Error executing process > 'BARPLOTS'

[WZo0o]

Hello GiantSpaceRobot
I can successfully run the group comparison analysis with your example file results, when I try to do the group comparison analysis based on our own data , I always meet Error executing process > 'BARPLOTS'.
can you help me to solve this issue?

executor > local (46)
[8c/8b9bf0] process > PREPARE_TRNA_GTF [100%] 1 of 1 ✔
[86/d7cf71] process > PREPARE_NCRNA_GTF [100%] 1 of 1 ✔
[be/6bb898] process > TRIM_READS (2) [100%] 2 of 2 ✔
[99/efe468] process > MAKE_STAR_DB [100%] 1 of 1 ✔
[1a/d89db6] process > STAR_ALIGN (2) [100%] 2 of 2 ✔
[20/5a4419] process > BAM_COLLAPSE (2) [100%] 2 of 2 ✔
[5c/bdc724] process > ADD_EMPTY_COUNTS (B14_1_L3_... [100%] 2 of 2 ✔
[73/d6e37c] process > BAM_SPLIT (2) [100%] 2 of 2 ✔
[e9/70ec03] process > FEATURE_COUNT_TRNA (B14_1_L... [100%] 2 of 2 ✔
[ae/030849] process > FEATURE_COUNT_NCRNA (B14_1_... [100%] 2 of 2 ✔
[ab/9b0c9b] process > SUM_COUNTS [100%] 1 of 1 ✔
[3f/d270fe] process > GENERATE_TRNA_DEPTH_FILES (... [100%] 2 of 2 ✔
[06/c0a6ce] process > GENERATE_MULTIMAPPER_TRNA_D... [100%] 2 of 2 ✔
[e1/bfde3b] process > GENERATE_NCRNA_DEPTH_FILES ... [100%] 2 of 2 ✔
[68/9e3b51] process > GENERATE_DEPTHFILE_STATS (B... [100%] 4 of 4 ✔
[18/1a499e] process > RAW_COUNTS_TO_PROPORTIONS (... [100%] 2 of 2 ✔
[fb/af50b8] process > RAW_COUNTS_TO_NORM_COUNTS (... [100%] 2 of 2 ✔
[2e/ba7d5c] process > COUNTS_TO_COLLAPSED_COUNTS ... [100%] 4 of 4 ✔
[26/2926af] process > PREDICT_TSRNA_TYPE [100%] 1 of 1 ✔
[78/45980d] process > PLOT_TRNA_ALIGNMENT_LENGTH ... [ 50%] 1 of 2
[3c/223f44] process > PLOT_TRNA_ALL_PLOTS (B14_1_... [100%] 2 of 2 ✔
[- ] process > GENERATE_RESULTS_PDF -
[a9/eff521] process > DESEQ2 [ 0%] 0 of 1
[36/7e4bf3] process > DATA_TRANSFORMATIONS [ 0%] 0 of 1
[- ] process > DISTRIBUTION_SCORE -
[- ] process > SLOPE_SCORE -
[- ] process > CLEAVAGE_SCORE -
[- ] process > FISHERS_METHOD -
[- ] process > RESULTS_TABLE -
[- ] process > COMBINED_SCORE -
[- ] process > PLOT_TSRNA_LENGTHS -
[- ] process > PLOT_TRNAS -
[- ] process > PLOT_NCRNAS -
[- ] process > VENN_DIAGRAM -
[29/e3ed1a] process > GENERATE_COUNT_DATAFRAME [100%] 1 of 1 ✔
[9e/6ff258] process > STACKED_BARPLOTS [100%] 1 of 1 ✔
[86/17119b] process > BARPLOTS [ 0%] 0 of 1
[- ] process > PREDICT_TSRNA_TYPE_GROUPS -
[- ] process > GENERATE_RESULTS_PDF_GROUPS -
[- ] process > ORGANISE_RESULTS_GROUPS -
[- ] process > PUBLISH_FILES -
Error executing process > 'BARPLOTS'

Caused by:
Process BARPLOTS terminated with an error exit status (1)

Command executed:

