[fd/0e2ca2] Submitted process > pipeline:reference_assembly:map_reads (1)
[b4/134dc4] Submitted process > pipeline:reference_assembly:map_reads (2)
[bf/d419f5] Submitted process > pipeline:reference_assembly:map_reads (3)
[fd/5091fb] Submitted process > pipeline:reference_assembly:map_reads (4)
[17/8cbc54] Submitted process > pipeline:reference_assembly:map_reads (5)
[dd/792bcb] Submitted process > pipeline:reference_assembly:map_reads (6)
[05/b709c0] Submitted process > pipeline:reference_assembly:map_reads (7)
[ab/4cba32] Submitted process > pipeline:reference_assembly:map_reads (8)
[28/a6ee2e] Submitted process > pipeline:reference_assembly:map_reads (9)
[91/a8637b] Submitted process > pipeline:reference_assembly:map_reads (10)
[28/d8d8d6] Submitted process > pipeline:reference_assembly:map_reads (11)
[69/971c06] Submitted process > pipeline:reference_assembly:map_reads (12)
[09/d089ee] Submitted process > pipeline:split_bam (1)
[e2/5b9e98] Submitted process > pipeline:split_bam (2)
[27/627e38] Submitted process > pipeline:split_bam (3)
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[df/86f607] Submitted process > pipeline:split_bam (9)
[08/51d423] Submitted process > pipeline:split_bam (10)
[8b/5db131] Submitted process > pipeline:split_bam (11)
[bf/b33b85] Submitted process > pipeline:assemble_transcripts (1)
[21/857c71] Submitted process > pipeline:assemble_transcripts (4)
[6c/b8831e] Submitted process > pipeline:assemble_transcripts (3)
[e1/0c79cf] Submitted process > pipeline:assemble_transcripts (2)
[ab/41548b] Submitted process > pipeline:assemble_transcripts (6)
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[ff/20cd1d] Submitted process > pipeline:assemble_transcripts (9)
[ed/807c67] Submitted process > pipeline:assemble_transcripts (7)
[c4/d69de0] Submitted process > pipeline:assemble_transcripts (10)
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[60/ed952a] Submitted process > pipeline:assemble_transcripts (20)
[ee/f1aea5] Submitted process > pipeline:assemble_transcripts (17)
[dc/1f0f78] Submitted process > pipeline:assemble_transcripts (18)
[b4/f53fb9] Submitted process > pipeline:assemble_transcripts (26)
[28/c4119b] Submitted process > pipeline:assemble_transcripts (23)
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[b1/e92c03] Submitted process > pipeline:assemble_transcripts (28)
[c2/dadcd1] Submitted process > pipeline:assemble_transcripts (24)
[5a/8e1680] Submitted process > pipeline:assemble_transcripts (29)
[ee/2e5caa] Submitted process > pipeline:assemble_transcripts (35)
[59/eae1b5] Submitted process > pipeline:assemble_transcripts (31)
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[a9/1feb51] Submitted process > pipeline:assemble_transcripts (32)
[a5/ce7527] Submitted process > pipeline:assemble_transcripts (33)
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[43/dd88c7] Submitted process > pipeline:assemble_transcripts (39)
[b8/e105ee] Submitted process > pipeline:assemble_transcripts (42)
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[b5/bcd006] Submitted process > pipeline:assemble_transcripts (55)
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[9c/54198e] Submitted process > pipeline:assemble_transcripts (59)
[f8/683117] Submitted process > pipeline:assemble_transcripts (62)
[80/dc5b87] Submitted process > pipeline:assemble_transcripts (60)
[65/133753] Submitted process > pipeline:split_bam (12)
[2f/699114] Submitted process > pipeline:assemble_transcripts (65)
[79/7251fd] Submitted process > pipeline:assemble_transcripts (66)
[ac/9780bf] Submitted process > pipeline:assemble_transcripts (63)
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[89/e9e403] Submitted process > pipeline:assemble_transcripts (64)
[0b/c83907] Submitted process > pipeline:assemble_transcripts (71)
[ee/0401bc] Submitted process > pipeline:assemble_transcripts (67)
[84/afd7b8] Submitted process > pipeline:merge_gff_bundles (8)
[ab/85e9df] Submitted process > pipeline:merge_gff_bundles (9)
[5a/7ec208] Submitted process > pipeline:merge_gff_bundles (2)
[5b/aa4cf3] Submitted process > pipeline:merge_gff_bundles (7)
[53/efa6e0] Submitted process > pipeline:merge_gff_bundles (11)
[1b/c29043] Submitted process > pipeline:merge_gff_bundles (1)
[c0/0232d1] Submitted process > pipeline:merge_gff_bundles (3)
[31/695b63] Submitted process > pipeline:merge_gff_bundles (4)
[ba/28940a] Submitted process > pipeline:merge_gff_bundles (6)
[57/0b8a14] Submitted process > pipeline:merge_gff_bundles (5)
[2b/cc6206] Submitted process > pipeline:merge_gff_bundles (10)
[5c/138e07] Submitted process > pipeline:merge_gff_bundles (12)
[e1/511b84] Submitted process > pipeline:run_gffcompare (1)
[44/603280] Submitted process > pipeline:run_gffcompare (2)
[7f/428000] Submitted process > pipeline:run_gffcompare (3)
[a7/70b3e8] Submitted process > pipeline:run_gffcompare (4)
[72/058764] Submitted process > pipeline:run_gffcompare (5)
[29/f2e3ff] Submitted process > pipeline:run_gffcompare (6)
[54/04d77d] Submitted process > pipeline:run_gffcompare (7)
[bc/9f79a3] Submitted process > pipeline:run_gffcompare (8)
[c8/ce96fc] Submitted process > pipeline:run_gffcompare (9)
[4d/591190] Submitted process > pipeline:run_gffcompare (10)
[bf/85923e] Submitted process > pipeline:run_gffcompare (11)
[e3/711763] Submitted process > pipeline:run_gffcompare (12)
[92/af2a83] Submitted process > output (1)
[9e/245452] Submitted process > pipeline:get_transcriptome (1)
[31/2a8f4b] Submitted process > output (2)
[78/7dbc4f] Submitted process > pipeline:get_transcriptome (2)
[bd/124b7d] Submitted process > output (3)
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[79/f0125f] Submitted process > output (4)
[79/7d7286] Submitted process > pipeline:get_transcriptome (4)
[38/b4924c] Submitted process > output (5)
[64/8f1f96] Submitted process > pipeline:get_transcriptome (5)
[97/afda4c] Submitted process > output (6)
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[6b/872f0a] Submitted process > output (7)
[64/571d5b] Submitted process > pipeline:get_transcriptome (7)
[d0/5ff35d] Submitted process > output (8)
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[a5/7c2241] Submitted process > output (9)
[81/b56122] Submitted process > pipeline:get_transcriptome (9)
[97/d981d4] Submitted process > output (10)
[78/841f94] Submitted process > pipeline:get_transcriptome (10)
[c6/bc875c] Submitted process > output (11)
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[a7/d76ec5] Submitted process > output (14)
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[a4/484a5a] Submitted process > output (24)
[af/1d9c99] Submitted process > pipeline:get_transcriptome (12)
[56/5aff6d] Submitted process > pipeline:makeReport (4)
[80/ee49f4] Submitted process > pipeline:makeReport (2)
[f8/d2709c] Submitted process > pipeline:makeReport (1)
[77/0c45f1] Submitted process > pipeline:makeReport (12)
[ea/70f3e1] Submitted process > pipeline:makeReport (6)
[b7/84dc1d] Submitted process > pipeline:makeReport (9)
[ab/658a6c] Submitted process > pipeline:makeReport (11)
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[10/58f153] Submitted process > pipeline:makeReport (3)
[f0/96aad4] Submitted process > pipeline:makeReport (8)
[18/c4e2f8] Submitted process > pipeline:makeReport (10)
[0d/be76d0] Submitted process > output (25)
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Invocation logs
[2023-04-28 00:16:06] [INFO] Initialising. 
[2023-04-28 00:16:06] [INFO] Reading launch data. 
[2023-04-28 00:16:06] [INFO] Acquiring database record. 
[2023-04-28 00:16:06] [INFO] Starting workflow invocation. 
[2023-04-28 00:16:06] [INFO] Connecting to app via IPC. 
[2023-04-28 00:16:06] [INFO] Launching nextflow subprocess. 
[2023-04-28 00:16:06] [INFO] Uplink established to app. 
[2023-04-28 04:45:41] [INFO] Subprocess closed. 
[2023-04-28 04:45:41] [INFO] Exiting.

