e5 closest
e5 deep dive expression - miRNA target prediction
I am redoing the miRNA target prediction analysis for the deep dive expression project. Kathleen reran the miRNA short stack with a different version of the software and the results changed slightly (see issue).
For miRNA target prediction, the 3’UTR sequences of the genes are needed. These typically aren’t annotated in coral genomes but I manually added them by adding 1000bp to the right flank of the gene and subtracting any overlap with nearby genes (see post). I already have the 3’UTR sequences for our species of interest woohoo. Download updated miRNA sequences onto Andromeda for target prediction with miranda software.
Note - miRNA sequences are not named yet as of 11/1/24.
Apulchra
Download new sequences and rename fasta files. Copied fasta sequences from here.
cd /data/putnamlab/jillashey/e5/mirna_seqs
nano Apul_updated_results.fasta # paste fasta sequences
grep -A 1 'mature' Apul_updated_results.fasta | grep -v '^--$' > Apul_updated_results_mature.fasta
zgrep -c ">" Apul_updated_results_mature.fasta
39
In original run (using old short stack version and Amil genome), 38 mature miRNAs were identified. With updated short stack and new Apul genome, 39 mature miRNAs were identified.
I already have the 3’UTR fasta file here: /data/putnamlab/jillashey/e5/refs/Apul/Apul_3UTR_1kb.fasta. In the scripts folder: nano miranda_strict_all_1kb_apul_updated.sh
#!/bin/bash -i
#SBATCH -t 48:00:00
#SBATCH --nodes=1 --ntasks-per-node=10
#SBATCH --export=NONE
#SBATCH --mem=500GB
#SBATCH --mail-type=BEGIN,END,FAIL #email you when job starts, stops and/or fails
#SBATCH --mail-user=jillashey@uri.edu #your email to send notifications
#SBATCH --account=putnamlab
#SBATCH -D /data/putnamlab/jillashey/e5/scripts
#SBATCH -o slurm-%j.out
#SBATCH -e slurm-%j.error
echo "Apul starting miranda run with all genes and miRNAs with energy cutoff <-20 and strict binding invoked"$(date)
echo "Using updated miRNAs from newer short stack version"
module load Miniconda3/4.9.2
conda activate /data/putnamlab/conda/miranda
miranda /data/putnamlab/jillashey/e5/mirna_seqs/Apul_updated_results_mature.fasta /data/putnamlab/jillashey/e5/refs/Apul/Apul_3UTR_1kb.fasta -en -20 -strict -out /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_apul_updated.tab
conda deactivate
echo "miranda run finished!"$(date)
echo "counting number of interactions attempted" $(date)
zgrep -c "Performing Scan" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_apul_updated.tab
echo "Parsing output" $(date)
grep -A 1 "Scores for this hit:" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_apul.tab | sort | grep '>' > /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_parsed_apul_updated.txt
echo "counting number of putative interactions predicted" $(date)
wc -l /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_parsed_apul_updated.txt
echo "Apul miranda script complete" $(date)
Submitted batch job 346351. Results:
counting number of interactions attempted Fri Nov 1 14:02:45 EDT 2024
2062788
Parsing output Fri Nov 1 14:02:46 EDT 2024
counting number of putative interactions predicted Fri Nov 1 14:02:46 EDT 2024
6109 /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_parsed_apul_updated.txt
With previous miRNAs and Amil genome, there were 4144 putative interactions predicted. With updated miRNAs and Apul genome, 6109 putative interactions were predicted.
It is possible that miRNAs also bind to coding regions as well. Perform miranda run using coding sequences as targets. First, extract sequences from genome using bedtools.
