Command-line blast example

• Command-line blast example
  • Making conda env
  • Making example files
  • Blasting
    • With few references
    • With many references

Making conda env

conda create -n blast -c conda-forge -c bioconda -c defaults blast

conda activate blast

Making example files

printf ">NR_024570.1 Escherichia coli strain U 5/41 16S ribosomal RNA, partial sequence
AGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGCAGCTTGCTGCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGCACAAAGAGGGGGACCTTAGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGA
>NR_169460.1 Pseudomonas aylmerensis strain S1E40 16S ribosomal RNA, partial sequence
CTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGA
" > refs.fa

printf ">Q1
AGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGCAGCTTGCGGGGGTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGCACAAAGAGGGGGACCTTAGGGCCTCTTGCCCCCCCATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGA
>Q2
CTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGCGGCGGACCCCCGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGGGGGGGTCGGATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGA
>Q3_exact
AGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGCAGCTTGCTGCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGCACAAAGAGGGGGACCTTAGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGA
>Q4_exact
CTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGA
" > queries.fa

Blasting

With few references

With a small number of references we want to blast against, it's fine to just provide the references as a fasta file like so:

blastn -query queries.fa -subject refs.fa > blast-results.txt

Typically we want things in table form, here's one way to run it for that and sort by bitscore:

blastn -query queries.fa -subject refs.fa -outfmt "6 qseqid qlen sseqid slen length pident evalue bitscore" -max_hsps 1 -max_target_seqs 1 | sort -nrk 8 > blast-results.tmp

# adding a header
cat <(printf "qseqid\tqlen\tsseqid\tslen\tlength\tpident\tevalue\tbitscore\n") blast-results.tmp > blast-results.tsv

rm blast-results.tmp

column -ts $'\t' blast-results.tsv

# qseqid    qlen  sseqid       slen  length  pident   evalue     bitscore
# Q4_exact  297   NR_169460.1  297   297     100.000  5.96e-161  549
# Q3_exact  297   NR_024570.1  297   297     100.000  5.96e-161  549
# Q2        297   NR_169460.1  297   297     97.306   1.31e-147  505
# Q1        297   NR_024570.1  297   297     97.306   1.31e-147  505

With many references

When we have many references, it is more efficient to create a blast database like so:

makeblastdb -dbtype nucl -in refs.fa -out refs-blast-db

Then we could run the blast like so:

blastn -query queries.fa -db refs-blast-db -outfmt "6 qseqid qlen sseqid slen length pident evalue bitscore" -max_hsps 1 -max_target_seqs 1 | sort -nrk 8 > blast-results2.tmp

# adding a header
cat <(printf "qseqid\tqlen\tsseqid\tslen\tlength\tpident\tevalue\tbitscore\n") blast-results2.tmp > blast-results2.tsv

rm blast-results2.tmp

column -ts $'\t' blast-results2.tsv

# qseqid    qlen  sseqid       slen  length  pident   evalue     bitscore
# Q4_exact  297   NR_169460.1  297   297     100.000  5.96e-161  549
# Q3_exact  297   NR_024570.1  297   297     100.000  5.96e-161  549
# Q2        297   NR_169460.1  297   297     97.306   1.31e-147  505
# Q1        297   NR_024570.1  297   297     97.306   1.31e-147  505