e5 closest
e5 deep dive - looking at closest genes to ncRNAs
The e5 deep dive project is examining ncRNA dynamics in 3 species of coral from Moorea, French Polynesia. The github for the project is here.
In this post, I will be assessing what genomic features are closest to the ncRNAs that we have identified through the deep dive. I will need gffs and fasta files for this task.
Important miRNA files
Important piRNA files
Important lncRNA files
- Apul lncRNA bed; Apul lncRNA fasta
- Peve lncRNA bed; Peve lncRNA fasta
- Ptuh lncRNA bed; Ptuh lncRNA fasta
Because I don’t own the deep dive repo, I copied all needed files onto my local computer and then put them on Andromeda. I also added the species prefix to each file.
cd /data/putnamlab/jillashey/e5
mkdir ncRNA_gff
cd ncRNA_gff
mkdir miRNA piRNA lncRNA
Acropora pulchra
Apul miRNA
Sort the genome gff file for Apul. We are using the Amil genome for reference.
cd /data/putnamlab/jillashey/genome/Amil_v2.01/
wget http://gannet.fish.washington.edu/seashell/snaps/GCF_013753865.1_Amil_v2.1_genomic.gff
sort -k1,1 -k4,4n GCF_013753865.1_Amil_v2.1_genomic.gff > GCF_013753865.1_Amil_v2.1_genomic_sorted.gff
Sort miRNA gff file and run bed closest.
cd /data/putnamlab/jillashey/e5/ncRNA_gff/miRNA
sort -k1,1 -k4,4n Apul_Results.gff3 > Apul_Results_sorted.gff3
interactive
module load BEDTools/2.30.0-GCC-11.3.0
bedtools closest -a Apul_Results_sorted.gff3 -b /data/putnamlab/jillashey/genome/Amil_v2.01/GCF_013753865.1_Amil_v2.1_genomic_sorted.gff > Apul_output.bed
wc -l Apul_output.bed
61599 Apul_output.bed
head Apul_output.bed
NC_058066.1 ShortStack Unknown_sRNA_locus 152483 152910 140 - . ID=Cluster_1;DicerCall=N;MIRNA=N NC_058066.1 RefSeq region 1 39361238 . + . ID=NC_058066.1:1..39361238;Dbxref=taxon:45264;Name=1;chromosome=1;collection-date=2017;country=Indonesia;gbkey=Src;genome=chromosome;isolate=JS-1;isolation-source=Whole tissue;mol_type=genomic DNA;tissue-type=Adult tissue
NC_058066.1 ShortStack Unknown_sRNA_locus 152483 152910 140 - . ID=Cluster_1;DicerCall=N;MIRNA=N NC_058066.1 Gnomon gene 92732 195229 . + . ID=gene-LOC114963509;Dbxref=GeneID:114963509;Name=LOC114963509;gbkey=Gene;gene=LOC114963509;gene_biotype=protein_coding
NC_058066.1 ShortStack Unknown_sRNA_locus 152483 152910 140 - . ID=Cluster_1;DicerCall=N;MIRNA=N NC_058066.1 Gnomon mRNA 92732 195229 . + . ID=rna-XM_044317725.1;Parent=gene-LOC114963509;Dbxref=GeneID:114963509,Genbank:XM_044317725.1;Name=XM_044317725.1;Note=The sequence of the model RefSeq transcript was modified relative to this genomic sequence to represent the inferred CDS: deleted 1 base in 1 codon;exception=unclassified transcription discrepancy;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114963509;model_evidence=Supporting evidence includes similarity to: 2 mRNAs%2C 3 ESTs%2C 12 Proteins%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 1 sample with support for all annotated introns;product=uncharacterized LOC114963509;transcript_id=XM_044317725.1
NC_058066.1 ShortStack Unknown_sRNA_locus 152483 152910 140 - . ID=Cluster_1;DicerCall=N;MIRNA=N NC_058066.1 Gnomon gene 145277 165521 . + . ID=gene-LOC122957574;Dbxref=GeneID:122957574;Name=LOC122957574;gbkey=Gene;gene=LOC122957574;gene_biotype=protein_coding
NC_058066.1 ShortStack Unknown_sRNA_locus 152483 152910 140 - . ID=Cluster_1;DicerCall=N;MIRNA=N NC_058066.1 Gnomon mRNA 145277 165521 . + . ID=rna-XM_044317744.1;Parent=gene-LOC122957574;Dbxref=GeneID:122957574,Genbank:XM_044317744.1;Name=XM_044317744.1;gbkey=mRNA;gene=LOC122957574;model_evidence=Supporting evidence includes similarity to: 5 Proteins%2C and 99%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 2 samples with support for all annotated introns;product=uncharacterized LOC122957574;transcript_id=XM_044317744.1
NC_058066.1 ShortStack Unknown_sRNA_locus 152483 152910 140 - . ID=Cluster_1;DicerCall=N;MIRNA=N NC_058066.1 Gnomon CDS 152268 152501 . + 2 ID=cds-XP_044173679.1;Parent=rna-XM_044317744.1;Dbxref=GeneID:122957574,Genbank:XP_044173679.1;Name=XP_044173679.1;gbkey=CDS;gene=LOC122957574;product=uncharacterized protein LOC122957574;protein_id=XP_044173679.1
NC_058066.1 ShortStack Unknown_sRNA_locus 152483 152910 140 - . ID=Cluster_1;DicerCall=N;MIRNA=N NC_058066.1 Gnomon exon 152268 152501 . + . ID=exon-XM_044317744.1-4;Parent=rna-XM_044317744.1;Dbxref=GeneID:122957574,Genbank:XM_044317744.1;gbkey=mRNA;gene=LOC122957574;product=uncharacterized LOC122957574;transcript_id=XM_044317744.1
NC_058066.1 ShortStack Unknown_sRNA_locus 161064 161674 549 . . ID=Cluster_2;DicerCall=N;MIRNA=N NC_058066.1 RefSeq region 1 39361238 . + . ID=NC_058066.1:1..39361238;Dbxref=taxon:45264;Name=1;chromosome=1;collection-date=2017;country=Indonesia;gbkey=Src;genome=chromosome;isolate=JS-1;isolation-source=Whole tissue;mol_type=genomic DNA;tissue-type=Adult tissue
NC_058066.1 ShortStack Unknown_sRNA_locus 161064 161674 549 . . ID=Cluster_2;DicerCall=N;MIRNA=N NC_058066.1 Gnomon gene 92732 195229 . + . ID=gene-LOC114963509;Dbxref=GeneID:114963509;Name=LOC114963509;gbkey=Gene;gene=LOC114963509;gene_biotype=protein_coding