interactive
cd /data/putnamlab/jillashey/e5/refs/Apul
module load BEDTools/2.30.0-GCC-11.3.0
awk '$3 == "transcript"' genome.fixed.gtf > Apul_transcripts.gtf
bedtools getfasta -fi /data/putnamlab/REFS/Apul/apul.hifiasm.s55_pa.p_ctg.fa.k32.w100.z1000.ntLink.5rounds.masked.fa -bed Apul_transcripts.gtf -fo apul_transcripts.fasta
grep -c ">" apul_transcripts.fasta
44371
wc -l Apul_transcripts.gtf
44371
Run miranda. In the scripts folder: nano miranda_strict_all_mrna_apul_updated.sh
#!/bin/bash -i
#SBATCH -t 48:00:00
#SBATCH --nodes=1 --ntasks-per-node=10
#SBATCH --export=NONE
#SBATCH --mem=500GB
#SBATCH --mail-type=BEGIN,END,FAIL #email you when job starts, stops and/or fails
#SBATCH --mail-user=jillashey@uri.edu #your email to send notifications
#SBATCH --account=putnamlab
#SBATCH -D /data/putnamlab/jillashey/e5/scripts
#SBATCH -o slurm-%j.out
#SBATCH -e slurm-%j.error
echo "Apul starting miranda run with mRNAs as targets and miRNAs with energy cutoff <-20 and strict binding invoked"$(date)
echo "Using updated miRNAs from newer short stack version"
module load Miniconda3/4.9.2
conda activate /data/putnamlab/conda/miranda
miranda /data/putnamlab/jillashey/e5/mirna_seqs/Apul_updated_results_mature.fasta /data/putnamlab/jillashey/e5/refs/Apul/apul_transcripts.fasta -en -20 -strict -out /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_apul_updated.tab
conda deactivate
echo "miranda run finished!"$(date)
echo "counting number of interactions attempted" $(date)
zgrep -c "Performing Scan" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_apul_updated.tab
echo "Parsing output" $(date)
grep -A 1 "Scores for this hit:" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_apul_updated.tab | sort | grep '>' > /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_parsed_apul_updated.txt
echo "counting number of putative interactions predicted" $(date)
wc -l /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_parsed_apul_updated.txt
echo "Apul miranda script complete" $(date)
Submitted batch job 361539
Pevermanni
Download new sequences and rename fasta files. Copied fasta sequences from here.
cd /data/putnamlab/jillashey/e5/mirna_seqs
nano Peve_updated_results.fasta # paste fasta sequences
grep -A 1 'mature' Peve_updated_results.fasta | grep -v '^--$' > Peve_updated_results_mature.fasta
zgrep -c ">" Peve_updated_results_mature.fasta
45
In original run (using old short stack version), 46 mature miRNAs were identified. With updated short stack, 45 mature miRNAs were identified.
I already have the 3’UTR fasta file here: /data/putnamlab/jillashey/genome/Peve/peve_3UTR_1kb.fasta. In the scripts folder: nano miranda_strict_all_1kb_peve_updated.sh
#!/bin/bash -i
#SBATCH -t 48:00:00
#SBATCH --nodes=1 --ntasks-per-node=10
#SBATCH --export=NONE
#SBATCH --mem=500GB
#SBATCH --mail-type=BEGIN,END,FAIL #email you when job starts, stops and/or fails
#SBATCH --mail-user=jillashey@uri.edu #your email to send notifications
#SBATCH --account=putnamlab
#SBATCH -D /data/putnamlab/jillashey/e5/scripts
#SBATCH -o slurm-%j.out
#SBATCH -e slurm-%j.error
echo "Peve starting miranda run with all genes and miRNAs with energy cutoff <-20 and strict binding invoked"$(date)
echo "Using updated miRNAs from newer short stack version"
module load Miniconda3/4.9.2
conda activate /data/putnamlab/conda/miranda
miranda /data/putnamlab/jillashey/e5/mirna_seqs/Peve_updated_results_mature.fasta /data/putnamlab/jillashey/genome/Peve/peve_3UTR_1kb.fasta -en -20 -strict -out /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_peve_updated.tab
conda deactivate
echo "miranda run finished!"$(date)
echo "counting number of interactions attempted" $(date)
zgrep -c "Performing Scan" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_peve_updated.tab
echo "Parsing output" $(date)
grep -A 1 "Scores for this hit:" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_peve_updated.tab | sort | grep '>' > /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_parsed_peve_updated.txt
echo "counting number of putative interactions predicted" $(date)
wc -l /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_parsed_peve_updated.txt
echo "Peve miranda script complete" $(date)
Submitted batch job 346352. Results:
counting number of interactions attempted Fri Nov 1 14:05:10 EDT 2024
1750500
Parsing output Fri Nov 1 14:05:17 EDT 2024
counting number of putative interactions predicted Fri Nov 1 14:05:17 EDT 2024
5067 /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_parsed_peve_updated.txt
With previous miRNAs, there were 7187 putative interactions predicted. With updated miRNAs, 5067 putative interactions were predicted.
