wget -r \
--no-directories --no-parent \
-P ../data/big/ \
-A "*sorted.bam" https://gannet.fish.washington.edu/Atumefaciens/20230821-cvir-stringtie-GCF_002022765.2-isoforms/65 - Exon Coverage
Following Li et al. Will take bams and get some data..
Trimmed reads were mapped to the Aiptasia genome using HISAT2 v2.1.0, and mapping coverage per position was extracted using BEDTools v2.17.0. To assess spurious transcription levels, we deter- mined the coverage per exon normalized across all six replicates (assuming every replicate had a coverage of 1 million sequences in total) and then calculated the average coverage ratios of exons 2 to 6 versus exon 1 for every gene
Individual BAMS and their corresponding index files are in each individual sample subirectory:
https://gannet.fish.washington.edu/Atumefaciens/20230821-cvir-stringtie-GCF_002022765.2-isoforms/
A merged BAM of all samples is here (79GB):
https://gannet.fish.washington.edu/Atumefaciens/20230821-cvir-stringtie-GCF_002022765.2-isoforms/20230821_cvir_stringtie_GCF_002022765-sorted-bams-merged.bam
Merged BAM index file (23MB):
https://gannet.fish.washington.edu/Atumefaciens/20230821-cvir-stringtie-GCF_002022765.2-isoforms/20230821_cvir_stringtie_GCF_002022765-sorted-bams-merged.bam.bai
Download BAMs
wget -r \
--no-directories --no-parent \
-P ../data/big/ \
-A "*sorted.bam.bai" https://gannet.fish.washington.edu/Atumefaciens/20230821-cvir-stringtie-GCF_002022765.2-isoforms/bedtools coverage
/home/shared/bedtools2/bin/bedtools coverage \
-a ../genome-features/GCF_002022765.2_C_virginica-3.0_genomic.gff \
-b ../data/big/S9M.sorted.bam \
> ../output/65-exon-coverage/S9gff_exon_coverage.txthead ../genome-features/GCF_002022765.2_C_virginica-3.0_genomic.gff##gff-version 3
#!gff-spec-version 1.21
#!processor NCBI annotwriter
#!genome-build C_virginica-3.0
#!genome-build-accession NCBI_Assembly:GCF_002022765.2
#!annotation-source NCBI Crassostrea virginica Annotation Release 100
##sequence-region NC_035780.1 1 65668440
##species https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=6565
NC_035780.1 RefSeq region 1 65668440 . + . ID=NC_035780.1:1..65668440;Dbxref=taxon:6565;Name=1;chromosome=1;collection-date=22-Mar-2015;country=USA;gbkey=Src;genome=chromosome;isolate=RU13XGHG1-28;isolation-source=Rutgers Haskin Shellfish Research Laboratory inbred lines (NJ);mol_type=genomic DNA;tissue-type=whole sample
NC_035780.1 Gnomon gene 13578 14594 . + . ID=gene-LOC111116054;Dbxref=GeneID:111116054;Name=LOC111116054;gbkey=Gene;gene=LOC111116054;gene_biotype=lncRNAcd ../data
/home/shared/datasets download genome accession GCF_002022765.2 --include gff3,gtfhead /home/shared/8TB_HDD_01/sr320/github/ceabigr/data/ncbi_dataset/data/GCF_002022765.2/genomic.gtf#gtf-version 2.2
#!genome-build C_virginica-3.0
#!genome-build-accession NCBI_Assembly:GCF_002022765.2
#!annotation-source NCBI Crassostrea virginica Annotation Release 100
NC_035780.1 Gnomon gene 13578 14594 . + . gene_id "LOC111116054"; db_xref "GeneID:111116054"; gbkey "Gene"; gene "LOC111116054"; gene_biotype "lncRNA";