NC_058066.1 ShortStack Unknown_sRNA_locus 161064 161674 549 . . ID=Cluster_2;DicerCall=N;MIRNA=N NC_058066.1 Gnomon mRNA 92732 195229 . + . ID=rna-XM_044317725.1;Parent=gene-LOC114963509;Dbxref=GeneID:114963509,Genbank:XM_044317725.1;Name=XM_044317725.1;Note=The sequence of the model RefSeq transcript was modified relative to this genomic sequence to represent the inferred CDS: deleted 1 base in 1 codon;exception=unclassified transcription discrepancy;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114963509;model_evidence=Supporting evidence includes similarity to: 2 mRNAs%2C 3 ESTs%2C 12 Proteins%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 1 sample with support for all annotated introns;product=uncharacterized LOC114963509;transcript_id=XM_044317725.1
Remove the unknown siRNA loci
awk 'BEGIN {OFS="\t"} $3 != "Unknown_sRNA_locus"' Apul_output.bed > filtered_Apul_output.bed
wc -l filtered_Apul_output.bed
755 filtered_Apul_output.bed
head filtered_Apul_output.bed
NC_058066.1 ShortStack siRNA24_locus 5224792 5225215 1264 + . ID=Cluster_184;DicerCall=24;MIRNA=N NC_058066.1 RefSeq region 1 39361238 . + . ID=NC_058066.1:1..39361238;Dbxref=taxon:45264;Name=1;chromosome=1;collection-date=2017;country=Indonesia;gbkey=Src;genome=chromosome;isolate=JS-1;isolation-source=Whole tissue;mol_type=genomic DNA;tissue-type=Adult tissue
NC_058066.1 ShortStack siRNA24_locus 5224792 5225215 1264 + . ID=Cluster_184;DicerCall=24;MIRNA=N NC_058066.1 Gnomon gene 5153290 5231353 . - . ID=gene-LOC114950433;Dbxref=GeneID:114950433;Name=LOC114950433;gbkey=Gene;gene=LOC114950433;gene_biotype=protein_coding
NC_058066.1 ShortStack siRNA24_locus 5224792 5225215 1264 + . ID=Cluster_184;DicerCall=24;MIRNA=N NC_058066.1 Gnomon mRNA 5153290 5231353 . - . ID=rna-XM_044310280.1;Parent=gene-LOC114950433;Dbxref=GeneID:114950433,Genbank:XM_044310280.1;Name=XM_044310280.1;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114950433;model_evidence=Supporting evidence includes similarity to: 15 mRNAs%2C 31 Proteins%2C and 99%25 coverage of the annotated genomic feature by RNAseq alignments;product=uncharacterized LOC114950433;transcript_id=XM_044310280.1
NC_058066.1 ShortStack siRNA21_locus 7563377 7563797 118 + . ID=Cluster_225;DicerCall=21;MIRNA=N NC_058066.1 RefSeq region 1 39361238 . + . ID=NC_058066.1:1..39361238;Dbxref=taxon:45264;Name=1;chromosome=1;collection-date=2017;country=Indonesia;gbkey=Src;genome=chromosome;isolate=JS-1;isolation-source=Whole tissue;mol_type=genomic DNA;tissue-type=Adult tissue
NC_058066.1 ShortStack siRNA21_locus 7563377 7563797 118 + . ID=Cluster_225;DicerCall=21;MIRNA=N NC_058066.1 Gnomon gene 7523354 7569602 . - . ID=gene-LOC114970982;Dbxref=GeneID:114970982;Name=LOC114970982;gbkey=Gene;gene=LOC114970982;gene_biotype=protein_coding
NC_058066.1 ShortStack siRNA21_locus 7563377 7563797 118 + . ID=Cluster_225;DicerCall=21;MIRNA=N NC_058066.1 Gnomon mRNA 7523354 7569602 . - . ID=rna-XM_044320669.1;Parent=gene-LOC114970982;Dbxref=GeneID:114970982,Genbank:XM_044320669.1;Name=XM_044320669.1;Note=The sequence of the model RefSeq transcript was modified relative to this genomic sequence to represent the inferred CDS: inserted 1 base in 1 codon;exception=unclassified transcription discrepancy;gbkey=mRNA;gene=LOC114970982;model_evidence=Supporting evidence includes similarity to: 6 Proteins%2C and 98%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 1 sample with support for all annotated introns;product=uncharacterized LOC114970982;transcript_id=XM_044320669.1
NC_058066.1 ShortStack siRNA22_locus 8905068 8905484 102 - . ID=Cluster_251;DicerCall=22;MIRNA=N NC_058066.1 RefSeq region 1 39361238 . + . ID=NC_058066.1:1..39361238;Dbxref=taxon:45264;Name=1;chromosome=1;collection-date=2017;country=Indonesia;gbkey=Src;genome=chromosome;isolate=JS-1;isolation-source=Whole tissue;mol_type=genomic DNA;tissue-type=Adult tissue
NC_058066.1 ShortStack MIRNA_hairpin 12757125 12757218 8293 - . ID=Cluster_316;DicerCall=23;MIRNA=Y NC_058066.1 RefSeq region 1 39361238 . + . ID=NC_058066.1:1..39361238;Dbxref=taxon:45264;Name=1;chromosome=1;collection-date=2017;country=Indonesia;gbkey=Src;genome=chromosome;isolate=JS-1;isolation-source=Whole tissue;mol_type=genomic DNA;tissue-type=Adult tissue
NC_058066.1 ShortStack MIRNA_hairpin 12757125 12757218 8293 - . ID=Cluster_316;DicerCall=23;MIRNA=Y NC_058066.1 Gnomon gene 12755159 12764546 . - . ID=gene-LOC114961148;Dbxref=GeneID:114961148;Name=LOC114961148;gbkey=Gene;gene=LOC114961148;gene_biotype=protein_coding
NC_058066.1 ShortStack MIRNA_hairpin 12757125 12757218 8293 - . ID=Cluster_316;DicerCall=23;MIRNA=Y NC_058066.1 Gnomon mRNA 12755159 12764546 . - . ID=rna-XM_029339755.2;Parent=gene-LOC114961148;Dbxref=GeneID:114961148,Genbank:XM_029339755.2;Name=XM_029339755.2;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114961148;model_evidence=Supporting evidence includes similarity to: 7 mRNAs%2C 8 Proteins%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 87 samples with support for all annotated introns;product=zinc finger MYND domain-containing protein 19-like;transcript_id=XM_029339755.2
Some of the closest features to the miRNAs are “regions”, which seems to be large sections of chromosomes or chromosomes themselves. Because I only care about which mRNAs are closest to the miRNA features, I’m going to subset the gff by mRNA.
