miRanda is a target prediction software, used to identify likely miRNA-mRNA interactions.
Inputs:
FASTA of A.pulchra lncRNAs, generated in 08-Apul-lncRNA
FASTA of A.pulchra mature miRNAs miRNA_mature-Apul.fasta. miRNAs identified in 04-Apul-sRNA-discovery-ShortStack, matures isolated for use in 06-Apul-miRNA-mRNA-RNAhybrid
# from lncRNA gtf
/home/shared/bedtools2/bin/bedtools getfasta -fi ../data/Apulchra-genome.fa -bed ../output/08-Apul-lncRNA/lncRNAs.gtf -fo ../output/10-Apul-lncRNA-miRNA-miRanda/lncRNAs.fa
# score cutoff >100
# energy cutoff <-10
# strict binding
/home/shared/miRanda-3.3a/src/miranda \
../data/06-Apul-miRNA-mRNA-RNAhybrid/miRNA_mature-Apul.fasta \
../output/10-Apul-lncRNA-miRNA-miRanda/lncRNAs.fa \
-sc 100 \
-en -10 \
-strict \
-out ../output/10-Apul-lncRNA-miRNA-miRanda/Apul-miRanda-lncRNA-strict_all.tabLet’s look at the output
echo "miranda run finished!"
echo "Counting number of interacting miRNA-lncRNA pairs"
zgrep -c "Performing Scan" ../output/10-Apul-lncRNA-miRNA-miRanda/Apul-miRanda-lncRNA-strict_all.tab
echo "Parsing output"
grep -A 1 "Scores for this hit:" ../output/10-Apul-lncRNA-miRNA-miRanda/Apul-miRanda-lncRNA-strict_all.tab | sort | grep '>' > ../output/10-Apul-lncRNA-miRNA-miRanda/Apul-miRanda-lncRNA-strict-parsed.txt
echo "counting number of putative interactions predicted (can include multiple interactions between single miRNA-lncRNA pair)"
wc -l ../output/10-Apul-lncRNA-miRNA-miRanda/Apul-miRanda-lncRNA-strict_all.tab
## miranda run finished!
## Counting number of interacting miRNA-lncRNA pairs
## 1233231
## Parsing output
## counting number of putative interactions predicted (can include multiple interactions between single miRNA-lncRNA pair)
## 12105116 ../output/10-Apul-lncRNA-miRNA-miRanda/Apul-miRanda-lncRNA-strict_all.tabThis is a lot of putative interactions! We can probably narrow it down though. In vertebrates, miRNA-mRNA binding only requires complementarity of an miRNA seed region of ~8 nucleotides. This requirement is built in to miRanda target prediction. In cnidarians, however, miRNA-mRNA binding is believed to require near-complete complementarity of the full mature miRNA, similarly to plants. While I couldn’t find any information on expected requirements for miRNA-lncRNA sponges, its possible the binding will function similarly to miRNA-mRNa binding. Let’s look at how many putative interactions are predicted for a binding length of at least 21 nucleotides (the length of our smallest mature miRNA).
echo "number of putative interactions of at least 21 nucleotides"
awk -F'\t' '$7 >= 21' ../output/10-Apul-lncRNA-miRNA-miRanda/Apul-miRanda-lncRNA-strict-parsed.txt | wc -l
echo ""
echo "check some:"
awk -F'\t' '$7 >= 21' ../output/10-Apul-lncRNA-miRNA-miRanda/Apul-miRanda-lncRNA-strict-parsed.txt | head -5## number of putative interactions of at least 21 nucleotides
## 24018
##
## check some:
## >Cluster_10452.mature::ptg000020l:10483758-10483779(-) ntLink_3:56720-62176 150.00 -18.45 2 21 351 375 22 63.64% 77.27%
## >Cluster_10452.mature::ptg000020l:10483758-10483779(-) ntLink_3:76351-81676 150.00 -18.45 2 21 220 244 22 63.64% 77.27%
## >Cluster_10452.mature::ptg000020l:10483758-10483779(-) ntLink_3:95704-100623 150.00 -18.45 2 21 378 402 22 63.64% 77.27%
## >Cluster_10452.mature::ptg000020l:10483758-10483779(-) ntLink_4:152868-157343 159.00 -17.21 2 21 2472 2495 21 66.67% 80.95%
## >Cluster_10452.mature::ptg000020l:10483758-10483779(-) ntLink_4:205718-210236 159.00 -17.21 2 21 2451 2474 21 66.67% 80.95%We can also see from the alignment percentages (last 2 entries) that this number includes alignments with multiple mismatches. Let’s filter again to reduce the number of permissible mismatches. Let’s say we want no more than 3 mismatches. For an alignment of 21 nucleotides, this would be an alignment rate of (21-3)/21 = 85.7%.
echo "number of putative interactions of at least 21 nucleotides, with at most 3 mismatches"
awk -F'\t' '$7 >= 21' ../output/10-Apul-lncRNA-miRNA-miRanda/Apul-miRanda-lncRNA-strict-parsed.txt | awk -F'\t' '$8 >= 85' | wc -l
echo ""
echo "check some:"
awk -F'\t' '$7 >= 21' ../output/10-Apul-lncRNA-miRNA-miRanda/Apul-miRanda-lncRNA-strict-parsed.txt | awk -F'\t' '$8 >= 85' | head -5## number of putative interactions of at least 21 nucleotides, with at most 3 mismatches
## 22
##
## check some:
## >Cluster_14532.mature::ptg000025l:7472581-7472603(-) ptg000021l:14212189-14212865 174.00 -23.17 2 22 384 406 21 85.71% 85.71%
## >Cluster_14532.mature::ptg000025l:7472581-7472603(-) ptg000023l:17245108-17245424 174.00 -18.76 2 22 49 71 21 85.71% 85.71%
## >Cluster_14532.mature::ptg000025l:7472581-7472603(-) ptg000024l:8680062-8686568 178.00 -21.90 2 22 3861 3883 21 85.71% 90.48%
## >Cluster_14532.mature::ptg000025l:7472581-7472603(-) ptg000024l:8680105-8686841 178.00 -21.90 2 22 3818 3840 21 85.71% 90.48%
## >Cluster_14532.mature::ptg000025l:7472581-7472603(-) ptg000026l:4000983-4003199 180.00 -21.97 2 22 185 208 21 85.71% 85.71%