Primer Design - C.gigas Alternative Oxidase AOX Variants Using Primer3

2025
Crassotrea gigas
Pacific oyster
Primer3
EMBOSS
PrimerSearch
Magallana gigas
primers
AOX
alternative oxidase
Author

Sam White

Published

January 17, 2025

Introduction

This document outlines the process of designing primers for the alternative oxidase (AOX) gene variants in Crassostrea gigas (Pacific oyster) using Primer3. The goal is to develop specific primers for qPCR to study the expression of AOX isoforms under different experimental conditions.

The AOX gene is involved in the mitochondrial electron transport chain and is known to be responsive to oxidative stress. By designing specific primers for different AOX isoforms, we can investigate their expression patterns and potential roles in oyster physiology and stress responses.

The following sections detail the steps taken to identify AOX isoforms, retrieve their sequences, design primers using Primer3, and validate the primers using EMBOSS PrimerSearch.

Objectives

  1. Identify AOX isoforms in Crassostrea gigas.
  2. Retrieve nucleotide sequences for the identified isoforms.
  3. Design specific primers for each isoform using Primer3.
  4. Validate primer specificity using EMBOSS PrimerSearch.

Methods

The methods section includes the following steps:

  1. Identification of AOX Isoforms: Using BLASTx to identify AOX isoforms in the Crassostrea gigas genome.
  2. Sequence Retrieval: Fetching nucleotide sequences for the identified isoforms using NCBI’s e-utilities.
  3. Primer Design: Using Primer3 to design specific primers for each AOX isoform.
  4. Primer Validation: Using EMBOSS PrimerSearch to validate the specificity of the designed primers against the Crassostrea gigas genome.

The detailed procedures and scripts used for each step are provided in the subsequent sections.

Note

The contents below are from markdown knitted from 20250117-cgig-aox-primer-design.Rmd (commit 75f0197).

1 Background

This notebook describes using Primer3 (Untergasser et al. 2012; Koressaar and Remm 2007) to reproducibly design qPCR primers for Crassostrea gigas (Magallana gigas) alternative oxidase (AOX) (Wikipedia) isoforms. Alternative oxidase is a mitochondrial gene/enzyme known to be responsive to oxidative stress, so this may be a useful biomarker that we haven’t previously considered.

Isoforms of AOX in Crassostrea gigas (Magallana gigas) were identified by running BLASTx using the Arabidopsis AOX1a nucleotide sequence (NM_113135.4) as a query against the Crassostrea gigas (Magallana gigas) genome using NCBI’s web-based BLAST interface.

BLASTx returned three matches:

1.1 Input files

  • Target FastA sequences

  • Genome FastA

1.2 Output files

  • emboss-primers.txt: Output from EMBOSS PrimerSearch containing primer pair matches within the genome.

  • *primer3-primers-default-format.txt: The default output format containing Primer3 primer identification info. Machine-readable format.

  • *primer3-primers.txt: Primer3 output file containing primer identification info. An easy-to-read format.

1.3 Required software

Utilizes NCBI’s e-utilities (Kans 2013) for fetching FastA sequences.

This process also utilizes pyfaidx (Shirley et al. 2015).

EMBOSS PrimerSearch (Rice, Longden, and Bleasby 2000) will be utilized to assess primer specificity across the genome.

2 CREATE BASH VARIABLES FILE

This allows usage of Bash variables across R Markdown chunks.

{
echo "#### Assign Variables ####"
echo ""

echo "# DATA DIRECTORIES"
echo 'export data_dir="../data"'
echo 'export output_top="../output"'
echo 'export genome_fasta_splits_dir="${data_dir}/fasta_splits"'
echo ""

echo "# SEQUENCES"
echo 'export sequence_IDs=(XM_066083499.1 XM_034462847.2 NM_001305360.1)'
echo ""

echo "# SEQUENCE REGIONS"
echo 'export left_buffer="500"'
echo 'export right_buffer="500"'
echo ""

echo "# INPUT FILES"
echo 'export genome_fasta="GCF_963853765.1_xbMagGiga1.1_genomic.fna"'
echo 'export genome_gff="GCF_963853765.1_xbMagGiga1.1_genomic.gff"'
echo 'export ncbi_gff_gz="GCF_963853765.1_xbMagGiga1.1_genomic.gff.gz"'
echo 'export ncbi_fasta_gz="GCF_963853765.1_xbMagGiga1.1_genomic.fna.gz"'
echo 'export ncbi_md5sums="md5checksums.txt"'
echo 'export ncbi_url="https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/963/853/765/GCF_963853765.1_xbMagGiga1.1/"'
echo 'export NM_001305360_fasta="NM_001305360.1.fasta"'
echo 'export XM_034462847_fasta="XM_034462847.2.fasta"'
echo 'export XM_066083499_fasta="XM_066083499.1.fasta"'


echo "# OUTPUT FILES"
echo ""

echo "# SET CPUS"
echo 'export threads=40'
echo ""

echo "# PROGRAMS"
echo 'esearch="/home/sam/edirect/esearch"'
echo 'efetch="/home/sam/edirect/efetch"'
echo 'export pyfaidx=/home/shared/pyfaidx-0.8.1.1'
echo 'export primer3_dir="/home/shared/primer3-2.6.1/src"'
echo 'export primer3="${primer3_dir}/primer3_core"'
echo 'export primer3_config="${primer3_dir}/primer3_config"'
echo 'export primersearch="/home/shared/EMBOSS-6.6.0/emboss/primersearch"'



