Mapping a short primer sequence to a whole genome

Question

[robert.murphy]

I have a degenerate primer pair that I want to independently (so each primer used as a reference) align to a whole genomes. From this I want to take the base number at the start of the alignment for the forward primer and the end value for the reverse primer. I will then take the sequence in-between these two sites and expand it ~100 bp in either direction. Essentially I want to so an in silico style PCR.

Reference primers

>A3F_primer_NRPS_AD_domain
GCSTACSYSATSTACACSTCSGG

>A7R_primer_NRPS_AD_domain
SASGTCVCCSGTSCGGTA

Alignment

I am wanting to/have used BBMap but the output SAM files seem to lack any indication of a a successful mapping? Secondly, why are the "Read Sequences" longer than the reference sequence?

tig00000006_np1212 724352_part_1152     4       *       0       0       *       *       0       0       CACCGCCATGGGCCCTTGCTAGCGGATCGGGCCCGCACTCCGCATCCCAGGGTGTCCTTACCGGCGGGGCCGGCCGCACCCGTCCGGTACGCCGTCCGCCCCCCGTCCCGCACGCCCCACCCCGCCCGCCGTA
tig00000006_np1212 724352_part_1153     4       *       0       0       *       *       0       0       GACGAGCCGGGGATCGTGGGGGCGGCGGCCCGGCTCGGGGTGCCGGTGCGCACCCATCCGGCCGGGGCGCTGGCCGCGGTCCGCGTGCCGCATCCGTCGGACGCGGTGGGCGCGGCGGTGGGGACCCCGTCGG
tig00000006_np1212 724352_part_1154     4       *       0       0       *       *       0       0       GTCGCTCGCCTCCCTCGCCGCCTACCCCGACGGGTCCTCCGCCCGCGTGGCGGTCGCCGACCGGCACGGGCTGCCGGCGGACCGGGTGCTGCTGACGGCGGGCGCGGCCGAGGCGTTCGTCCTGATCGCGCGG
tig00000006_np1212 724352_part_1155     4       *       0       0       *       *       0       0       GGATCGGCTACGTGCTGGCCGATCCGGAGACGGTGGCGCTGCTGGCCGACGCGCAGCCGCTGTGGCCCGTCTCGACCCCGGCGCTCGCGGCGGCCGAGGCGTGCATGGAGCCGCGGGCGCTGGTGGAGGCGGC
tig00000006_np1212 724352_part_1156     4       *       0       0       *       *       0       0       GAAGGGGCCGGGCCGACGCGGTTCGCGTACGAGTGGCGGTGGACGGGGCGGCAGGGCGCCGAGGCCGTTCCCCTGACGGCCGGGGAGGGGCGGGGCCCGGTGTCGGGCCCCGCCCCGGACGCGCTACGGCGTC
tig00000006_np1212 724352_part_1157     4       *       0       0       *       *       0       0       CGGGGTCTCGCAGGTGGCCTCGGCGAGGGTGACCTCGCCGTTCACCTCGGCCACGTTCAGCTTGAGCGGGTTGACCGCGACCTTGAGCTGGAGCGCCGTCGCGGCCGCCGTCCGCTCGGTGGTCACCGTCTTG
tig00000006_np1212 724352_part_1158     4       *       0       0       *       *       0       0       CCTCGTTGAGCGTGGCCTTGAGGGGCACGTGGACGGACTTGTTGAGGAGCGAGACGTTGAGCCCGGTGCGGAGCACGACCGCGCTCGCCCGGCCCTCGCCCTTGCCGGGCGTCGCCGGGGCTGCGTGCGCGGG
tig00000006_np1212 724352_part_1159     4       *       0       0       *       *       0       0       TCGTCAACACGGCTCCCCGCAGCGCGCCGCGTCCGCTCACCCGACGGCGCCGCCGTTCCGGGTCACCCGACCACCCGCCCTTCGAGCACGACGTGCCGCGGGGCCGCCAGCACCCGCACGTCCGCGCGCGGGT

[cpad0112]

https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi ?

[robert.murphy]

This seems to be a tool for generating primer pairs?

[cpad0112]

also for searching user provided primer sequences in user provided sequence with restrictions on degeneracy. For test primer set on test sequence:

[robert.murphy]

How did you achieve this? When I give only an input fasta and the two primer sequences I get the error indicating it does not allow for degeneracy in the primers I think: Exception error: Multiple templates are currently not supported! .

Answer 1

[shenwei356]

Try seqkit amplicon - for retrieving amplicon (or specific region around it) via primer(s).