NC_035780.1 Gnomon exon 13578 13603 . + . gene_id "LOC111116054"; transcript_id "XR_002636969.1"; db_xref "GeneID:111116054"; gbkey "ncRNA"; gene "LOC111116054"; model_evidence "Supporting evidence includes similarity to: 100% coverage of the annotated genomic feature by RNAseq alignments, including 1 sample with support for all annotated introns"; product "uncharacterized LOC111116054"; exon_number "1";
NC_035780.1 Gnomon exon 14237 14290 . + . gene_id "LOC111116054"; transcript_id "XR_002636969.1"; db_xref "GeneID:111116054"; gbkey "ncRNA"; gene "LOC111116054"; model_evidence "Supporting evidence includes similarity to: 100% coverage of the annotated genomic feature by RNAseq alignments, including 1 sample with support for all annotated introns"; product "uncharacterized LOC111116054"; exon_number "2";
NC_035780.1 Gnomon exon 14557 14594 . + . gene_id "LOC111116054"; transcript_id "XR_002636969.1"; db_xref "GeneID:111116054"; gbkey "ncRNA"; gene "LOC111116054"; model_evidence "Supporting evidence includes similarity to: 100% coverage of the annotated genomic feature by RNAseq alignments, including 1 sample with support for all annotated introns"; product "uncharacterized LOC111116054"; exon_number "3";
NC_035780.1 Gnomon gene 28961 33324 . + . gene_id "LOC111126949"; db_xref "GeneID:111126949"; gbkey "Gene"; gene "LOC111126949"; gene_biotype "protein_coding";
NC_035780.1 Gnomon exon 28961 29073 . + . gene_id "LOC111126949"; transcript_id "XM_022471938.1"; db_xref "GeneID:111126949"; gbkey "mRNA"; gene "LOC111126949"; model_evidence "Supporting evidence includes similarity to: 3 Proteins, and 100% coverage of the annotated genomic feature by RNAseq alignments, including 21 samples with support for all annotated introns"; product "UNC5C-like protein"; exon_number "1"; cd ../genome-features
curl -O https://eagle.fish.washington.edu/Cvirg_tracks/C_virginica-3.0_Gnomon_exon.bedhead ../genome-features/C_virginica-3.0_Gnomon_exon.bedNC_035780.1 13578 13603
NC_035780.1 14237 14290
NC_035780.1 14557 14594
NC_035780.1 28961 29073
NC_035780.1 30524 31557
NC_035780.1 31736 31887
NC_035780.1 31977 32565
NC_035780.1 32959 33324
NC_035780.1 66869 66897
NC_035780.1 64123 64334Lets get bam that overlaps with exons
bedtools intersect -a sorted.bam -b exons.gtf > exon_reads.bam
bedtools multicov -bams exon_reads.bam -bed exons.gtf > exon_expression_counts.txt
/home/shared/bedtools2/bin/bedtools multicov \
-bams ../data/big/S77F.sorted.bam \
-bed ../data/ncbi_dataset/data/GCF_002022765.2/genomic.gtf \
> ../output/65-exon-coverage/S77_exon_exp_counts.txttail -20 ../output/65-exon-coverage/S77_exon_exp_counts.txt/home/shared/bedtools2/bin/bedtools coverage \
-a ../genome-features/GCF_902806645.1_cgigas_uk_roslin_v1_genomic-mito.gtf \
-b ../data/big/S9M.sorted.bam \
> ../output/65-exon-coverage/S9gtf_exon_coverage.txthead -1 ../output/65-exon-coverage/S9gtf_exon_coverage.txtNC_047559.1 Gnomon gene 9839 11386 . + . gene_id "LOC117693020"; db_xref "GeneID:117693020"; gbkey "Gene"; gene "LOC117693020"; gene_biotype "lncRNA"; 0 0 1548 0.0000000loops
for bamfile in ../data/big/*sorted.bam; do
# Calculate coverage using BEDTools
/home/shared/bedtools2/bin/bedtools coverage -a ../genome-features/GCF_02022765.2_C_virginica- -b $bamfile > ../65-exon-coverage/${bamfile%.bam}_exon_coverage.txt
done