get_amplicons_and_reads.py – Generates predicted amplicons and reads from primers

Description:

Using hits files (generated with analyze_primers.py), this module will look at an individual primer or a specified primer pair to generate amplicons and reads. By default, the weighted score is used to determine if a particular primer will amplify, but other results (3’ mismatches, overall mismatches) can be used to determine if a primer will amplify. Every fasta file used to generate the hits file must be passed to this module (multiple fastas should be separated by a colon). If a single hits file is specified, the amplicon will be the entire sequence following the 3’ end of the primer.

Usage: get_amplicons_and_reads.py [options]

Input Arguments:

Note

[REQUIRED]

-i, --primer_hits

Target primer hits files. Separate multiple files with a colon.

-f, --fasta_fps

Fasta filepaths. Must match the fasta files used in the analyze_primers module. Multiple fasta files can be passed, separated with a colon. Order not important.

[OPTIONAL]

-o, --output_dir

Specify output directory for amplicons and reads. [default: .]

-s, --score_type

Value to use from primer hits file to determine a given primer’s amplification success. Valid choices are weighted_score, overall_mismatches, tp_mismatches. Gibbs energy scores not currently implemented [default: weighted_score]

-t, --score_threshold

If primer has score at or below this parameter, the primer amplification is considered to be successful [default: 1.0]

-m, --min_seq_len

Sets minimum sequence length of amplicon to be included in the output amplicon file [default: 100]

-d, --read_direction

Direction of reads generated. Can be forward (f), reverse (r), or paired end (p). [default: r]

-R, --read_len

Length of reads to generate. Should be set according to sequencing technology/reagents used. [default: 250]

Output:

An amplicon file (in fasta format) will be written for the forward and reverse primer pair, with a name based upon the name of the primers (taken from hits file name, e.g. 27f_338r_amplicons.fasta). The reads file will be generated in the same manner, with an “_reads” instead of “_amplicons”.

Standard Example usage (examine pair of primer hits files):

get_amplicons_and_reads.py [options] {-f fasta_filepath -i primer_hits_filepath1:primer_hits_filepath2 [required] }

Use total mismatches instead of weighted score to determine if a given primer will amplify:

get_amplicons_and_reads.py -f fasta_filepath -i primer_hits_filepath1:primer_hits_filepath2 -s total_mismatches