} > .bashvars

cat .bashvars
#### Assign Variables ####

# DATA DIRECTORIES
export data_dir="../data"
export output_top="../output"
export genome_fasta_splits_dir="${data_dir}/fasta_splits"

# SEQUENCES
export sequence_IDs=(XM_066083499.1 XM_034462847.2 NM_001305360.1)

# SEQUENCE REGIONS
export left_buffer="500"
export right_buffer="500"

# INPUT FILES
export genome_fasta="GCF_963853765.1_xbMagGiga1.1_genomic.fna"
export genome_gff="GCF_963853765.1_xbMagGiga1.1_genomic.gff"
export ncbi_gff_gz="GCF_963853765.1_xbMagGiga1.1_genomic.gff.gz"
export ncbi_fasta_gz="GCF_963853765.1_xbMagGiga1.1_genomic.fna.gz"
export ncbi_md5sums="md5checksums.txt"
export ncbi_url="https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/963/853/765/GCF_963853765.1_xbMagGiga1.1/"
export NM_001305360_fasta="NM_001305360.1.fasta"
export XM_034462847_fasta="XM_034462847.2.fasta"
export XM_066083499_fasta="XM_066083499.1.fasta"
# OUTPUT FILES

# SET CPUS
export threads=40

# PROGRAMS
esearch="/home/sam/edirect/esearch"
efetch="/home/sam/edirect/efetch"
export pyfaidx=/home/shared/pyfaidx-0.8.1.1
export primer3_dir="/home/shared/primer3-2.6.1/src"
export primer3="${primer3_dir}/primer3_core"
export primer3_config="${primer3_dir}/primer3_config"
export primersearch="/home/shared/EMBOSS-6.6.0/emboss/primersearch"

3 DOWNLOAD NCBI GENOME FILES

3.1 Download the files

# Load bash variables into memory
source .bashvars

for file in ${ncbi_gff_gz} ${ncbi_fasta_gz} ${ncbi_md5sums}
do
  wget \
  --no-check-certificate \
  --continue \
  --quiet \
  --directory-prefix=${data_dir} \
  ${ncbi_url}${file}
done

ls -lh "${data_dir}"
total 1.1G
drwxr-xr-x 2 sam sam 4.0K Jan 21 16:59 fasta_splits
-rw-r--r-- 1 sam sam 545M Jun 28  2024 GCF_963853765.1_xbMagGiga1.1_genomic.fna
-rw-r--r-- 1 sam sam 1.1K Jan 21 10:28 GCF_963853765.1_xbMagGiga1.1_genomic.fna.fai
-rw-r--r-- 1 sam sam 167M Jun 28  2024 GCF_963853765.1_xbMagGiga1.1_genomic.fna.gz
-rw-r--r-- 1 sam sam 373M Jun 28  2024 GCF_963853765.1_xbMagGiga1.1_genomic.gff
-rw-r--r-- 1 sam sam  15M Jun 28  2024 GCF_963853765.1_xbMagGiga1.1_genomic.gff.gz
-rw-r--r-- 1 sam sam  20K Oct 27 16:10 md5checksums.txt
-rw-r--r-- 1 sam sam 1.4K Jan 18 23:18 NM_001305360.1.fasta
-rw-rw-r-- 1 sam sam 104K Jan 21 13:50 NM_001305360.1-NCBI-BLASTn-alignment.png
-rw-rw-r-- 1 sam sam 1.1K Jan 18 23:18 XM_034462847.2.fasta
-rw-rw-r-- 1 sam sam 119K Jan 21 13:50 XM_034462847.2-NCBI-BLASTn-alignment.png
-rw-rw-r-- 1 sam sam 1.5K Jan 18 23:18 XM_066083499.1.fasta
-rw-rw-r-- 1 sam sam 147K Jan 21 13:50 XM_066083499.1-NCBI-BLASTn-alignment.png

3.2 Check MD5 Checkums

# Load bash variables into memory
source .bashvars

cd "${data_dir}"

for file in *.gz
do
  grep "${file}" ${ncbi_md5sums} | md5sum -c -
done
./GCF_963853765.1_xbMagGiga1.1_genomic.fna.gz: OK
./GCF_963853765.1_xbMagGiga1.1_genomic.gff.gz: OK