cd /data/putnamlab/jillashey/genome/Amil_v2.01
awk '$3 == "mRNA"' GCF_013753865.1_Amil_v2.1_genomic_sorted.gff > GCF_013753865.1_Amil_v2.1_genomic_sorted_mRNA.gff
Run bed closest
cd /data/putnamlab/jillashey/e5/ncRNA_gff/miRNA
interactive
module load BEDTools/2.30.0-GCC-11.3.0
bedtools closest -a Apul_Results_sorted.gff3 -b /data/putnamlab/jillashey/genome/Amil_v2.01/GCF_013753865.1_Amil_v2.1_genomic_sorted_mRNA.gff > Apul_output_mRNA_only.bed
wc -l Apul_output.bed
24039 Apul_output_mRNA_only.bed
Remove the unknown siRNA loci
awk 'BEGIN {OFS="\t"} $3 != "Unknown_sRNA_locus"' Apul_output_mRNA_only.bed > filtered_Apul_output_mRNA_only.bed
Apul piRNA
The gff file is already sorted above and the piRNA bed file is also already sorted. Run bed closest
cd /data/putnamlab/jillashey/e5/ncRNA_gff/piRNA
interactive
module load BEDTools/2.30.0-GCC-11.3.0
bedtools closest -a APUL.merged.clusters.bed -b /data/putnamlab/jillashey/genome/Amil_v2.01/GCF_013753865.1_Amil_v2.1_genomic_sorted_mRNA.gff > Apul_piRNA_output_mRNA_only.bed
wc -l Apul_piRNA_output_mRNA_only.bed
143 Apul_piRNA_output_mRNA_only.bed
head Apul_piRNA_output_mRNA_only.bed
NC_058066.1 17726050 17734960 NC_058066.1 Gnomon mRNA 17729627 17732829 . + . ID=rna-XM_029347425.2;Parent=gene-LOC114967414;Dbxref=GeneID:114967414,Genbank:XM_029347425.2;Name=XM_029347425.2;gbkey=mRNA;gene=LOC114967414;model_evidence=Supporting evidence includes similarity to: 8 mRNAs%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments;product=zinc finger protein 862-like;transcript_id=XM_029347425.2
NC_058066.1 27441463 27447983 NC_058066.1 Gnomon mRNA 27462364 27464454 . + . ID=rna-XM_029327730.2;Parent=gene-LOC114951564;Dbxref=GeneID:114951564,Genbank:XM_029327730.2;Name=XM_029327730.2;gbkey=mRNA;gene=LOC114951564;model_evidence=Supporting evidence includes similarity to: 3 Proteins;product=zinc finger protein 862-like;transcript_id=XM_029327730.2
NC_058066.1 28121256 28125982 NC_058066.1 Gnomon mRNA 28126954 28127481 . + . ID=rna-XM_044328014.1;Parent=gene-LOC122964458;Dbxref=GeneID:122964458,Genbank:XM_044328014.1;Name=XM_044328014.1;gbkey=mRNA;gene=LOC122964458;model_evidence=Supporting evidence includes similarity to: 100%25 coverage of the annotated genomic feature by RNAseq alignments;product=uncharacterized protein K02A2.6-like;transcript_id=XM_044328014.1
NC_058066.1 28290198 28297001 NC_058066.1 Gnomon mRNA 28275708 28276278 . + . ID=rna-XM_029347200.2;Parent=gene-LOC114967202;Dbxref=GeneID:114967202,Genbank:XM_029347200.2;Name=XM_029347200.2;gbkey=mRNA;gene=LOC114967202;model_evidence=Supporting evidence includes similarity to: 1 mRNA%2C and 16%25 coverage of the annotated genomic feature by RNAseq alignments;product=piggyBac transposable element-derived protein 4-like;transcript_id=XM_029347200.2
NC_058066.1 28445323 28452636 NC_058066.1 Gnomon mRNA 28444999 28446236 . + . ID=rna-XM_029353270.2;Parent=gene-LOC114972849;Dbxref=GeneID:114972849,Genbank:XM_029353270.2;Name=XM_029353270.2;gbkey=mRNA;gene=LOC114972849;model_evidence=Supporting evidence includes similarity to: 67%25 coverage of the annotated genomic feature by RNAseq alignments;product=uncharacterized LOC114972849;transcript_id=XM_029353270.2
NC_058066.1 28445323 28452636 NC_058066.1 Gnomon mRNA 28446503 28451426 . - . ID=rna-XM_044328060.1;Parent=gene-LOC114972848;Dbxref=GeneID:114972848,Genbank:XM_044328060.1;Name=XM_044328060.1;gbkey=mRNA;gene=LOC114972848;model_evidence=Supporting evidence includes similarity to: 2 mRNAs%2C 1 Protein%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 2 samples with support for all annotated introns;product=uncharacterized LOC114972848;transcript_id=XM_044328060.1
NC_058066.1 29297022 29310880 NC_058066.1 Gnomon mRNA 29313159 29314285 . + . ID=rna-XM_044317430.1;Parent=gene-LOC114972063;Dbxref=GeneID:114972063,Genbank:XM_044317430.1;Name=XM_044317430.1;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114972063;model_evidence=Supporting evidence includes similarity to: 1 Protein%2C and 84%25 coverage of the annotated genomic feature by RNAseq alignments;product=uncharacterized LOC114972063;transcript_id=XM_044317430.1
NC_058067.1 14128407 14136847 NC_058067.1 Gnomon mRNA 14118443 14130434 . + . ID=rna-XM_044316573.1;Parent=gene-LOC122956872;Dbxref=GeneID:122956872,Genbank:XM_044316573.1;Name=XM_044316573.1;gbkey=mRNA;gene=LOC122956872;model_evidence=Supporting evidence includes similarity to: 3 mRNAs%2C 3 ESTs%2C 1 Protein%2C and 99%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 35 samples with support for all annotated introns;product=uncharacterized LOC122956872;transcript_id=XM_044316573.1
NC_058067.1 14155111 14163931 NC_058067.1 Gnomon mRNA 14157497 14159293 . + . ID=rna-XM_044316312.1;Parent=gene-LOC122956629;Dbxref=GeneID:122956629,Genbank:XM_044316312.1;Name=XM_044316312.1;gbkey=mRNA;gene=LOC122956629;model_evidence=Supporting evidence includes similarity to: 4 Proteins;product=uncharacterized protein K02A2.6-like;transcript_id=XM_044316312.1
NC_058067.1 30302082 30308566 NC_058067.1 Gnomon mRNA 30314470 30317762 . + . ID=rna-XM_044317247.1;Parent=gene-LOC114948565;Dbxref=GeneID:114948565,Genbank:XM_044317247.1;Name=XM_044317247.1;gbkey=mRNA;gene=LOC114948565;model_evidence=Supporting evidence includes similarity to: 7 mRNAs%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments;product=uncharacterized LOC114948565;transcript_id=XM_044317247.1
Apul lncRNA
The gff file is already sorted above. Sort lncRNa bed file.
