[IGBF-3809] RNA- Quast deeper dive and explore - JIRA UNCC

Details

Type: Task
Status: Closed (View Workflow)
Priority: Minor
Resolution: Done
Affects Version/s: None
Fix Version/s: None
Labels:
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Story Points:
2
Epic Link:
Support NSF pollen grant
Sprint:
Summer 4, Summer 5, Summer 6, Summer 7

Description

Goal:

To dive deeper into RNA-Quast and determine what our results are.

The tool works on the cluster and Brandon has got it to run on the de novo assemblies.

So while we got results, we need to be able to interpret them.
And figure out how to harness this tool.

So that means reading the original paper and exploring how other papers (rnaSpades paper) use this as a metric for assessing their assemblies.

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Robert Reid added a comment - 02/Jul/24 2:15 PM

The paper about the tool:
https://academic.oup.com/bioinformatics/article/32/14/2210/1743439

rnaQUAST: a quality assessment tool for de novo transcriptome assemblies
Elena Bushmanova, Dmitry Antipov, Alla Lapidus, Vladimir Suvorov, Andrey D. Prjibelski Author Notes
Bioinformatics, Volume 32, Issue 14, July 2016, Pages 2210–2212, https://doi.org/10.1093/bioinformatics/btw218
Published: 23 April 2016

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Robert Reid added a comment - 02/Jul/24 2:15 PM The paper about the tool: https://academic.oup.com/bioinformatics/article/32/14/2210/1743439 rnaQUAST: a quality assessment tool for de novo transcriptome assemblies Elena Bushmanova, Dmitry Antipov, Alla Lapidus, Vladimir Suvorov, Andrey D. Prjibelski Author Notes Bioinformatics, Volume 32, Issue 14, July 2016, Pages 2210–2212, https://doi.org/10.1093/bioinformatics/btw218 Published: 23 April 2016

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Robert Reid added a comment - 02/Jul/24 2:19 PM

"By comparing the resulting alignments with the gene database, rnaQUAST calculates various statistics and generates a summary report."

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Robert Reid added a comment - 02/Jul/24 2:19 PM "By comparing the resulting alignments with the gene database, rnaQUAST calculates various statistics and generates a summary report."

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Robert Reid added a comment - 07/Aug/24 1:11 PM

Scripts from initial rna-quast run are located here:

/projects/tomato_genome/fnb/dataprocessing/brandon_work/tam/tam-rnaquast
/projects/tomato_genome/fnb/dataprocessing/brandon_work/nag/nag-rnaquast
/projects/tomato_genome/fnb/dataprocessing/brandon_work/mal/mal-rnaquast
/projects/tomato_genome/fnb/dataprocessing/brandon_work/hei/hei-rnaquast

Within each of these folders are

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Robert Reid added a comment - 07/Aug/24 1:11 PM Scripts from initial rna-quast run are located here: /projects/tomato_genome/fnb/dataprocessing/brandon_work/tam/tam-rnaquast /projects/tomato_genome/fnb/dataprocessing/brandon_work/nag/nag-rnaquast /projects/tomato_genome/fnb/dataprocessing/brandon_work/mal/mal-rnaquast /projects/tomato_genome/fnb/dataprocessing/brandon_work/hei/hei-rnaquast Within each of these folders are

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Robert Reid added a comment - 07/Aug/24 1:11 PM

RnaQuast is now a module on the cluster also:

module load rnaquast
Loading rnaquast/2.3.0

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Robert Reid added a comment - 07/Aug/24 1:11 PM RnaQuast is now a module on the cluster also: module load rnaquast Loading rnaquast/2.3.0

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Robert Reid added a comment - 07/Aug/24 1:23 PM - edited

The actual python rnaquast script is buried deep in the env.