Instance parameters
{
fastq:/mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/08032023_12DG_all_purifications_on_one_Flow_cell/fastq_pass_08032023_all_purification_on_one_flow_cell
transcriptome_source:reference-guided
ref_genome:/mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/Vadim and Adithi/gencode/GRCh38.p13.genome.fa
ref_transcriptome:/mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/Vadim and Adithi/gencode/gencode.v42.transcripts_corrected.fa
ref_annotation:/mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/Vadim and Adithi/gencode/gencode.v42.chr_patch_hapl_scaff.annotation.gtf
out_dir:/mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs/instances/wf-transcriptomes_f8ffb00a-7e22-49ec-b292-3ea56c4ad79e/output
sample_sheet:/mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/08032023_12DG_all_purifications_on_one_Flow_cell/sample_sheet_purificationtestallononeflowcell_changed_v1_with_adithi.txt
gffcompare_opts:-R
minimap_index_opts:-k14
minimap2_opts:-uf
minimum_mapping_quality:40
poly_context:24
max_poly_run:8
stringtie_opts:--conservative
isOnClust2_batch_size:-1
isOnClust2_sort_options:--kmer-size 11 --window-size 15 --min-shared 5 --min-qual 7.0 --mapped-threshold 0.65 --aligned-threshold 0.2 --min-fraction 0.8 --min-prob-no-hits 0.0 -M -1 -P 500 -g 50 -c 150 -F 2
jaffal_genome:hg38
jaffal_annotation:genCode22
jaffal_dir:/mnt/c/home/epi2melabs/JAFFA
condition_sheet:/mnt/wsl/docker-desktop-bind-mounts/Ubuntu/d2da218f1d9fc4b179b486f52dd5b84a962b3db801c5cbf78b9765ef6aad4f33/epi2melabs-data/Test with all documents/08032023_12DG_all_purifications_on_one_Flow_cell/condition_sheet_purificationtestallononeflowcell_changed_with_adithi.txt
min_gene_expr:10
min_feature_expr:3
min_samps_gene_expr:3
min_samps_feature_expr:1
threads:2
pychopper_opts:-m edlib
isoform_table_nrows:5000
wf.agent:epi2melabs/4.1.1
}
sarahjeeeze commented 1 year ago

Hi, Thanks for this feedback. I confirm that I also see a discrepancy when I run the workflow, we will investigate this and get back to you.

SamanthaStewart commented 1 year ago

Hi Sarahjeeeze, have you already found a possible issue?

sarahjeeeze commented 11 months ago

Hi, thanks for the reminder. Yes I think I found the issue, it's how the data is sorted in the report, will add a fix very soon - in the meantime it is the ...read_aln_stats.tsv files which have the correct values.

sarahjeeeze commented 11 months ago

Hi, we added a fix for this in the latest version.

sarahjeeeze commented 10 months ago

Closing, as should be resolved and no response for a while.