Details
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Type:
Task
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Status: Closed (View Workflow)
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Priority:
Minor
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Resolution: Done
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Affects Version/s: None
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Fix Version/s: None
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Labels:None
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Story Points:4
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Epic Link:
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Sprint:Summer 2, Summer 3, Summer 4, Summer 5, Summer 6, Summer 7, Fall 1
Description
GOAL:
To set up and run an nf-core/rna-seq pipeline to align our RNA-Seq sequences against the de novo created contigs (Trinity, Spades).
Next flow requires a GTF and a bed file along with the contigs. There are various ways to generate these and it is possible issues will arise from these files.
Otherwise, we can follow Molly's protocol:
https://docs.google.com/document/d/1ig9ET-ykXF5nAX3P487cXWmZDGUlQpcwrvFXpbyP5vw/edit?usp=sharing
Attachments
Issue Links
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- To-Do
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Activity
Looks like we now have the proper tsv salmon count files needed for the next step.
Lots of them.
rreid2@str-i2:/projects/tomato_genome/fnb/dataprocessing/brandon_work/NEXTFLOW/start_fresh$ ls Tam-run-2/results-3.14.0/star_salmon/*tsv
Tam-run-2/results-3.14.0/star_salmon/salmon.merged.gene_counts_length_scaled.tsv Tam-run-2/results-3.14.0/star_salmon/salmon.merged.transcript_counts.tsv
Tam-run-2/results-3.14.0/star_salmon/salmon.merged.gene_counts_scaled.tsv Tam-run-2/results-3.14.0/star_salmon/salmon.merged.transcript_lengths.tsv
Tam-run-2/results-3.14.0/star_salmon/salmon.merged.gene_counts.tsv Tam-run-2/results-3.14.0/star_salmon/salmon.merged.transcript_tpm.tsv
Tam-run-2/results-3.14.0/star_salmon/salmon.merged.gene_lengths.tsv Tam-run-2/results-3.14.0/star_salmon/tx2gene.tsv
Tam-run-2/results-3.14.0/star_salmon/salmon.merged.gene_tpm.tsv
rreid2@str-i2:/projects/tomato_genome/fnb/dataprocessing/brandon_work/NEXTFLOW/start_fresh$ ls Mal-run-2/results-3.14.0/star_salmon/*tsv
Mal-run-2/results-3.14.0/star_salmon/salmon.merged.gene_counts_length_scaled.tsv Mal-run-2/results-3.14.0/star_salmon/salmon.merged.transcript_counts.tsv
Mal-run-2/results-3.14.0/star_salmon/salmon.merged.gene_counts_scaled.tsv Mal-run-2/results-3.14.0/star_salmon/salmon.merged.transcript_lengths.tsv
Mal-run-2/results-3.14.0/star_salmon/salmon.merged.gene_counts.tsv Mal-run-2/results-3.14.0/star_salmon/salmon.merged.transcript_tpm.tsv
Mal-run-2/results-3.14.0/star_salmon/salmon.merged.gene_lengths.tsv Mal-run-2/results-3.14.0/star_salmon/tx2gene.tsv
Mal-run-2/results-3.14.0/star_salmon/salmon.merged.gene_tpm.tsv
rreid2@str-i2:/projects/tomato_genome/fnb/dataprocessing/brandon_work/NEXTFLOW/start_fresh$ ls Nag-run-2/results-3.14.0/star_salmon/*tsv
Nag-run-2/results-3.14.0/star_salmon/salmon.merged.gene_counts_length_scaled.tsv Nag-run-2/results-3.14.0/star_salmon/salmon.merged.transcript_counts.tsv
Nag-run-2/results-3.14.0/star_salmon/salmon.merged.gene_counts_scaled.tsv Nag-run-2/results-3.14.0/star_salmon/salmon.merged.transcript_lengths.tsv
Nag-run-2/results-3.14.0/star_salmon/salmon.merged.gene_counts.tsv Nag-run-2/results-3.14.0/star_salmon/salmon.merged.transcript_tpm.tsv
Nag-run-2/results-3.14.0/star_salmon/salmon.merged.gene_lengths.tsv Nag-run-2/results-3.14.0/star_salmon/tx2gene.tsv
Nag-run-2/results-3.14.0/star_salmon/salmon.merged.gene_tpm.tsv
Results are found in /projects/tomato_genome/fnb/dataprocessing/brandon_work/NEXTFLOW/start_fresh
for Nag, Tam, and Mal
In Mal, Hei, Nag, Tam, there are the full compliment of bam files and bai index files.
And the sizes appear correct.
For tsv salmon counts, it looks as though only Heinz has the tsv file of counts, the other 3 runs do not.
Something is amiss !
In mal, there is an error along the nextflow path:
Command executed:
inner_distance.py \
-i M.25C.0hr.U.1.sorted.bam \
-r blat-mal.bed \
-o M.25C.0hr.U.1 \
\
> stdout.txt
head -n 2 stdout.txt > M.25C.0hr.U.1.inner_distance_mean.txt
cat <<-END_VERSIONS > versions.yml
"NFCORE_RNASEQ:RNASEQ:BAM_RSEQC:RSEQC_INNERDISTANCE":
rseqc: $(inner_distance.py --version | sed -e "s/inner_distance.py //g")
END_VERSIONS
And in Tam, there is a totally different error, it gets further along before error occurs.
These are nasty in that they do not crash the pipeline but let it continue rolling along.
We need to back up a few steps and rerun Mal, Tam and Nag.
Rob needs to review that all 4 runs appear complete.
Will check for proper bam sizes, bai, and bedgraphs, etc.
And that the salmon table exists.
To make a bed file:
cat ../postblat/blat-heinz-bestLongHit.fna | awk '$0 ~ "^>"
{name=substr($0, 2); printf name"\t1\t"}$0 !~ "^>"
{printf length($0)"\t"name"\n"}' > blat-heinz.bed
And all the bam files exist:
To check:
/projects/tomato_genome/fnb/dataprocessing/brandon_work/NEXTFLOW/start_fresh$ ls -lrt Tam-run-2/results-3.14.0/star_salmon/*bam
There are some size discrepancies across the bam files, the smallest being 800MB and the largest 5GB. But no idea if that is anything to be concerned about.
Next phase: Create 1 large salmon counts file using the results from these 4 salmon files. That means new ticket!