#Vermillion A bioinformatics package for the processing of targeted DNA sequencing to discover novel endogenous retrovirus insertion sites.
#Workflow
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Blast targetted sequences against your transposable element of interest
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Make a BLAST database for your transposable element of interest (e.g. ALVE-1)
makeblastdb -in transposable_element.fasta -dbtype nucl -out transposable_element_db -parse_seqids -
Notes:
- Ensure your input targetted sequencing files are in fasta format (not fastq)
- Blast DB Options: culling_limit 1 (to keep top hit only) outfmt 6 (tab delimited output)
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BLAST against pair 1
blastn -query -db transposable_element_db -culling_limit 1 -out blast_output_pair_1.txt -outfmt 6 -
BLAST against pair 2
blastn -query -db transposable_element_db -culling_limit 1 -out blast_output_pair_2.txt -outfmt 6
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Filter for informative sequences and trim away transposable element (leaving only genome sequence > 20 nt)
python trimInternalGenome.py blast_output_pair_1.txt blast_output_for_pair_2.txt targetted_seqs_pair_1.fasta targetted_seqs_pair_2.fasta informative_seqs.fasta -
Blast informative and trimmed sequences against the host genome
makeblastdb -in genome.fasta -dbtype nucl -out genome_db name -parse_seqids blastn -query informative_seqs.fasta -db genome_db -evalue 1e-30 -out genome_hits_raw.txt -outfmt 6-
Do some slight formatting to the output file before clustering
sed 's/_1//g;s/chr//g' genome_hits_raw.txt > genome_hits.txt
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Cluster the reads which are aligning to the same region of the genome
python cluster_insertion_sites.py genome_hits.txt clusters.txt -
Output original sequences for each cluster to identify genome insertions sites and for possible PCR primer design
python cluster_sequence_files.py clusters.txt file_prefix targetted_seqs_pair_1.fasta targetted_seqs_pair_2.fasta