cd /data/putnamlab/jillashey/e5/ncRNA_gff/lncRNA
sort -k1,1 -k2,2n -k3,3n Apul_lncRNA.bed > Apul_lncRNA_sorted.bed
There were some issues with negative numbers being present in the starting coordinate position in some of the bed files. When I look at the fasta files for these specific lncRNAs, it says that the start position is 0. I’m going to change any instances of negative numbers to a 0
awk '{if ($2 < 0) $2 = 0; print $1 "\t" $2 "\t" $3}' Apul_lncRNA_sorted.bed > Apul_lncRNA_sorted_fixed.bed
Run bed closest
interactive
module load BEDTools/2.30.0-GCC-11.3.0
bedtools closest -a Apul_lncRNA_sorted_fixed.bed -b /data/putnamlab/jillashey/genome/Amil_v2.01/GCF_013753865.1_Amil_v2.1_genomic_sorted_mRNA.gff > Apul_lncRNA_output_mRNA_only.bed
wc -l Apul_lncRNA_output_mRNA_only.bed
18475 Apul_lncRNA_output_mRNA_only.bed
head Apul_lncRNA_output_mRNA_only.bed
head Apul_lncRNA_output_mRNA_only.bed
NC_058066.1 393116 393357 NC_058066.1 Gnomon mRNA 393884 399722 . + . ID=rna-XM_029329017.2;Parent=gene-LOC114952935;Dbxref=GeneID:114952935,Genbank:XM_029329017.2;Name=XM_029329017.2;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114952935;model_evidence=Supporting evidence includes similarity to: 2 mRNAs%2C 3 Proteins%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 61 samples with support for all annotated introns;product=uncharacterized LOC114952935;transcript_id=XM_029329017.2
NC_058066.1 468617 469943 NC_058066.1 Gnomon mRNA 470084 472938 . + . ID=rna-XM_029329050.2;Parent=gene-LOC114952957;Dbxref=GeneID:114952957,Genbank:XM_029329050.2;Name=XM_029329050.2;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114952957;model_evidence=Supporting evidence includes similarity to: 1 mRNA%2C 23 Proteins%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments;product=trace amine-associated receptor 1-like;transcript_id=XM_029329050.2
NC_058066.1 574074 574816 NC_058066.1 Gnomon mRNA 566968 573222 . - . ID=rna-XM_029328984.2;Parent=gene-LOC114952908;Dbxref=GeneID:114952908,Genbank:XM_029328984.2;Name=XM_029328984.2;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114952908;model_evidence=Supporting evidence includes similarity to: 3 mRNAs%2C 2 ESTs%2C 8 Proteins%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 88 samples with support for all annotated introns;product=titin-like%2C transcript variant X1;transcript_id=XM_029328984.2
NC_058066.1 574074 574816 NC_058066.1 Gnomon mRNA 566968 573222 . - . ID=rna-XM_044317851.1;Parent=gene-LOC114952908;Dbxref=GeneID:114952908,Genbank:XM_044317851.1;Name=XM_044317851.1;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114952908;model_evidence=Supporting evidence includes similarity to: 1 EST%2C 7 Proteins%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 51 samples with support for all annotated introns;product=titin-like%2C transcript variant X2;transcript_id=XM_044317851.1
NC_058066.1 852086 852315 NC_058066.1 Gnomon mRNA 814602 850828 . - . ID=rna-XM_029328850.2;Parent=gene-LOC114952824;Dbxref=GeneID:114952824,Genbank:XM_029328850.2;Name=XM_029328850.2;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114952824;model_evidence=Supporting evidence includes similarity to: 16 mRNAs%2C 1 EST%2C 9 Proteins%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 48 samples with support for all annotated introns;product=ubiquitin carboxyl-terminal hydrolase 24-like;transcript_id=XM_029328850.2
NC_058066.1 853114 853820 NC_058066.1 Gnomon mRNA 854617 868846 . + . ID=rna-XM_029328890.2;Parent=gene-LOC114952850;Dbxref=GeneID:114952850,Genbank:XM_029328890.2;Name=XM_029328890.2;Note=The sequence of the model RefSeq transcript was modified relative to this genomic sequence to represent the inferred CDS: deleted 1 base in 1 codon;exception=unclassified transcription discrepancy;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114952850;model_evidence=Supporting evidence includes similarity to: 1 mRNA%2C 2 ESTs%2C 20 Proteins%2C and 99%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 84 samples with support for all annotated introns;product=endoplasmic reticulum metallopeptidase 1-like;transcript_id=XM_029328890.2
NC_058066.1 946276 946580 NC_058066.1 Gnomon mRNA 947261 949367 . + . ID=rna-XM_029329051.2;Parent=gene-LOC114952958;Dbxref=GeneID:114952958,Genbank:XM_029329051.2;Name=XM_029329051.2;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114952958;model_evidence=Supporting evidence includes similarity to: 8 mRNAs%2C 5 Proteins%2C and 100%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 16 samples with support for all annotated introns;product=dynein light chain Tctex-type 5-B-like;transcript_id=XM_029329051.2
NC_058066.1 1132235 1134678 NC_058066.1 Gnomon mRNA 1088762 1114844 . + . ID=rna-XM_044318173.1;Parent=gene-LOC114952875;Dbxref=GeneID:114952875,Genbank:XM_044318173.1;Name=XM_044318173.1;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114952875;model_evidence=Supporting evidence includes similarity to: 6 mRNAs%2C 2 ESTs%2C 10 Proteins%2C and 99%25 coverage of the annotated genomic feature by RNAseq alignments;product=transient receptor potential cation channel subfamily A member 1-like;transcript_id=XM_044318173.1
NC_058066.1 1135314 1144814 NC_058066.1 Gnomon mRNA 1088762 1114844 . + . ID=rna-XM_044318173.1;Parent=gene-LOC114952875;Dbxref=GeneID:114952875,Genbank:XM_044318173.1;Name=XM_044318173.1;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114952875;model_evidence=Supporting evidence includes similarity to: 6 mRNAs%2C 2 ESTs%2C 10 Proteins%2C and 99%25 coverage of the annotated genomic feature by RNAseq alignments;product=transient receptor potential cation channel subfamily A member 1-like;transcript_id=XM_044318173.1
NC_058066.1 1144882 1148491 NC_058066.1 Gnomon mRNA 1088762 1114844 . + . ID=rna-XM_044318173.1;Parent=gene-LOC114952875;Dbxref=GeneID:114952875,Genbank:XM_044318173.1;Name=XM_044318173.1;experiment=COORDINATES: polyA evidence [ECO:0006239];gbkey=mRNA;gene=LOC114952875;model_evidence=Supporting evidence includes similarity to: 6 mRNAs%2C 2 ESTs%2C 10 Proteins%2C and 99%25 coverage of the annotated genomic feature by RNAseq alignments;product=transient receptor potential cation channel subfamily A member 1-like;transcript_id=XM_044318173.1
Porites evermanni
Peve miRNA
Sort Peve genome gff file
cd /data/putnamlab/jillashey/genome/Peve/
sort -k1,1 -k4,4n Porites_evermanni_v1.annot.gff > Porites_evermanni_v1.annot_sorted.gff
Sort miRNA gff file and run bed closest.