It is possible that miRNAs also bind to coding regions as well. Perform miranda run using coding sequences as targets. First, extract sequences from genome using bedtools.
interactive
cd /data/putnamlab/jillashey/genome/Peve
module load BEDTools/2.30.0-GCC-11.3.0
bedtools getfasta -fi Porites_evermanni_v1.fa -bed peve_GFFannotation.mRNA_sorted.gff -fo peve_mRNA.fasta
grep -c ">" peve_mRNA.fasta
40389
wc -l peve_GFFannotation.mRNA_sorted.gff
40389
Run miranda. In the scripts folder: nano miranda_strict_all_mrna_peve_updated.sh
#!/bin/bash -i
#SBATCH -t 48:00:00
#SBATCH --nodes=1 --ntasks-per-node=10
#SBATCH --export=NONE
#SBATCH --mem=500GB
#SBATCH --mail-type=BEGIN,END,FAIL #email you when job starts, stops and/or fails
#SBATCH --mail-user=jillashey@uri.edu #your email to send notifications
#SBATCH --account=putnamlab
#SBATCH -D /data/putnamlab/jillashey/e5/scripts
#SBATCH -o slurm-%j.out
#SBATCH -e slurm-%j.error
echo "Peve starting miranda run with mRNAs as targets and miRNAs with energy cutoff <-20 and strict binding invoked"$(date)
echo "Using updated miRNAs from newer short stack version"
module load Miniconda3/4.9.2
conda activate /data/putnamlab/conda/miranda
miranda /data/putnamlab/jillashey/e5/mirna_seqs/Peve_updated_results_mature.fasta /data/putnamlab/jillashey/genome/Peve/peve_mRNA.fasta -en -20 -strict -out /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_peve_updated.tab
conda deactivate
echo "miranda run finished!"$(date)
echo "counting number of interactions attempted" $(date)
zgrep -c "Performing Scan" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_peve_updated.tab
echo "Parsing output" $(date)
grep -A 1 "Scores for this hit:" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_peve_updated.tab | sort | grep '>' > /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_parsed_peve_updated.txt
echo "counting number of putative interactions predicted" $(date)
wc -l /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_parsed_peve_updated.txt
echo "Peve miranda script complete" $(date)
Submitted batch job 361535
Ptuahiniensis
Download new sequences and rename fasta files. Copied fasta sequences from here.
cd /data/putnamlab/jillashey/e5/mirna_seqs
nano Ptuh_updated_results.fasta # paste fasta sequences
grep -A 1 'mature' Ptuh_updated_results.fasta | grep -v '^--$' > Ptuh_updated_results_mature.fasta
zgrep -c ">" Ptuh_updated_results_mature.fasta
37
In original run (using old short stack version), 37 mature miRNAs were identified. With updated short stack, 37 mature miRNAs were identified.
