[IGBF-3772] Run NEXTFLOW on contigs that are NOT SL4 or SL5 - JIRA UNCC

Robert Reid created issue - 11/Jun/24 9:02 AM

Robert Reid made changes - 11/Jun/24 9:02 AM

Field	Original Value	New Value
Epic Link		IGBF-2993 [ 21429 ]

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Robert Reid added a comment - 11/Jun/24 9:39 AM

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

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Robert Reid added a comment - 11/Jun/24 9:39 AM 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

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Robert Reid added a comment - 11/Jun/24 9:57 AM

Trinity has a tool to make a GTF via perl.

perl /apps/pkg/trinity/2.14.0/util/misc/cdna_fasta_file_to_transcript_gtf.pl tamaulipas-trinity.Trinity.fasta | grep -w "exon" - > Trinity.gtf

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Robert Reid added a comment - 11/Jun/24 9:57 AM Trinity has a tool to make a GTF via perl. perl /apps/pkg/trinity/2.14.0/util/misc/cdna_fasta_file_to_transcript_gtf.pl tamaulipas-trinity.Trinity.fasta | grep -w "exon" - > Trinity.gtf

Robert Reid made changes - 12/Jun/24 11:59 AM

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Robert Reid added a comment - 12/Jun/24 12:00 PM

Location:
/projects/tomato_genome/fnb/dataprocessing/trinity/NEXTFLOW/hei-run1

Initial run:

./doIt.sh heinz-SRP499796-files.csv ../heinz-trinity.Trinity.fasta Trinity.gtf blat-heinz.bed tomato.config 1> out.txt 2> err.txt

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Robert Reid added a comment - 12/Jun/24 12:00 PM Location: /projects/tomato_genome/fnb/dataprocessing/trinity/NEXTFLOW/hei-run1 Initial run: ./doIt.sh heinz-SRP499796-files.csv ../heinz-trinity.Trinity.fasta Trinity.gtf blat-heinz.bed tomato.config 1> out.txt 2> err.txt

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Robert Reid added a comment - 17/Jun/24 4:35 PM

Wrong heinz fasta file. Tried using the full 800,000 contigs as the reference file. It crashed, samtools could not even index it.

Using the blat-bestlonghit.fna file instead. This is the fasta sequences that had the best matches to the original Heinz reference via blat.

/projects/tomato_genome/fnb/dataprocessing/trinity/NEXTFLOW/hei-run1$ seqinfo blat-heinz-bestLongHit.fna

File blat-heinz-bestLongHit.fna

Number of sequences 38857

Residue counts:
Total 102938111

Sequence lengths:
Minimum 184
Maximum 28109
Average 2649.15
N50 3592

Rerunning script as follows:
./doIt.sh heinz-SRP499796-files.csv blat-heinz-bestLongHit.fna Trinity.gtf blat-heinz.bed tomato.config 1> out.txt 2> err.txt1

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Robert Reid added a comment - 17/Jun/24 4:35 PM Wrong heinz fasta file. Tried using the full 800,000 contigs as the reference file. It crashed, samtools could not even index it. Using the blat-bestlonghit.fna file instead. This is the fasta sequences that had the best matches to the original Heinz reference via blat. /projects/tomato_genome/fnb/dataprocessing/trinity/NEXTFLOW/hei-run1$ seqinfo blat-heinz-bestLongHit.fna File blat-heinz-bestLongHit.fna Number of sequences 38857 Residue counts: Total 102938111 Sequence lengths: Minimum 184 Maximum 28109 Average 2649.15 N50 3592 Rerunning script as follows: ./doIt.sh heinz-SRP499796-files.csv blat-heinz-bestLongHit.fna Trinity.gtf blat-heinz.bed tomato.config 1> out.txt 2> err.txt1

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Robert Reid added a comment - 17/Jun/24 4:36 PM

Mental note for Rob:

Add the bloody modules!

srun --partition Draco --cpus-per-task 16 --mem-per-cpu 12000 --time 60:00:00 --pty bash

module load nf-core
module load singularity

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Robert Reid added a comment - 17/Jun/24 4:36 PM Mental note for Rob: Add the bloody modules! srun --partition Draco --cpus-per-task 16 --mem-per-cpu 12000 --time 60:00:00 --pty bash module load nf-core module load singularity

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Robert Reid added a comment - 18/Jun/24 11:34 AM

cat blat-heinz-bestLongHit.fna | awk '$0 ~ "^>"

{name=substr($0, 2); printf name"\t1\t"}

' > blat-heinz.bed

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Robert Reid added a comment - 18/Jun/24 11:34 AM cat blat-heinz-bestLongHit.fna | awk '$0 ~ "^>" {name=substr($0, 2); printf name"\t1\t"} ' > blat-heinz.bed

Ann Loraine made changes - 21/Jun/24 8:25 PM

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Robert Reid made changes - 28/Jun/24 10:02 AM