module load rnaquast
/apps/pkg/anaconda3/envs/rnaquast-2.3.0/bin/rnaQUAST.py

Sample command:
(https://github.com/ablab/rnaquast?tab=readme-ov-file#sec2)

python /apps/pkg/anaconda3/envs/rnaquast-2.3.0/bin/rnaQUAST.py \
--transcripts /PATH/TO/transcripts1.fasta /PATH/TO/ANOTHER/transcripts2.fasta /PATH/TO/MULTIPLE/*.fasta [...] \
--reference /PATH/TO/reference_genome.fasta --gtf /PATH/TO/gene_coordinates.gtf

All of the options:
python /apps/pkg/anaconda3/envs/rnaquast-2.3.0/bin/rnaQUAST.py
usage: /apps/pkg/anaconda3/envs/rnaquast-2.3.0/bin/rnaQUAST.py [-h] [~~r REFERENCE [REFERENCE ...]] [~~-gtf GTF [GTF ...]] [--gene_db GENE_DB] [-c TRANSCRIPTS [TRANSCRIPTS ...]] [-psl ALIGNMENT [ALIGNMENT ...]] [-sam READS_ALIGNMENT] [-1 LEFT_READS]
[-2 RIGHT_READS] [-s SINGLE_READS] [--gmap_index GMAP_INDEX] [-o OUTPUT_DIR] [--test] [-d] [-t THREADS] [-l LABELS [LABELS ...]] [-ss] [--min_alignment MIN_ALIGNMENT] [--no_plots]
[--blat] [--gene_mark] [--meta] [--lower_threshold LOWER_THRESHOLD] [--upper_threshold UPPER_THRESHOLD] [--disable_infer_genes] [--disable_infer_transcripts] [--busco BUSCO]
[--prokaryote]

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Robert Reid added a comment - 07/Aug/24 1:23 PM - edited The actual python rnaquast script is buried deep in the env. module load rnaquast /apps/pkg/anaconda3/envs/rnaquast-2.3.0/bin/rnaQUAST.py Sample command: ( https://github.com/ablab/rnaquast?tab=readme-ov-file#sec2 ) python /apps/pkg/anaconda3/envs/rnaquast-2.3.0/bin/rnaQUAST.py \ --transcripts /PATH/TO/transcripts1.fasta /PATH/TO/ANOTHER/transcripts2.fasta /PATH/TO/MULTIPLE/*.fasta [...] \ --reference /PATH/TO/reference_genome.fasta --gtf /PATH/TO/gene_coordinates.gtf All of the options: python /apps/pkg/anaconda3/envs/rnaquast-2.3.0/bin/rnaQUAST.py usage: /apps/pkg/anaconda3/envs/rnaquast-2.3.0/bin/rnaQUAST.py [-h] [ r REFERENCE [REFERENCE ...] ] [ -gtf GTF [GTF ...] ] [--gene_db GENE_DB] [-c TRANSCRIPTS [TRANSCRIPTS ...] ] [-psl ALIGNMENT [ALIGNMENT ...] ] [-sam READS_ALIGNMENT] [-1 LEFT_READS] [-2 RIGHT_READS] [-s SINGLE_READS] [--gmap_index GMAP_INDEX] [-o OUTPUT_DIR] [--test] [-d] [-t THREADS] [-l LABELS [LABELS ...] ] [-ss] [--min_alignment MIN_ALIGNMENT] [--no_plots] [--blat] [--gene_mark] [--meta] [--lower_threshold LOWER_THRESHOLD] [--upper_threshold UPPER_THRESHOLD] [--disable_infer_genes] [--disable_infer_transcripts] [--busco BUSCO] [--prokaryote]

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Robert Reid added a comment - 09/Aug/24 9:22 AM

Tried out a test run.

Slurm script for this run can be found here:
/projects/tomato_genome/scripts/rob/rnaquastRun.slurm

Reported run log says it was a success!
Results are here:
/projects/tomato_genome/fnb/dataprocessing/trinity/rnaquast/rnaQUAST_results/

We get a lot for a short run!