cd /data/putnamlab/jillashey/e5/ncRNA_gff/miRNA
sort -k1,1 -k4,4n Peve_Results.gff3 > Peve_Results_sorted.gff3
interactive
module load BEDTools/2.30.0-GCC-11.3.0
bedtools closest -a Peve_Results_sorted.gff3 -b /data/putnamlab/jillashey/genome/Peve/Porites_evermanni_v1.annot_sorted.gff > Peve_output.bed
wc -l Peve_output.bed
32806 Peve_output.bed
head Peve_output.bed
Porites_evermani_scaffold_1 ShortStack Unknown_sRNA_locus 45711 46131 88 + . ID=Cluster_1;DicerCall=N;MIRNA=N Porites_evermani_scaffold_1 Gmove mRNA 32616 67628 399 - . ID=Peve_00000122;Name=Peve_00000122;start=1;stop=1;cds_size=399
Porites_evermani_scaffold_1 ShortStack Unknown_sRNA_locus 201507 201931 58 - . ID=Cluster_2;DicerCall=N;MIRNA=N Porites_evermani_scaffold_1 Gmove CDS 205241 208000 . + . Parent=Peve_00000106
Porites_evermani_scaffold_1 ShortStack Unknown_sRNA_locus 201507 201931 58 - . ID=Cluster_2;DicerCall=N;MIRNA=N Porites_evermani_scaffold_1 Gmove mRNA 205241 208000 276 + . ID=Peve_00000106;Name=Peve_00000106;start=1;stop=1;cds_size=2760
Porites_evermani_scaffold_1 ShortStack Unknown_sRNA_locus 313446 313846 50 - . ID=Cluster_3;DicerCall=N;MIRNA=N Porites_evermani_scaffold_1 Gmove mRNA 307343 313927 924.6 - . ID=Peve_00000114;Name=Peve_00000114;start=1;stop=1;cds_size=1206
Porites_evermani_scaffold_1 ShortStack Unknown_sRNA_locus 313446 313846 50 - . ID=Cluster_3;DicerCall=N;MIRNA=N Porites_evermani_scaffold_1 Gmove UTR 313287 313927 . - . Parent=Peve_00000114
Porites_evermani_scaffold_1 ShortStack Unknown_sRNA_locus 406146 406734 175 - . ID=Cluster_4;DicerCall=N;MIRNA=N Porites_evermani_scaffold_1 Gmove mRNA 384175 413351 1590.6 - . ID=Peve_00000121;Name=Peve_00000121;start=1;stop=1;cds_size=1860
Porites_evermani_scaffold_1 ShortStack Unknown_sRNA_locus 409839 410269 169 - . ID=Cluster_5;DicerCall=N;MIRNA=N Porites_evermani_scaffold_1 Gmove mRNA 384175 413351 1590.6 - . ID=Peve_00000121;Name=Peve_00000121;start=1;stop=1;cds_size=1860
Porites_evermani_scaffold_1 ShortStack Unknown_sRNA_locus 465244 465668 169 - . ID=Cluster_6;DicerCall=N;MIRNA=N Porites_evermani_scaffold_1 Gmove mRNA 462457 477071 1669.32 - . ID=Peve_00000006;Name=Peve_00000006;start=1;stop=1;cds_size=2034
Porites_evermani_scaffold_1 ShortStack Unknown_sRNA_locus 465244 465668 169 - . ID=Cluster_6;DicerCall=N;MIRNA=N Porites_evermani_scaffold_1 Gmove CDS 465272 465508 . - . Parent=Peve_00000006
Porites_evermani_scaffold_1 ShortStack Unknown_sRNA_locus 468473 468950 91900 - . ID=Cluster_7;DicerCall=N;MIRNA=N Porites_evermani_scaffold_1 Gmove mRNA 462457 477071 1669.32 - . ID=Peve_00000006;Name=Peve_00000006;start=1;stop=1;cds_size=2034
Remove anything that is unknown
awk 'BEGIN {OFS="\t"} $3 != "Unknown_sRNA_locus"' Peve_output.bed > filtered_Peve_output.bed
wc -l filtered_Peve_output.bed
449 filtered_Peve_output.bed
head filtered_Peve_output.bed
Porites_evermani_scaffold_1 ShortStack MIRNA_hairpin 1404250 1404342 9574 - . ID=Cluster_29;DicerCall=N;MIRNA=Y Porites_evermani_scaffold_1 Gmove mRNA 1380413 1416448 1270.53 - . ID=Peve_00000077;Name=Peve_00000077;start=1;stop=1;cds_size=1851
Porites_evermani_scaffold_1 ShortStack mature_miRNA 1404272 1404293 3403 - . ID=Cluster_29.mature;Parent=Cluster_29 Porites_evermani_scaffold_1 Gmove mRNA 1380413 1416448 1270.53 - . ID=Peve_00000077;Name=Peve_00000077;start=1;stop=1;cds_size=1851
Porites_evermani_scaffold_1 ShortStack miRNA-star 1404301 1404322 23 - . ID=Cluster_29.star;Parent=Cluster_29 Porites_evermani_scaffold_1 Gmove mRNA 1380413 1416448 1270.53 - . ID=Peve_00000077;Name=Peve_00000077;start=1;stop=1;cds_size=1851
