# How to do BLASTP for short sequences (<20 aa) effectively?

I am trying to find sequence homology between viral sequences and my protein of interest. I have the sequences of their epitopes which varies from 5 to 500 amino acids long. For shorter sequences, it is recommended to use blastp-short but I am confused until which length it is okay to run blastp?

For shorter sequences, what are the parameters needs to be added for effective results?

blastp -task blastp-short -query POI.fasta -db virus.fasta -out output.txt -outfmt 6 -evalue 20000

You should increase the -evalue 10 default option to a higher value because this value it's adjusted to the sequence database size. Check this table, maybe this can help you to decide which parameters are the best option for you.

    OPTION      TASK          DEFAUL    DESCRIPTION
word_size   blastp        3         Word size of initial match.
word_size   blastp-short  2         Word size of initial match.
gapopen     blastp        1         Cost to open a gap.
gapopen     blastp-short  9         Cost to open a gap.
gapextend   blastp        1         Cost to extend a gap.
gapextend   blastp-short  1         Cost to extend a gap.
matrix      blastp        BLOSUM62  Scoring matrix name.
matrix      blastp-short  PAM30     Scoring matrix name.
threshold   blastp        11        Minimum score to add a word to the BLAST lookup table.
threshold   blastp-short  16        Minimum score to add a word to the BLAST lookup table.
window_size blastp        40        Multiple hits window size, use 0 to specify 1-hit algorithm.
window_size blastp-short  15        Multiple hits window size, use 0 to specify 1-hit algorithm.

• Thanks! Can you please explain what is the meaning of word_size and window_size in the parameters?
– Riq
Mar 21 '20 at 20:40
• The word_size parameter is used in the initial search when BLAST makes a table of all possible "words" present in the query sequence. This first search is not only for identical "words" but for all "words" that would yield an alignment with a score above a threshold, with this step, BLASTp increases speed at the expense of only a small loss in sensitivity by only trying to extend a "hit" if there is a second "hit" within a certain window_size. Mar 21 '20 at 21:20
• How does word_size effects my BLASTp result? If I want to find 5 or 6 amino acid match at least then increasing word_size to 5 or 6 will help? It means it will try to find 5 or 6 amino acids initial match?
– Riq
Mar 22 '20 at 23:43