Details
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Type:
Task
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Status: Closed (View Workflow)
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Priority:
Trivial
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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:3
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Epic Link:
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Sprint:Spring 6, Spring 7, Spring 8, Spring 9, Spring 10
Description
GOAL: to set up a script to run Trinity on Kelsey data.
Eventually get this script integrated into bitbucket for future purposes.
This dataset could then be aligned as contigs to SL4 and SL5.
This dataset could also be fed into IGB as a "reference" and we can look at how the raw sequences align the newly created contigs.
Attachments
Activity
Blat runs were a success.
Going to close this task and start a number of related new ones.
- Need to have these runs tested. New student Brandon starts this week, so this is a good task for him to learn the HPC cluster and how to learn slurm.
- Need a new task to start a google slide deck outlining this project. This will suitable for a biweekly tomato meeting. This will also allow me to ponder the next best steps.
- Need a new task to parse the results generated.
#
First blat run was a success.
As per Ann's suggestion to use pslx to get the sequence also, I will run again like so:
blat /projects/tomato_genome/db/SL5/SL5.cds.fa ./infile.fa blat-$
{file}.pslx \-ooc=${file}
.11.ooc \
-t=dna -q=dna \
-maxIntron=10000 \
-out=pslx
This slrum script can be found at:
/projects/tomato_genome/fnb/dataprocessing/trinity
Once this works, we will repeat for the other 3 varieties. And then create new ticket for the next sprint to parse the results, create a refined set of contigs with good annotations to become the NEW reference genome and prepare NETFLOW.
Success on all 4 trinity runs.
Locations of the assembled contigs:
- /projects/tomato_genome/fnb/dataprocessing/trinity/mal/malintka-trinity.Trinity.fasta
- /projects/tomato_genome/fnb/dataprocessing/trinity/hei/heinz-trinity.Trinity.fasta
- /projects/tomato_genome/fnb/dataprocessing/trinity/nag/nagcarlang-trinity.Trinity.fasta
- /projects/tomato_genome/fnb/dataprocessing/trinity/tam/tamaulipas-trinity.Trinity.fasta
Now need to decide on next steps.
Blat to get annotations.
Star align expt reads to these contigs or a subset to get the read counts. (Nextflow / salmon)
We expect 35,000 genes. We HAVE MANY more than that.
File tamaulipas-trinity.Trinity.fasta
Number of sequences 797,447
Hopefully we have isoforms. In reality we will have many chimeras that are not biologically true.
Will start step 2, the blat script to align reads back to the Heinz de novo transcripts.
For Tam run, totally different error than above.
Transcript polishing issue......stay tuned.
1 of 4 runs are completed.
Got an error in 2 of the runs that I have never seen before.
Parafly error.
Apparently it is related to multithreading.
The first bit of advice is to simply relaunch the runs.
Will try this first. And then work backwards from there.
MANY steps in the process have been completed.
The command line for a Trinity de novo run is:
Trinity --seqType fq --max_memory 400G --output foo-trinity \
--left foo_r1_paired.fq \
--right foo_r2_paired.fq --CPU 24
Ran into a SNAFU. My mega collection of reads might be corupted. Trinity does not like the files.
Using a smaller subset to ensure that the code works correctly.
And this smaller subset works great, creating 158,000 contigs.
Now to redo the making of the larger read set to be used......
Putting notes related to the Trinity reference free strategy.
This location will contain random notes related to Trinity.
https://drive.google.com/drive/folders/1yMG7gWB67-falaLF1o-kDCQIK_E8mwgu?usp=drive_link
Where this project will reside on HPC:
/projects/tomato_genome/fnb/dataprocessing/trinity
Ok, this next step is a bit too crazy for a one line command line.
Bash script needed!
So Rob as run the scrips a few years ago!
/projects/tomato_genome/scripts/rob/scripts/
rwx-w--- 1 rreid2 tomato_genome 2.7K Jun 30 2021 trinity-tam.slurm
rwx-w--- 1 rreid2 tomato_genome 2.7K Jun 30 2021 trinity-mal.slurm
rwx-w--- 1 rreid2 tomato_genome 2.7K Jun 30 2021 trinity-hei.slurm
rwx-w--- 1 rreid2 tomato_genome 2.7K Jul 7 2021 trinity-nag.slurm
These 4 scripts are for each of the 4 original varieties.
And are a good starting point going forward.
Plan:
Make a copy of script.
gather the previous sequence data of marks for TMNH varieties PLUS Kelseys data and make a MEGA large raw sequence file.
Run Trinity on each variety to make a set of de novo contigs.
Script to parse the blast results.
~/scripts/python/identifyBlatMatches.py ./infile.fa ./blat-tamaulipas.pslx
Command for later to summarize the Trinity contigs.
{ OFS = "\t" }/projects/tomato_genome/fnb/dataprocessing/trinity/tam/blat$ awk -F "_i" 'BEGIN
{ print $1 }' tmp2