Porites_evermani_scaffold_10 ShortStack siRNA21_locus 565492 565912 76 + . ID=Cluster_353;DicerCall=21;MIRNA=N Porites_evermani_scaffold_10 Gmove mRNA 562195 564790 948 + . ID=Peve_00000127;Name=Peve_00000127;start=1;stop=1;cds_size=474
Porites_evermani_scaffold_10 ShortStack siRNA21_locus 565492 565912 76 + . ID=Cluster_353;DicerCall=21;MIRNA=N Porites_evermani_scaffold_10 Gmove UTR 564704 564790 . + . Parent=Peve_00000127
Porites_evermani_scaffold_1005 ShortStack siRNA23_locus 126975 127397 168 + . ID=Cluster_9183;DicerCall=23;MIRNA=N Porites_evermani_scaffold_1005 Gmove mRNA 114473 133413 606 + . ID=Peve_00000326;Name=Peve_00000326;start=0;stop=1;cds_size=606
Porites_evermani_scaffold_1060 ShortStack siRNA22_locus 77378 77799 99 + . ID=Cluster_9583;DicerCall=22;MIRNA=N Porites_evermani_scaffold_1060 Gmove mRNA 64377 75861 817.5 + . ID=Peve_00001166;Name=Peve_00001166;start=1;stop=1;cds_size=1275
Porites_evermani_scaffold_1060 ShortStack siRNA22_locus 77378 77799 99 + . ID=Cluster_9583;DicerCall=22;MIRNA=N Porites_evermani_scaffold_1060 Gmove CDS 75822 75861 . + . Parent=Peve_00001166
Porites_evermani_scaffold_108 ShortStack siRNA24_locus 306073 306496 53 - . ID=Cluster_2199;DicerCall=24;MIRNA=N Porites_evermani_scaffold_108 Gmove mRNA 278980 311091 2217.57 - . ID=Peve_00001444;Name=Peve_00001444;start=1;stop=1;cds_size=4026
Porites_evermani_scaffold_108 ShortStack siRNA24_locus 306073 306496 53 - . ID=Cluster_2199;DicerCall=24;MIRNA=N Porites_evermani_scaffold_108 Gmove CDS 306249 306300 . - . Parent=Peve_00001444
Peve piRNA
The gff file is already sorted above. Sort piRNA bed file and run bed closest
cd /data/putnamlab/jillashey/e5/ncRNA_gff/piRNA
interactive
module load BEDTools/2.30.0-GCC-11.3.0
bedtools sort -i PEVE.merged.clusters.bed > PEVE.merged.clusters_sorted.bed
bedtools closest -a PEVE.merged.clusters_sorted.bed -b /data/putnamlab/jillashey/genome/Peve/Porites_evermanni_v1.annot_sorted.gff > Peve_piRNA_output.bed
wc -l Peve_piRNA_output.bed
475 Peve_piRNA_output.bed
head Peve_piRNA_output.bed
Porites_evermani_scaffold_100 452587 464012 Porites_evermani_scaffold_100 Gmove CDS 467740 467796 . + . Parent=Peve_00000202
Porites_evermani_scaffold_100 452587 464012 Porites_evermani_scaffold_100 Gmove mRNA 467740 468875 273 + . ID=Peve_00000202;Name=Peve_00000202;start=1;stop=1;cds_size=273
Porites_evermani_scaffold_1011 75258 81284 Porites_evermani_scaffold_1011 Gmove mRNA 42046 82245 666.253 - . ID=Peve_00000418;Name=Peve_00000418;start=1;stop=1;cds_size=924
Porites_evermani_scaffold_1011 75258 81284 Porites_evermani_scaffold_1011 Gmove CDS 78877 78941 . + . Parent=Peve_00000424
Porites_evermani_scaffold_1011 75258 81284 Porites_evermani_scaffold_1011 Gmove mRNA 78877 80087 1125 + . ID=Peve_00000424;Name=Peve_00000424;start=0;stop=1;cds_size=1125
Porites_evermani_scaffold_1011 75258 81284 Porites_evermani_scaffold_1011 Gmove CDS 79012 79034 . + . Parent=Peve_00000424
Porites_evermani_scaffold_1011 75258 81284 Porites_evermani_scaffold_1011 Gmove CDS 79051 80087 . + . Parent=Peve_00000424
Porites_evermani_scaffold_1024 29005 37949 Porites_evermani_scaffold_1024 Gmove mRNA 28469 29122 195 - . ID=Peve_00000604;Name=Peve_00000604;start=0;stop=1;cds_size=195
Porites_evermani_scaffold_1024 29005 37949 Porites_evermani_scaffold_1024 Gmove CDS 29089 29122 . - . Parent=Peve_00000604
Porites_evermani_scaffold_1024 29005 37949 Porites_evermani_scaffold_1024 Gmove CDS 30636 30770 . - . Parent=Peve_00000611
Peve lncRNA
The gff file is already sorted above. Sort lncRNa bed file.