Barplots.R
All-Features_raw-counts.tsv
human_ncRNAs_relative_cdhit.gtf
Layout.csv

Command exit status:
1

Command output:
(empty)

Command error:
Note: Using an external vector in selections is ambiguous.
ℹ Use all_of(ReplicateNumber1) instead of ReplicateNumber1 to silence this message.
ℹ See https://tidyselect.r-lib.org/reference/faq-external-vector.html.
This message is displayed once per session.
Error in t.test.default(my.column.condition1, my.column.condition2) :
not enough 'x' observations
Calls: t.test -> t.test.default
Execution halted

Work dir:
/lustre/wangzh/SC/tsrna_test/combinde_test/work/86/17119be4163e8ce0eeecd1b20aab30

Tip: you can replicate the issue by changing to the process work dir and entering the command bash .command.run

WARN: Killing pending tasks (3)

executor > local (46)
[8c/8b9bf0] process > PREPARE_TRNA_GTF [100%] 1 of 1 ✔
[86/d7cf71] process > PREPARE_NCRNA_GTF [100%] 1 of 1 ✔
[be/6bb898] process > TRIM_READS (2) [100%] 2 of 2 ✔
[99/efe468] process > MAKE_STAR_DB [100%] 1 of 1 ✔
[1a/d89db6] process > STAR_ALIGN (2) [100%] 2 of 2 ✔
[20/5a4419] process > BAM_COLLAPSE (2) [100%] 2 of 2 ✔
[5c/bdc724] process > ADD_EMPTY_COUNTS (B14_1_L3_... [100%] 2 of 2 ✔
[73/d6e37c] process > BAM_SPLIT (2) [100%] 2 of 2 ✔
[e9/70ec03] process > FEATURE_COUNT_TRNA (B14_1_L... [100%] 2 of 2 ✔
[ae/030849] process > FEATURE_COUNT_NCRNA (B14_1_... [100%] 2 of 2 ✔
[ab/9b0c9b] process > SUM_COUNTS [100%] 1 of 1 ✔
[3f/d270fe] process > GENERATE_TRNA_DEPTH_FILES (... [100%] 2 of 2 ✔
[06/c0a6ce] process > GENERATE_MULTIMAPPER_TRNA_D... [100%] 2 of 2 ✔
[e1/bfde3b] process > GENERATE_NCRNA_DEPTH_FILES ... [100%] 2 of 2 ✔
[68/9e3b51] process > GENERATE_DEPTHFILE_STATS (B... [100%] 4 of 4 ✔
[18/1a499e] process > RAW_COUNTS_TO_PROPORTIONS (... [100%] 2 of 2 ✔
[fb/af50b8] process > RAW_COUNTS_TO_NORM_COUNTS (... [100%] 2 of 2 ✔
[2e/ba7d5c] process > COUNTS_TO_COLLAPSED_COUNTS ... [100%] 4 of 4 ✔
[26/2926af] process > PREDICT_TSRNA_TYPE [100%] 1 of 1 ✔
[78/45980d] process > PLOT_TRNA_ALIGNMENT_LENGTH ... [100%] 1 of 1
[3c/223f44] process > PLOT_TRNA_ALL_PLOTS (B14_1_... [100%] 2 of 2 ✔
[- ] process > GENERATE_RESULTS_PDF -
[- ] process > DESEQ2 -
[- ] process > DATA_TRANSFORMATIONS -
[- ] process > DISTRIBUTION_SCORE -
[- ] process > SLOPE_SCORE -
[- ] process > CLEAVAGE_SCORE -
[- ] process > FISHERS_METHOD -
[- ] process > RESULTS_TABLE -
[- ] process > COMBINED_SCORE -
[- ] process > PLOT_TSRNA_LENGTHS -
[- ] process > PLOT_TRNAS -
[- ] process > PLOT_NCRNAS -
[- ] process > VENN_DIAGRAM -
[29/e3ed1a] process > GENERATE_COUNT_DATAFRAME [100%] 1 of 1 ✔
[9e/6ff258] process > STACKED_BARPLOTS [100%] 1 of 1 ✔
[86/17119b] process > BARPLOTS [100%] 1 of 1, failed: 1 ✘
[- ] process > PREDICT_TSRNA_TYPE_GROUPS -
[- ] process > GENERATE_RESULTS_PDF_GROUPS -
[- ] process > ORGANISE_RESULTS_GROUPS -
[- ] process > PUBLISH_FILES -
Error executing process > 'BARPLOTS'