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Robert Reid made changes - 28/Jun/24 10:03 AM

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Robert Reid [ robertreid ]

Brandon Bendickson [ bbendick ]

Ann Loraine made changes - 30/Jun/24 8:52 AM

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Ann Loraine made changes - 01/Jul/24 10:36 AM

Description

GOAL:
To set up and run nextflow on our various 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

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

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Ann Loraine added a comment - 01/Jul/24 10:39 AM - edited

Notes from Ann:

Use the latest version of the nf-core/rnaseq pipeline, or other pipeline as appropriate given the goals. Ann asked the URC team to install a newer version of the nextflow, which the latest rnaseq pipeline requires.
Rob: Investigate whether there is another nf-core pipeline that is better for what we want to do: "Count the number of aligned reads for each assembled contig, where the contigs are hypothetical transcript sequences"
Rob: Also, before doing anything further, check results from contig quality checking - "RNA-quast"

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Ann Loraine added a comment - 01/Jul/24 10:39 AM - edited Notes from Ann: Use the latest version of the nf-core/rnaseq pipeline, or other pipeline as appropriate given the goals. Ann asked the URC team to install a newer version of the nextflow, which the latest rnaseq pipeline requires. Rob: Investigate whether there is another nf-core pipeline that is better for what we want to do: "Count the number of aligned reads for each assembled contig, where the contigs are hypothetical transcript sequences" Rob: Also, before doing anything further, check results from contig quality checking - "RNA-quast"

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Robert Reid added a comment - 05/Jul/24 10:11 AM

Ann's run of Nextflow on the tardigrade recently.

https://jira.bioviz.org/browse/IGBF-3790

This is a bit better than Molly's doc (Only took 2 months to become antiquated!!!!)

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Robert Reid added a comment - 05/Jul/24 10:11 AM Ann's run of Nextflow on the tardigrade recently. https://jira.bioviz.org/browse/IGBF-3790 This is a bit better than Molly's doc (Only took 2 months to become antiquated!!!!)

Brandon Bendickson made changes - 11/Jul/24 9:37 AM

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Ann Loraine made changes - 14/Jul/24 2:40 PM

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Ann Loraine made changes - 14/Jul/24 2:40 PM

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Robert Reid made changes - 15/Jul/24 9:24 AM

Link

This issue blocks IGBF-3817 [ IGBF-3817 ]

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Robert Reid added a comment - 17/Jul/24 8:42 AM

Update on these Nextflow runs.

Brandon is running into an additional tmp folder write / remove issue that needs resolving.
Otherwise it is VERY close to the end.
Hopefully we will meet today and hash out any other problems that arise.

Once to the end, we will rerun on the other 3 varieties to confirm all is working ok.

Also, this ticket needs to be moved back to in progress but I was not able to, only have the option to move it in the forward direction.

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Robert Reid added a comment - 17/Jul/24 8:42 AM Update on these Nextflow runs. Brandon is running into an additional tmp folder write / remove issue that needs resolving. Otherwise it is VERY close to the end. Hopefully we will meet today and hash out any other problems that arise. Once to the end, we will rerun on the other 3 varieties to confirm all is working ok. Also, this ticket needs to be moved back to in progress but I was not able to, only have the option to move it in the forward direction.

Brandon Bendickson made changes - 18/Jul/24 3:15 PM

Comment

[ Besthits.fna file had many duplicates causing errors with the samtools sort command, resulting in the pipeline dying on me. I made a tmp directory so I could troubleshoot. I removed all duplicates using this command: awk '!seen[$1]++' heinz_rna_spades.gtf > heinz_bestlonghit_no_dupes.fna. Nextflow was completed successfully after using the no dupes file as my fasta file.

Results: /projects/tomato_genome/fnb/dataprocessing/brandon_work/NEXTFLOW/Hei-run-1/tmp ]

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Brandon Bendickson added a comment - 19/Jul/24 1:13 PM

I am running into issues with the bed file. Heinz ran without error, despite having an empty bed file. I tried using the commands in an earlier comment to generate a bed file but resulted in an error and the death of the run. I also tried using an empty bed file, like for Heinz, but also resulted in an error.

INFO: Converting SIF file to temporary sandbox...
[E::idx_find_and_load] Could not retrieve index file for 'M.25C.3hr.U.3.sorted.bam'
Get exon regions from blat-mal.bed ...
Traceback (most recent call last):
File "/usr/local/bin/inner_distance.py", line 95, in <module>
main()
File "/usr/local/bin/inner_distance.py", line 87, in main
obj.mRNA_inner_distance(outfile=options.output_prefix,low_bound=options.lower_bound_size,up_bound=options.upper_bound_size,step=options.step_size,refbed=options.ref_gene,sample_size=options.sampleSize, q_cut = options.map_qual)
File "/usr/local/lib/python3.9/site-packages/qcmodule/SAM.py", line 3580, in mRNA_inner_distance
for exn in bed_obj.getExon():
File "/usr/local/lib/python3.9/site-packages/qcmodule/BED.py", line 478, in getExon
chrom_start = int(f[1])
ValueError: invalid literal for int() with base 10: 'Solyc08T000468.1'
INFO: Cleaning up image...

