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Batch Primer Design with primer3_batch_design

Purpose

Primers are designed for each template sequence using primer3, including predicting secondary structure formation for adapter-ligated primers.

Usage

For primer3_batch_design to run, primer3.sh must be found in the same folder and settings files for Primer3 must be in the multiplex_wormhole/settings folder under Primer3_Base_NoSecondaryFilters.txt and Primer3_Broad_NoSecondaryFilters.txt.

Python syntax

import os

os.chdir('/multiplex_wormhole')

from scripts.primer3_batch_design import main as primer3BatchDesign

primer3BatchDesign(IN_CSV, OUTDIR, PRIMER3_PATH)

Command line syntax

cd multiplex_wormhole/scripts

python3 primer3_batch_design.py IN_CSV OUTDIR PRIMER3_PATH

Arguments

IN_CSV : Path to CSV file containing DNA template sequences in the following format (including headers):

SEQUENCE_ID SEQUENCE_TEMPLATE SEQUENCE_TARGET
CLocus_704 TCAGAGAC... 53,1
... ... ...

The sequence target is in <POSITION,LENGTH> format. In the example, there is a SNP at basepair 53 within the locus 704.

OUTDIR : Directory where primer output files will be saved. This directory must already exist.

PRIMER3_PATH : Path to primer3_core file.

Outputs

For each template sequence, a Primer3 output file and error file will be saved with naming based on the template SEQUENCE_ID: <SEQUENCE_ID>.out and <SEQUENCE_ID>.err. All outputs will be saved to OUTDIR. See the primer3 Manual for interpreting output files.

Defaults

Default settings are found in the Primer3_Base_NoSecondaryFilters.txt and Primer3_Broad_NoSecondaryFilters.txt under multiplex_wormhole/primer3_settings. These settings can be manually changed in the text files. For details on primer3 setting options and definitions, see the primer3 Manual. Settings can also be explored in Primer3Plus and settings file saved. Make sure that SEQUENCE_ID=, SEQUENCE_TEMPLATE=, and SEQUENCE_TARGET= remain blank before inputting to the script.

These default settings follow Eriksson et al. 2020 and are intended for amplifying DNA for SNP-based genotyping assays of wildlife from degraded samples (specifically, noninvasive genetic samples such as scats). Specifically, some of the important defaults include:

Illumina Nextera i5 and i7 adapters are added to 5'ends of output primers

  • SEQUENCE_OVERHANG_LEFT=tcgtcggcagcgtcagatgtgtataagagacag
  • SEQUENCE_OVERHANG_RIGHT=gtctcgtgggctcggagatgtgtataagagacag

Primer annealing temp is 52 Celsius

  • PRIMER_ANNEALING_TEMP=52

Amplicon size is 70-120 base pairs, with an optimal of 100 bp

  • PRIMER_PRODUCT_SIZE_RANGE=70-120
  • PRIMER_PRODUCT_OPT_SIZE=100

Primer size range is 18-26 bp (optimal 20 bp)

  • PRIMER_OPT_SIZE=20
  • PRIMER_MIN_SIZE=18
  • PRIMER_MAX_SIZE=26

Primer Tm range from 57-63 (optimal 60)

  • PRIMER_MIN_TM=57
  • PRIMER_MAX_TM=63
  • PRIMER_OPT_TM=60

Primer GC content must be between 30-70% (optimal 50%)

  • PRIMER_MAX_GC=70
  • PRIMER_MIN_GC=30
  • PRIMER_OPT_GC_PERCENT=50

Primers must have at least one G or C (a GC clamp) at the 3' end

  • PRIMER_GC_CLAMP=1

At most, primers can have 4 Gs or Gcs at the ends

  • PRIMER_MAX_END_GC=4

At most, primers can have 4 repeats of the same base

  • PRIMER_MAX_POLY_X=4

Primer concentrations are 0.25 nM in the final PCR reaction

  • PRIMER_DNTP_CONCENTRATOIN=0.25

DNA template concentration is 50 nM in the final PCR reaction

  • PRIMER_DNA_CONCENTRATION=50

Salt concentrations are 3.8 mM for divalent cations and 50 mM for monovalenet cations

  • PRIMER_SALT_DIVALENT=3.8
  • PRIMER_SALT_MONOVALENT=50

If primers can't be found for the above settings, constraints are relaxed:

  • Primer GC content: 20-80%
  • GC Clamp: 0
  • Max End GC: 5
  • Max Poly X: 5

Citations

Eriksson, CE, Ruprecht J, Levi T. 2020. More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing. Molecular Ecology Resources 20(4): 10.1111/1755-0998.13208.