Caused by:
Process BARPLOTS terminated with an error exit status (1)

Command executed:

Barplots.R
All-Features_raw-counts.tsv
human_ncRNAs_relative_cdhit.gtf
Layout.csv

Command exit status:
1

Command output:
(empty)

Command error:
Note: Using an external vector in selections is ambiguous.
ℹ Use all_of(ReplicateNumber1) instead of ReplicateNumber1 to silence this message.
ℹ See https://tidyselect.r-lib.org/reference/faq-external-vector.html.
This message is displayed once per session.
Error in t.test.default(my.column.condition1, my.column.condition2) :
not enough 'x' observations
Calls: t.test -> t.test.default
Execution halted

Work dir:
/lustre/wangzh/SC/tsrna_test/combinde_test/work/86/17119be4163e8ce0eeecd1b20aab30

Tip: you can replicate the issue by changing to the process work dir and entering the command bash .command.run

[GiantSpaceRobot]

Hi there,
I can't replicate this issue as I don't have your data, but I would imagine there are not enough tRNA-derived reads in your FASTQ files. If you want to, you can send the following file to my email address (pauldonovandonegal@gmail.com):
/lustre/wangzh/SC/tsrna_test/combinde_test/work/86/17119be4163e8ce0eeecd1b20aab30/All-Features_raw-counts.tsv
I can test the error with this file and give you a definitive answer.

[WZo0o]

Hi there,
Thanks for your help, I have sent the following file to your email address(pauldonovandonegal@gmail.com):
/lustre/wangzh/SC/tsrna_test/combinde_test/work/86/17119be4163e8ce0eeecd1b20aab30/All-Features_raw-counts.tsv

[WZo0o]

Hi there,
I am wondering whether you have received my email about my data?
I would very much like to receive a definitive response about this issue.

[GiantSpaceRobot]

Hi there,

Apologies for not getting back to you. I am not able to spend much time on this tool as I am no longer in academic research.
I have updated the barplots script to allow it to deal with single replicates per group. Please replace your version of Barplots.R with the newest one on the repo. Let me know if this works for you.

[WZo0o]