~~rw-r~~r- 1 rreid2 tomato_genome 0 Aug 8 19:30 blat_tamaulipas_bestLongHit.unaligned.fasta
~~rw-r~~r- 1 rreid2 tomato_genome 13M Aug 8 19:30 blat_tamaulipas_bestLongHit.paralogs.fasta
~~rw-r~~r- 1 rreid2 tomato_genome 2.8M Aug 8 19:30 blat_tamaulipas_bestLongHit.misassembled.fasta
~~rw-r~~r- 1 rreid2 tomato_genome 3.5M Aug 8 19:30 blat_tamaulipas_bestLongHit.misassembled.blat.fasta
~~rw-r~~r- 1 rreid2 tomato_genome 46M Aug 8 19:30 blat_tamaulipas_bestLongHit.misassembled.blast.fasta
~~rw-r~~r- 1 rreid2 tomato_genome 51M Aug 8 19:30 blat_tamaulipas_bestLongHit.correct.fasta
~~rw-r~~r- 1 rreid2 tomato_genome 839K Aug 8 19:30 blat_tamaulipas_bestLongHit.unannotated.fasta
~~rw-r~~r- 1 rreid2 tomato_genome 217K Aug 8 19:30 blat_tamaulipas_bestLongHit.95%-assembled.list
~~rw-r~~r- 1 rreid2 tomato_genome 318K Aug 8 19:30 blat_tamaulipas_bestLongHit.50%-assembled.list

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Robert Reid added a comment - 09/Aug/24 9:22 AM Tried out a test run. Slurm script for this run can be found here: /projects/tomato_genome/scripts/rob/rnaquastRun.slurm Reported run log says it was a success! Results are here: /projects/tomato_genome/fnb/dataprocessing/trinity/rnaquast/rnaQUAST_results/ We get a lot for a short run! rw-r r - 1 rreid2 tomato_genome 0 Aug 8 19:30 blat_tamaulipas_bestLongHit.unaligned.fasta rw-r r - 1 rreid2 tomato_genome 13M Aug 8 19:30 blat_tamaulipas_bestLongHit.paralogs.fasta rw-r r - 1 rreid2 tomato_genome 2.8M Aug 8 19:30 blat_tamaulipas_bestLongHit.misassembled.fasta rw-r r - 1 rreid2 tomato_genome 3.5M Aug 8 19:30 blat_tamaulipas_bestLongHit.misassembled.blat.fasta rw-r r - 1 rreid2 tomato_genome 46M Aug 8 19:30 blat_tamaulipas_bestLongHit.misassembled.blast.fasta rw-r r - 1 rreid2 tomato_genome 51M Aug 8 19:30 blat_tamaulipas_bestLongHit.correct.fasta rw-r r - 1 rreid2 tomato_genome 839K Aug 8 19:30 blat_tamaulipas_bestLongHit.unannotated.fasta rw-r r - 1 rreid2 tomato_genome 217K Aug 8 19:30 blat_tamaulipas_bestLongHit.95%-assembled.list rw-r r - 1 rreid2 tomato_genome 318K Aug 8 19:30 blat_tamaulipas_bestLongHit.50%-assembled.list

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Robert Reid added a comment - 09/Aug/24 9:29 AM

SHORT SUMMARY REPORT

METRICS/TRANSCRIPTS blat_tamaulipas_bestLongHit blat_malintka_bestLongHit blat_nagcarlang_bestLongHit blat_heinz_bestLongHit SL5.cds

