error in DESeq2 step

[m-jahani]

Hi Hoelzer,

I am running RNAFLOW on a small dataset with two different conditions and two replicates per condition. I am encountering the following error in the DESeq2 step. Could you please help me understand what might be causing this issue? Thank you.

Error Message:

CPUs to use: 64, maximal CPUs to use: 64

R N A F L O W : R N A - S E Q A S S E M B L Y & D I F F E R E N T I A L G E N E E X P R E S S I O N A N A L Y S I S
= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =
Output path: results
Strandedness unstranded
Read mode: paired-end
TPM threshold: 1
Comparisons: /home/mjahani/scratch/RNA_SEQ_QU/files/comparisons_test.csv
Nanopore mode: false

executor > local (38)
[18/9f59d0] process > concat_genome [100%] 1 of 1 ✔
[41/dc262b] process > concat_annotation [100%] 1 of 1 ✔
executor > local (38)
[18/9f59d0] process > concat_genome [100%] 1 of 1 ✔
[41/dc262b] process > concat_annotation [100%] 1 of 1 ✔
[skipped ] process > download_sortmerna:sortmernaGet [100%] 1 of 1, stored: 1 ✔
[ac/de9a14] process > preprocess_illumina:fastqcPre (FCT040004_Week16_Ileum_rep2) [100%] 4 of 4 ✔
[4e/a629c7] process > preprocess_illumina:fastp (FCT040004_Week16_Rectum_rep1) [100%] 4 of 4 ✔
[7a/4c35ea] process > preprocess_illumina:fastqcPost (FCT040004_Week16_Ileum_rep2) [100%] 4 of 4 ✔
[dd/f7be0d] process > preprocess_illumina:extract_tar_bz2 [100%] 1 of 1 ✔
[c2/c405c1] process > preprocess_illumina:sortmerna (FCT040004_Week16_Rectum_rep1) [100%] 4 of 4 ✔
[ea/42d24f] process > preprocess_illumina:hisat2index [100%] 1 of 1 ✔
[fb/a7ec81] process > preprocess_illumina:hisat2 (FCT040004_Week16_Rectum_rep1) [100%] 4 of 4 ✔
[69/33e5c3] process > preprocess_illumina:index_bam (FCT040004_Week16_Ileum_rep1) [100%] 4 of 4 ✔
[d6/9cf0b0] process > expression_reference_based:featurecounts (FCT040004_Week16_Rectum_rep1) [100%] 4 of 4 ✔
[32/7262de] process > expression_reference_based:format_annotation_gene_rows [100%] 1 of 1 ✔
[30/f0c033] process > expression_reference_based:format_annotation [100%] 1 of 1 ✔
[81/fa1fe6] process > expression_reference_based:tpm_filter [100%] 1 of 1 ✔
[4c/5d9af2] process > expression_reference_based:deseq2 (1) [100%] 1 of 1, failed: 1 ✘
[- ] process > expression_reference_based:piano -
[- ] process > expression_reference_based:webgestalt -
[77/69501b] process > expression_reference_based:multiqc_sample_names (1) [100%] 1 of 1 ✔
[- ] process > expression_reference_based:multiqc (1) -
ERROR ~ Error executing process > 'expression_reference_based:deseq2 (1)'

Caused by:
Process expression_reference_based:deseq2 (1) terminated with an error exit status (1)
Command executed:
R CMD BATCH --no-save --no-restore '--args c(".") c("FCT040004_Week16_Ileum_rep1.counts.filtered.formated.tsv","FCT040004_Week16_Ileum_rep2.counts.filtered.formated.tsv","FCT040004_Week16_Rectum_rep1.counts.filtered.formated.tsv","FCT040004_Week16_Rectum_rep2.counts.filtered.formated.tsv") c("Ileum","Ileum","Rectum","Rectum") c("FCT040004_Week16_Ileum_rep1","FCT040004_Week16_Ileum_rep2","FCT040004_Week16_Rectum_rep1","FCT040004_Week16_Rectum_rep2") c("Ileum","Rectum") c("Ileum:Rectum") c("annotation.id2details") c("annotation.exon.gtf") c("FCT040004","FCT040004","FCT040004","FCT040004") c("") c("regionReport_DESeq2Exploration_custom.Rmd") c(64) c("ensembl_gene_id")' deseq2.R

Command exit status:
1

Command output:
(empty)

Work dir:
/lustre07/scratch/mjahani/RNA_SEQ_QU/run2/work/4c/5d9af2dde3076adf28c1ff2f5edc4f

Tip: You can try to figure out what's wrong by changing to the process work dir and showing the script file named .command.sh
-- Check '.nextflow.log' file for details

[hoelzer]

Hey @m-jahani !

That's hard to say. Can you provide a bit more information from the folder /lustre07/scratch/mjahani/RNA_SEQ_QU/run2/work/4c/5d9af2dde3076adf28c1ff2f5edc4f/. There should be a file called something like DESeq2.Rout. Does it have more detailed information on the error?

Besides, having two replicates per condition is anyway a difficult setup. You should have at least three biological replicates, better more. Maybe this is also part of the problem and DESeq2's error.

[m-jahani]

Here is the DESeq2.Rout log. It seems like one of the packages is not installed. I will fix that and try again.
How should I deal with having only two replications?

---

R version 4.3.1 (2023-06-16) -- "Beagle Scouts"
Copyright (C) 2023 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu (64-bit)

R is free software and comes with ABSOLUTELY NO WARRANTY.
You are welcome to redistribute it under certain conditions.
Type 'license()' or 'licence()' for distribution details.

Natural language support but running in an English locale

R is a collaborative project with many contributors.
Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.

Type 'demo()' for some demos, 'help()' for on-line help, or
'help.start()' for an HTML browser interface to help.
Type 'q()' to quit R.

library("DESeq2")
Error in library("DESeq2") : there is no package called ‘DESeq2’
Execution halted
deseq2.Rout (END)
R version 4.3.1 (2023-06-16) -- "Beagle Scouts"
Copyright (C) 2023 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu (64-bit)

R is free software and comes with ABSOLUTELY NO WARRANTY.
You are welcome to redistribute it under certain conditions.
Type 'license()' or 'licence()' for distribution details.

Natural language support but running in an English locale

R is a collaborative project with many contributors.
Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.

Type 'demo()' for some demos, 'help()' for on-line help, or
'help.start()' for an HTML browser interface to help.
Type 'q()' to quit R.

library("DESeq2")
Error in library("DESeq2") : there is no package called ‘DESeq2’
Execution halted"

[hoelzer]

Hey,

What is your nextflow run command? Are you using conda or docker/singularity? It is strange that the package DESeq2 can not be found. If you are using conda, can you please try docker instead? (or singularity if you are on a cluster or restricted machine with no option for installing docker)

How should I deal with having only two replications?

Sequence more : )

When this is not possible, you can still run tools such as DESeq2 or edgeR but you should be very careful with the resulting p-values.

Depending on your experimental design you could also think about combining samples from different conditions (e.g. time points) to have more replicates. But then this should be also reflected in the DESeq2 design matrix. For this we provide a "Source" column in the input, see https://github.com/hoelzer-lab/rnaflow/#input-files

However, this really depends on your experimental design. Having more replicates would be the best.