Hi there,
Thanks for your help.
When I replaced the version of Barplots.R , it worked fine. But I meet Error executing process > 'DESEQ2.
can you help me to solve this issue again?
executor > local (46)
[fb/6f9e42] process > PREPARE_TRNA_GTF [100%] 1 of 1 ✔
[2f/2c96f8] process > PREPARE_NCRNA_GTF [100%] 1 of 1 ✔
[7e/0ba33a] process > TRIM_READS (2) [100%] 2 of 2 ✔
[fc/d44f17] process > MAKE_STAR_DB [100%] 1 of 1 ✔
[65/eeea00] process > STAR_ALIGN (2) [100%] 2 of 2 ✔
[35/113e06] process > BAM_COLLAPSE (2) [100%] 2 of 2 ✔
[b1/38903e] process > ADD_EMPTY_COUNTS (B14_1_L3_... [100%] 2 of 2 ✔
[d1/8923f0] process > BAM_SPLIT (2) [100%] 2 of 2 ✔
[5b/12ddfc] process > FEATURE_COUNT_TRNA (B14_1_L... [100%] 2 of 2 ✔
[d2/4d6fd7] process > FEATURE_COUNT_NCRNA (B14_1_... [100%] 2 of 2 ✔
[40/fa0d5b] process > SUM_COUNTS [100%] 1 of 1 ✔
[d2/3820e5] process > GENERATE_TRNA_DEPTH_FILES (... [100%] 2 of 2 ✔
[62/75d69d] process > GENERATE_MULTIMAPPER_TRNA_D... [100%] 2 of 2 ✔
[96/032ff6] process > GENERATE_NCRNA_DEPTH_FILES ... [100%] 2 of 2 ✔
[c3/c9c13d] process > GENERATE_DEPTHFILE_STATS (B... [100%] 4 of 4 ✔
[b4/275f9a] process > RAW_COUNTS_TO_PROPORTIONS (... [100%] 2 of 2 ✔
[c4/7196b9] process > RAW_COUNTS_TO_NORM_COUNTS (... [100%] 2 of 2 ✔
[ba/ebb37c] process > COUNTS_TO_COLLAPSED_COUNTS ... [100%] 4 of 4 ✔
[07/67f353] process > PREDICT_TSRNA_TYPE [100%] 1 of 1 ✔
[1f/17a6c9] process > PLOT_TRNA_ALIGNMENT_LENGTH ... [100%] 1 of 1
[09/54d58c] process > PLOT_TRNA_ALL_PLOTS (B14_1_... [100%] 2 of 2 ✔
[- ] process > GENERATE_RESULTS_PDF -
[5a/04e777] process > DESEQ2 [100%] 1 of 1, failed: 1 ✘
[- ] process > DATA_TRANSFORMATIONS -
[- ] process > DISTRIBUTION_SCORE -
[- ] process > SLOPE_SCORE -
[- ] process > CLEAVAGE_SCORE -
[- ] process > FISHERS_METHOD -
[- ] process > RESULTS_TABLE -
[- ] process > COMBINED_SCORE -
[- ] process > PLOT_TSRNA_LENGTHS -
[- ] process > PLOT_TRNAS -
[- ] process > PLOT_NCRNAS -
[- ] process > VENN_DIAGRAM -
[c7/ac2b91] process > GENERATE_COUNT_DATAFRAME [100%] 1 of 1 ✔
[14/8325c7] process > STACKED_BARPLOTS [100%] 1 of 1 ✔
[ef/2a3acb] process > BARPLOTS [100%] 1 of 1 ✔
[- ] process > PREDICT_TSRNA_TYPE_GROUPS -
[- ] process > GENERATE_RESULTS_PDF_GROUPS -
[- ] process > ORGANISE_RESULTS_GROUPS -
[- ] process > PUBLISH_FILES -
Error executing process > 'DESEQ2'

Caused by:
Missing output file(s) *.pdf expected by process DESEQ2

Command executed:

condition1=$( awk -F ',' '{print $2}' Layout.csv | uniq | head -n 1 ) # Get the first condition name
condition2=$( awk -F ',' '{print $2}' Layout.csv | uniq | tail -n 1 ) # Get the second condition name
echo Conditions are $condition1 and $condition2
DESeq2_tsRNAsearch.R ./ "${condition1}vs${condition2}" human_ncRNAs_relative_cdhit.gtf Layout.csv

Create empty results files if none were generated

touch -a ${condition1}vs${condition2}_DESeq2-output.csv
touch -a ${condition1}vs${condition2}_DESeq2-output-upregulated.csv
touch -a ${condition1}vs${condition2}_DESeq2-output-downregulated.csv
cat *regulated.csv | grep -v ^, | sort -t',' -k7,7g | awk -F ',' '{print $1}' | awk -F ' ' '{print $1}'
> DEGs_names-only.txt
touch -a DEGs_names-only.txt

Command exit status:
0

Command output:
Conditions are B141 and B142
NULL
NULL
[1] "-----DESeq2 analysis underway-----"
[1] "Control group:B141"
[1] "Treatment group:B142"
[1] "Checkpoint 1"
[1] "Checkpoint 2"
[1] "Checkpoint 3"
[1] " Single replicate analysis, skipping formal DESeq2 analysis and carrying out single Log2 fold comparison instead..."

Command error:
A .csv file was provided, and the directory provided exists.

Work dir:
/lustre/wangzh/SC/tsrna_test/combinde_test/work/5a/04e7770ec8c1dffa0e47fb389c40ef

Tip: when you have fixed the problem you can continue the execution adding the option -resume to the run command line