I already have the 3’UTR fasta file here: /data/putnamlab/jillashey/genome/Pmea/Pmea_3UTR_1kb.fasta. In the scripts folder: nano miranda_strict_all_1kb_ptuh_updated.sh
#!/bin/bash -i
#SBATCH -t 48:00:00
#SBATCH --nodes=1 --ntasks-per-node=10
#SBATCH --export=NONE
#SBATCH --mem=500GB
#SBATCH --mail-type=BEGIN,END,FAIL #email you when job starts, stops and/or fails
#SBATCH --mail-user=jillashey@uri.edu #your email to send notifications
#SBATCH --account=putnamlab
#SBATCH -D /data/putnamlab/jillashey/e5/scripts
#SBATCH -o slurm-%j.out
#SBATCH -e slurm-%j.error
echo "Ptuh starting miranda run with all genes and miRNAs with energy cutoff <-20 and strict binding invoked"$(date)
echo "Using updated miRNAs from newer short stack version"
module load Miniconda3/4.9.2
conda activate /data/putnamlab/conda/miranda
miranda /data/putnamlab/jillashey/e5/mirna_seqs/Ptuh_updated_results_mature.fasta /data/putnamlab/jillashey/genome/Pmea/Pmea_3UTR_1kb.fasta -en -20 -strict -out /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_ptuh_updated.tab
conda deactivate
echo "miranda run finished!"$(date)
echo "counting number of interactions attempted" $(date)
zgrep -c "Performing Scan" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_ptuh_updated.tab
echo "Parsing output" $(date)
grep -A 1 "Scores for this hit:" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_ptuh_updated.tab | sort | grep '>' > /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_parsed_ptuh_updated.txt
echo "counting number of putative interactions predicted" $(date)
wc -l /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_parsed_ptuh_updated.txt
echo "Peve miranda script complete" $(date)
Submitted batch job 346353. Results:
counting number of interactions attempted Fri Nov 1 14:04:16 EDT 2024
1204979
Parsing output Fri Nov 1 14:04:19 EDT 2024
counting number of putative interactions predicted Fri Nov 1 14:04:19 EDT 2024
4105 /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_1kb_parsed_ptuh_updated.txt
With previous miRNAs, there were 3863 putative interactions predicted. With updated miRNAs, 4105 putative interactions were predicted.
It is possible that miRNAs also bind to coding regions as well. Perform miranda run using coding sequences as targets. First, extract sequences from genome using bedtools.
interactive
cd /data/putnamlab/jillashey/genome/Pmea
module load BEDTools/2.30.0-GCC-11.3.0
bedtools getfasta -fi Pocillopora_meandrina_HIv1.assembly.fasta -bed Pmea_transcript.gtf -fo pmea_mrna.fasta
grep -c ">" pmea_mrna.fasta
31840
wc -l Pmea_transcript.gtf
31840
Run miranda. In the scripts folder: nano miranda_strict_all_mrna_ptuh_updated.sh
#!/bin/bash -i
#SBATCH -t 48:00:00
#SBATCH --nodes=1 --ntasks-per-node=10
#SBATCH --export=NONE
#SBATCH --mem=500GB
#SBATCH --mail-type=BEGIN,END,FAIL #email you when job starts, stops and/or fails
#SBATCH --mail-user=jillashey@uri.edu #your email to send notifications
#SBATCH --account=putnamlab
#SBATCH -D /data/putnamlab/jillashey/e5/scripts
#SBATCH -o slurm-%j.out
#SBATCH -e slurm-%j.error
echo "Pmea starting miranda run with mRNAs as targets and miRNAs with energy cutoff <-20 and strict binding invoked"$(date)
echo "Using updated miRNAs from newer short stack version"
module load Miniconda3/4.9.2
conda activate /data/putnamlab/conda/miranda
miranda /data/putnamlab/jillashey/e5/mirna_seqs/Ptuh_updated_results_mature.fasta /data/putnamlab/jillashey/genome/Pmea/pmea_mrna.fasta -en -20 -strict -out /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_ptuh_updated.tab
conda deactivate
echo "miranda run finished!"$(date)
echo "counting number of interactions attempted" $(date)
zgrep -c "Performing Scan" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_ptuh_updated.tab
echo "Parsing output" $(date)
grep -A 1 "Scores for this hit:" /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_ptuh_updated.tab | sort | grep '>' > /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_parsed_ptuh_updated.txt
echo "counting number of putative interactions predicted" $(date)
wc -l /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_parsed_ptuh_updated.txt
echo "Pmea miranda script complete" $(date)
Submitted batch job 361538. Ran in a little under an hour. Results:
counting number of interactions attempted Thu Feb 13 11:34:11 EST 2025
1178080
Parsing output Thu Feb 13 11:34:14 EST 2025
counting number of putative interactions predicted Thu Feb 13 11:34:15 EST 2025
26311 /data/putnamlab/jillashey/e5/output/miranda/miranda_strict_all_mrna_parsed_ptuh_updated.txt
WAY may putative interactions predicted using mRNAs than 3’UTR.