cd /data/putnamlab/jillashey/e5/ncRNA_gff/lncRNA
sort -k1,1 -k2,2n -k3,3n Peve_lncRNA.bed > Peve_lncRNA_sorted.bed
There were some issues with negative numbers being present in the starting coordinate position in some of the bed files. When I look at the fasta files for these specific lncRNAs, it says that the start position is 0. I’m going to change any instances of negative numbers to a 0
awk '{if ($2 < 0) $2 = 0; print $1 "\t" $2 "\t" $3}' Peve_lncRNA_sorted.bed > Peve_lncRNA_sorted_fixed.bed
Run bed closest
interactive
module load BEDTools/2.30.0-GCC-11.3.0
bedtools closest -a Peve_lncRNA_sorted_fixed.bed -b /data/putnamlab/jillashey/genome/Peve/Porites_evermanni_v1.annot_sorted.gff > Peve_lncRNA_output.bed
wc -l Peve_lncRNA_output.bed
14288 Peve_lncRNA_output.bed
wc -l Peve_lncRNA_output.bed
14288 Peve_lncRNA_output.bed
(base) [jillashey@n065 lncRNA]$ head Peve_lncRNA_output.bed
Porites_evermani_scaffold_1 372244 372449 Porites_evermani_scaffold_1 Gmove mRNA 358046 370091 825.429 + ID=Peve_00000118;Name=Peve_00000118;start=1;stop=1;cds_size=963
Porites_evermani_scaffold_1 372244 372449 Porites_evermani_scaffold_1 Gmove CDS 370050 370091 . + Parent=Peve_00000118
Porites_evermani_scaffold_1 422642 423512 Porites_evermani_scaffold_1 Gmove CDS 424479 425361 . - Parent=Peve_00000002
Porites_evermani_scaffold_1 422642 423512 Porites_evermani_scaffold_1 Gmove mRNA 424479 429034 2439.63 - ID=Peve_00000002;Name=Peve_00000002;start=1;stop=1;cds_size=2019
Porites_evermani_scaffold_1 683877 684280 Porites_evermani_scaffold_1 Gmove CDS 685640 686293 . + Parent=Peve_00000028
Porites_evermani_scaffold_1 683877 684280 Porites_evermani_scaffold_1 Gmove mRNA 685640 686293 65.4 + ID=Peve_00000028;Name=Peve_00000028;start=1;stop=1;cds_size=654
Porites_evermani_scaffold_1 1084866 1089422 Porites_evermani_scaffold_1 Gmove CDS 1093419 1095324 . + Parent=Peve_00000059
Porites_evermani_scaffold_1 1084866 1089422 Porites_evermani_scaffold_1 Gmove mRNA 1093419 1096995 10488 + ID=Peve_00000059;Name=Peve_00000059;start=1;stop=1;cds_size=2622
Porites_evermani_scaffold_1 1202043 1202328 Porites_evermani_scaffold_1 Gmove CDS 1206473 1206925 . + Parent=Peve_00000063
Porites_evermani_scaffold_1 1202043 1202328 Porites_evermani_scaffold_1 Gmove mRNA 1206473 1206925 45.3 + ID=Peve_00000063;Name=Peve_00000063;start=1;stop=0;cds_size=453
Ptuh miRNA
Sort Ptuh genome gff file; in this project, we used the Pmea genome.
cd /data/putnamlab/jillashey/genome/Pmea
sort -k1,1 -k4,4n Pocillopora_meandrina_HIv1.genes.gff3 > Pocillopora_meandrina_HIv1.genes_sorted.gff3
Sort miRNA gff file and run bed closest.
cd /data/putnamlab/jillashey/e5/ncRNA_gff/miRNA
sort -k1,1 -k4,4n Ptuh_Results.gff3 > Ptuh_Results_sorted.gff3
interactive
module load BEDTools/2.30.0-GCC-11.3.0
bedtools closest -a Ptuh_Results_sorted.gff3 -b /data/putnamlab/jillashey/genome/Pmea/Pocillopora_meandrina_HIv1.genes_sorted.gff3 > Ptuh_output.bed
Success!!!
wc -l Ptuh_output.bed
21604 Ptuh_output.bed
head Ptuh_output.bed
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack Unknown_sRNA_locus 9092 9521 10813 + . ID=Cluster_1;DicerCall=N;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 10771 11117 . + 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20902.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack Unknown_sRNA_locus 9092 9521 10813 + . ID=Cluster_1;DicerCall=N;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS exon 10771 11117 . + 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20902.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack Unknown_sRNA_locus 9092 9521 10813 + . ID=Cluster_1;DicerCall=N;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript 10771 23652 . + . ID=Pocillopora_meandrina_HIv1___RNAseq.g20902.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack Unknown_sRNA_locus 53578 53997 287 + . ID=Cluster_2;DicerCall=N;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript 52050 53624 . - . ID=Pocillopora_meandrina_HIv1___RNAseq.g20906.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack Unknown_sRNA_locus 53578 53997 287 + . ID=Cluster_2;DicerCall=N;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 53573 53624 . - 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20906.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack Unknown_sRNA_locus 53578 53997 287 + . ID=Cluster_2;DicerCall=N;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS exon 53573 53624 . - 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20906.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack Unknown_sRNA_locus 150243 150718 2549 - . ID=Cluster_3;DicerCall=N;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript 143552 155669 . - . ID=Pocillopora_meandrina_HIv1___RNAseq.g20914.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack Unknown_sRNA_locus 150243 150718 2549 - . ID=Cluster_3;DicerCall=N;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 150290 150371 . - 1 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20914.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack Unknown_sRNA_locus 150243 150718 2549 - . ID=Cluster_3;DicerCall=N;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS exon 150290 150371 . - 1 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20914.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack Unknown_sRNA_locus 150243 150718 2549 - . ID=Cluster_3;DicerCall=N;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 150573 150661 . - 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20914.t1
Remove everything that is unknown.
awk 'BEGIN {OFS="\t"} $3 != "Unknown_sRNA_locus"' Ptuh_output.bed > filtered_Ptuh_output.bed
wc -l filtered_Ptuh_output.bed
405 filtered_Ptuh_output.bed
head filtered_Ptuh_output.bed
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack siRNA22_locus 173728 174150 1257 + . ID=Cluster_4;DicerCall=22;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 174509 175333 . - 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20918.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack siRNA22_locus 173728 174150 1257 + . ID=Cluster_4;DicerCall=22;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS exon 174509 175333 . - 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20918.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack siRNA22_locus 173728 174150 1257 + . ID=Cluster_4;DicerCall=22;MIRNA=N Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript 174509 176444 . - . ID=Pocillopora_meandrina_HIv1___RNAseq.g20918.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack MIRNA_hairpin 818027 818120 12096 + . ID=Cluster_19;DicerCall=23;MIRNA=Y Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript 816355 820160 . + . ID=Pocillopora_meandrina_HIv1___RNAseq.g21001.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack mature_miRNA 818049 818070 3240 + . ID=Cluster_19.mature;Parent=Cluster_19 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript 816355 820160 . + . ID=Pocillopora_meandrina_HIv1___RNAseq.g21001.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack miRNA-star 818079 818100 9 + . ID=Cluster_19.star;Parent=Cluster_19 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript 816355 820160 . + . ID=Pocillopora_meandrina_HIv1___RNAseq.g21001.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack MIRNA_hairpin 2872019 2872110 177 + . ID=Cluster_34;DicerCall=21;MIRNA=Y Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript 2868586 2871318 . + . ID=Pocillopora_meandrina_HIv1___TS.g25957.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack MIRNA_hairpin 2872019 2872110 177 + . ID=Cluster_34;DicerCall=21;MIRNA=Y Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 2870522 2871318 . + 2 Parent=Pocillopora_meandrina_HIv1___TS.g25957.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack MIRNA_hairpin 2872019 2872110 177 + . ID=Cluster_34;DicerCall=21;MIRNA=Y Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS exon 2870522 2871318 . + 2 Parent=Pocillopora_meandrina_HIv1___TS.g25957.t1
Pocillopora_meandrina_HIv1___Sc0000000 ShortStack mature_miRNA 2872041 2872061 110 + . ID=Cluster_34.mature;Parent=Cluster_34 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript 2868586 2871318 . + . ID=Pocillopora_meandrina_HIv1___TS.g25957.t1
Also renaming the output files with miRNA at the beginning of the file name.