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Brandon Bendickson added a comment - 19/Jul/24 1:13 PM I am running into issues with the bed file. Heinz ran without error, despite having an empty bed file. I tried using the commands in an earlier comment to generate a bed file but resulted in an error and the death of the run. I also tried using an empty bed file, like for Heinz, but also resulted in an error. INFO: Converting SIF file to temporary sandbox... [E::idx_find_and_load] Could not retrieve index file for 'M.25C.3hr.U.3.sorted.bam' Get exon regions from blat-mal.bed ... Traceback (most recent call last): File "/usr/local/bin/inner_distance.py", line 95, in <module> main() File "/usr/local/bin/inner_distance.py", line 87, in main obj.mRNA_inner_distance(outfile=options.output_prefix,low_bound=options.lower_bound_size,up_bound=options.upper_bound_size,step=options.step_size,refbed=options.ref_gene,sample_size=options.sampleSize, q_cut = options.map_qual) File "/usr/local/lib/python3.9/site-packages/qcmodule/SAM.py", line 3580, in mRNA_inner_distance for exn in bed_obj.getExon(): File "/usr/local/lib/python3.9/site-packages/qcmodule/BED.py", line 478, in getExon chrom_start = int(f [1] ) ValueError: invalid literal for int() with base 10: 'Solyc08T000468.1' INFO: Cleaning up image...

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Brandon Bendickson added a comment - 24/Jul/24 11:42 AM

The final run on Nag just completed. All results are found in /projects/tomato_genome/fnb/dataprocessing/brandon_work/NEXTFLOW

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Brandon Bendickson added a comment - 24/Jul/24 11:42 AM The final run on Nag just completed. All results are found in /projects/tomato_genome/fnb/dataprocessing/brandon_work/NEXTFLOW

Ann Loraine made changes - 28/Jul/24 12:04 PM

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Summer 2, Summer 3, Summer 4, Summer 5 [ 196, 197, 198, 199 ]

Summer 2, Summer 3, Summer 4, Summer 5, Summer 6 [ 196, 197, 198, 199, 200 ]

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Ann Loraine made changes - 11/Aug/24 5:11 PM

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Summer 2, Summer 3, Summer 4, Summer 5, Summer 6 [ 196, 197, 198, 199, 200 ]

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Robert Reid made changes - 13/Aug/24 9:43 AM

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Robert Reid [ robertreid ]

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Robert Reid added a comment - 13/Aug/24 9:44 AM

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.

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Robert Reid added a comment - 13/Aug/24 9:44 AM 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.

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Robert Reid added a comment - 15/Aug/24 9:27 AM

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.

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Robert Reid added a comment - 15/Aug/24 9:27 AM 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.

Robert Reid made changes - 15/Aug/24 9:28 AM

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Robert Reid [ robertreid ]

Brandon Bendickson [ bbendick ]

Robert Reid made changes - 15/Aug/24 9:28 AM

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Robert Reid made changes - 15/Aug/24 9:32 AM

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Robert Reid made changes - 15/Aug/24 9:32 AM

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Brandon Bendickson made changes - 22/Aug/24 10:13 AM

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Brandon Bendickson added a comment - 22/Aug/24 10:18 AM

Results are found in /projects/tomato_genome/fnb/dataprocessing/brandon_work/NEXTFLOW/start_fresh
for Nag, Tam, and Mal

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Brandon Bendickson added a comment - 22/Aug/24 10:18 AM Results are found in /projects/tomato_genome/fnb/dataprocessing/brandon_work/NEXTFLOW/start_fresh for Nag, Tam, and Mal

Robert Reid made changes - 26/Aug/24 9:28 AM

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Brandon Bendickson [ bbendick ]

Robert Reid [ robertreid ]

Ann Loraine made changes - 26/Aug/24 10:32 AM

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Summer 2, Summer 3, Summer 4, Summer 5, Summer 6, Summer 7 [ 196, 197, 198, 199, 200, 201 ]

Summer 2, Summer 3, Summer 4, Summer 5, Summer 6, Summer 7, Fall 1 [ 196, 197, 198, 199, 200, 201, 202 ]

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Robert Reid added a comment - 27/Aug/24 9:02 AM

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

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Robert Reid added a comment - 27/Aug/24 9:02 AM 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

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Robert Reid added a comment - 27/Aug/24 9:47 AM

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!

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Robert Reid added a comment - 27/Aug/24 9:47 AM 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!