== DATABASE METRICS ==
Genes 36648 36648 36648 36648 36648

Avg. number of exons per isoform 5.888 5.888 5.888 5.888 5.888

== BASIC TRANSCRIPTS METRICS ==
Transcripts 27867 27208 27100 27380 43752

Transcripts > 500 bp 25328 24941 25013 25142 29854

Transcripts > 1000 bp 21769 21590 21789 21935 18306

== ALIGNMENT METRICS ==
Aligned 27867 27201 27087 27374 43752

Uniquely aligned 25077 24169 23922 24258 43646

Multiply aligned 4819 5131 5177 51
39 105
Unaligned 0 7 13 6 0

== ALIGNMENT METRICS FOR NON-MISASSEMBLED TRANSCRIPTS ==
Avg. aligned fraction 0.968 0.982 0.982 0.982 0.999

Avg. alignment length 2379.194 2398.298 2643.803 2550.996 1119.497

Avg. mismatches per transcript 4.863 4.179 4.506 3.893 0.062

== ALIGNMENT METRICS FOR MISASSEMBLED (CHIMERIC) TRANSCRIPTS ==
Misassemblies 816 603 693 668 0

== ASSEMBLY COMPLETENESS (SENSITIVITY) ==
Database coverage 0.496 0.514 0.509 0.512 0.75

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Robert Reid added a comment - 09/Aug/24 9:29 AM SHORT SUMMARY REPORT METRICS/TRANSCRIPTS blat_tamaulipas_bestLongHit blat_malintka_bestLongHit blat_nagcarlang_bestLongHit blat_heinz_bestLongHit SL5.cds == DATABASE METRICS == Genes 36648 36648 36648 36648 36648 Avg. number of exons per isoform 5.888 5.888 5.888 5.888 5.888 == BASIC TRANSCRIPTS METRICS == Transcripts 27867 27208 27100 27380 43752 Transcripts > 500 bp 25328 24941 25013 25142 29854 Transcripts > 1000 bp 21769 21590 21789 21935 18306 == ALIGNMENT METRICS == Aligned 27867 27201 27087 27374 43752 Uniquely aligned 25077 24169 23922 24258 43646 Multiply aligned 4819 5131 5177 51 39 105 Unaligned 0 7 13 6 0 == ALIGNMENT METRICS FOR NON-MISASSEMBLED TRANSCRIPTS == Avg. aligned fraction 0.968 0.982 0.982 0.982 0.999 Avg. alignment length 2379.194 2398.298 2643.803 2550.996 1119.497 Avg. mismatches per transcript 4.863 4.179 4.506 3.893 0.062 == ALIGNMENT METRICS FOR MISASSEMBLED (CHIMERIC) TRANSCRIPTS == Misassemblies 816 603 693 668 0 == ASSEMBLY COMPLETENESS (SENSITIVITY) == Database coverage 0.496 0.514 0.509 0.512 0.75

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Robert Reid added a comment - 09/Aug/24 9:30 AM

Well this is great and wonderful and all that.
A new ticket shall be created to take these results where we export off of the cluster and come up with a cleaner way to present the findings!

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Robert Reid added a comment - 09/Aug/24 9:30 AM Well this is great and wonderful and all that. A new ticket shall be created to take these results where we export off of the cluster and come up with a cleaner way to present the findings! R

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Robert Reid added a comment - 09/Aug/24 9:32 AM

Let's have Brandon do a review of the script and folders and then close this ticket.

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Robert Reid added a comment - 09/Aug/24 9:32 AM Let's have Brandon do a review of the script and folders and then close this ticket.

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Robert Reid added a comment - 13/Aug/24 9:42 AM

Brandon and I sat and walked through the script and quickly reviewed the results.

The next step is to run this on the RNASpades results so that we can compare those.
Closing this ticket and making a new one!

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Robert Reid added a comment - 13/Aug/24 9:42 AM Brandon and I sat and walked through the script and quickly reviewed the results. The next step is to run this on the RNASpades results so that we can compare those. Closing this ticket and making a new one!

People

Assignee:

Brandon Bendickson

Reporter:

Robert Reid

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Dates

Created:

02/Jul/24 9:38 AM

Updated:

13/Aug/24 9:43 AM

Resolved:

13/Aug/24 9:43 AM