Ptuh piRNA
The gff file is already sorted above and the piRNA bed file is also sorted. Run bed closest
cd /data/putnamlab/jillashey/e5/ncRNA_gff/piRNA
interactive
module load BEDTools/2.30.0-GCC-11.3.0
bedtools closest -a PMEA.merged.clusters.bed -b /data/putnamlab/jillashey/genome/Pmea/Pocillopora_meandrina_HIv1.genes_sorted.gff3 > Ptuh_piRNA_output.bed
wc -l Ptuh_piRNA_output.bed
647 Ptuh_piRNA_output.bed
head Ptuh_piRNA_output.bed
head Ptuh_piRNA_output.bed
Pocillopora_meandrina_HIv1___Sc0000000 10376955 10381586 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 10377338 10377849 Parent=Pocillopora_meandrina_HIv1___RNAseq.g21904.t1
Pocillopora_meandrina_HIv1___Sc0000000 10376955 10381586 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS exon 10377338 10377849 Parent=Pocillopora_meandrina_HIv1___RNAseq.g21904.t1
Pocillopora_meandrina_HIv1___Sc0000000 10376955 10381586 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript 10377338 10381414 . - . ID=Pocillopora_meandrina_HIv1___RNAseq.g21904.t1
Pocillopora_meandrina_HIv1___Sc0000000 10376955 10381586 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 10380139 10381414 Parent=Pocillopora_meandrina_HIv1___RNAseq.g21904.t1
Pocillopora_meandrina_HIv1___Sc0000000 10376955 10381586 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS exon 10380139 10381414 Parent=Pocillopora_meandrina_HIv1___RNAseq.g21904.t1
Pocillopora_meandrina_HIv1___Sc0000001 7491180 7496937 Pocillopora_meandrina_HIv1___Sc0000001 AUGUSTUS CDS 7491795 7493045 . + 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g19232.t1
Pocillopora_meandrina_HIv1___Sc0000001 7491180 7496937 Pocillopora_meandrina_HIv1___Sc0000001 AUGUSTUS exon 7491795 7493045 . + 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g19232.t1
Pocillopora_meandrina_HIv1___Sc0000001 7491180 7496937 Pocillopora_meandrina_HIv1___Sc0000001 AUGUSTUS transcript 7491795 7493045 . + . ID=Pocillopora_meandrina_HIv1___RNAseq.g19232.t1
Pocillopora_meandrina_HIv1___Sc0000001 7491180 7496937 Pocillopora_meandrina_HIv1___Sc0000001 AUGUSTUS CDS 7494081 7495580 . - 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g19233.t1
Pocillopora_meandrina_HIv1___Sc0000001 7491180 7496937 Pocillopora_meandrina_HIv1___Sc0000001 AUGUSTUS exon 7494081 7495580 . - 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g19233.t1
Ptuh lncRNA
The gff file is already sorted above. Sort the bed file
cd /data/putnamlab/jillashey/e5/ncRNA_gff/lncRNA
sort -k1,1 -k2,2n -k3,3n Pmea_lncRNA.bed > Pmea_lncRNA_sorted.bed
There were some issues with negative numbers being present in the starting coordinate position in some of the bed files. When I look at the fasta files for these specific lncRNAs, it says that the start position is 0. I’m going to change any instances of negative numbers to a 0
awk '{if ($2 < 0) $2 = 0; print $1 "\t" $2 "\t" $3}' Pmea_lncRNA_sorted.bed > Pmea_lncRNA_sorted_fixed.bed
Run bed closest
interactive
module load BEDTools/2.30.0-GCC-11.3.0
bedtools closest -a Pmea_lncRNA_sorted_fixed.bed -b /data/putnamlab/jillashey/genome/Pmea/Pocillopora_meandrina_HIv1.genes_sorted.gff3 > Pmea_lncRNA_output.bed
wc -l Pmea_lncRNA_output.bed
40367 Pmea_lncRNA_output.bed
head Pmea_lncRNA_output.bed
Pocillopora_meandrina_HIv1___Sc0000000 122573 123665 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 124026 124169 . + 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20912.t1
Pocillopora_meandrina_HIv1___Sc0000000 122573 123665 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS exon 124026 124169 . + 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20912.t1
Pocillopora_meandrina_HIv1___Sc0000000 122573 123665 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript124026 129612 . + . ID=Pocillopora_meandrina_HIv1___RNAseq.g20912.t1
Pocillopora_meandrina_HIv1___Sc0000000 164390 165433 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 165477 165611 . - 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20917.t1
Pocillopora_meandrina_HIv1___Sc0000000 164390 165433 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS exon 165477 165611 . - 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20917.t1
Pocillopora_meandrina_HIv1___Sc0000000 164390 165433 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript165477 165840 . - . ID=Pocillopora_meandrina_HIv1___RNAseq.g20917.t1
Pocillopora_meandrina_HIv1___Sc0000000 164761 165433 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 165477 165611 . - 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20917.t1
Pocillopora_meandrina_HIv1___Sc0000000 164761 165433 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS exon 165477 165611 . - 0 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20917.t1
Pocillopora_meandrina_HIv1___Sc0000000 164761 165433 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS transcript165477 165840 . - . ID=Pocillopora_meandrina_HIv1___RNAseq.g20917.t1
Pocillopora_meandrina_HIv1___Sc0000000 182909 183240 Pocillopora_meandrina_HIv1___Sc0000000 AUGUSTUS CDS 186383 188139 . - 2 Parent=Pocillopora_meandrina_HIv1___RNAseq.g20919.t1
Written on August 19, 2024