3.3 Decompress NCBI files

# Load bash variables into memory
source .bashvars

cd "${data_dir}"

for file in *.gz
do
  gunzip "${file}"
done

ls -lh
gzip: GCF_963853765.1_xbMagGiga1.1_genomic.fna already exists;  not overwritten
gzip: GCF_963853765.1_xbMagGiga1.1_genomic.gff already exists;  not overwritten
total 1.1G
drwxr-xr-x 2 sam sam 4.0K Jan 21 16:59 fasta_splits
-rw-r--r-- 1 sam sam 545M Jun 28  2024 GCF_963853765.1_xbMagGiga1.1_genomic.fna
-rw-r--r-- 1 sam sam 1.1K Jan 21 10:28 GCF_963853765.1_xbMagGiga1.1_genomic.fna.fai
-rw-r--r-- 1 sam sam 167M Jun 28  2024 GCF_963853765.1_xbMagGiga1.1_genomic.fna.gz
-rw-r--r-- 1 sam sam 373M Jun 28  2024 GCF_963853765.1_xbMagGiga1.1_genomic.gff
-rw-r--r-- 1 sam sam  15M Jun 28  2024 GCF_963853765.1_xbMagGiga1.1_genomic.gff.gz
-rw-r--r-- 1 sam sam  20K Oct 27 16:10 md5checksums.txt
-rw-r--r-- 1 sam sam 1.4K Jan 18 23:18 NM_001305360.1.fasta
-rw-rw-r-- 1 sam sam 104K Jan 21 13:50 NM_001305360.1-NCBI-BLASTn-alignment.png
-rw-rw-r-- 1 sam sam 1.1K Jan 18 23:18 XM_034462847.2.fasta
-rw-rw-r-- 1 sam sam 119K Jan 21 13:50 XM_034462847.2-NCBI-BLASTn-alignment.png
-rw-rw-r-- 1 sam sam 1.5K Jan 18 23:18 XM_066083499.1.fasta
-rw-rw-r-- 1 sam sam 147K Jan 21 13:50 XM_066083499.1-NCBI-BLASTn-alignment.png

4 RETRIEVE GENE SEQUENCES

Note

The below would not execute in Rstudio/Rstudio Server. I tried on two different computers and the chunk would just “run” indefinitly. Code executes without issue in a terminal.

for ID in "${sequence_IDs[@]}"
do
  ${esearch} -db nuccore -query "${ID}" \
  | ${efetch} -format fasta \
  > "${data_dir}"/"${ID}".fasta
done

5 PRIMER DESIGN USING PRIMER3

5.1 Design primers

Quick explanation: Primer3 requires a specially formatted input file. The file must be formatted similarly to this:

SEQUENCE_ID=${seq_id}
SEQUENCE_TEMPLATE=${sequence}
PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=3
PRIMER_PICK_RIGHT_PRIMER=3
PRIMER_OPT_SIZE=18
PRIMER_MIN_SIZE=15
PRIMER_MAX_SIZE=21
PRIMER_MAX_NS_ACCEPTED=1
PRIMER_PRODUCT_SIZE_RANGE=75-150
P3_FILE_FLAG=1
PRIMER_EXPLAIN_FLAG=1
=

Values after the = on each line can be changed to whatever values the user decides. The ${sequence} must be a nucleotide sequence on a single line, with no line breaks.

The code in the chunk below uses a heredoc to write this information to a file. Use of a heredoc allows the variables specified in the Primer3 config to expand to their actual values. Everything between the following two lines gets printed (via cat) as shown and then redirected to the indicated file (primer3-params.txt):

cat << EOF > ${output_top}/primer3-params.txt
This text will end up in the file.
So will this.
And this.
EOF

Primer3 is run with the --format_output to make a nice, human-readable output format.

I’ve also set Primer3 to look for sequencing primers and have defined the SEQUENCE_TARGET.

5.1.0.1 NCBI BLASTn alignments

Each sequence was BLASTn’d against C.gigas nucleotides to identify unique regions in each of the three variants to improve our ability to detect each variant’s expression level.

5.1.0.1.1 NM_001305360.1

NM_001305360.1 BLASTn alignment
5.1.0.1.2 XM_034462847.2

XM_034462847.2 BLASTn alignment
5.1.0.1.3 XM_066083499.1

XM_066083499.1 BLASTn alignment

5.1.1 NM_001305360.1

# Load bash variables into memory
source .bashvars

# Get sequence only, by skipping the first record
# Remove newlines so sequence is on a single line
sequence=$(awk 'NR > 1' "${data_dir}/${NM_001305360_fasta}" | tr -d '\n')

sequence_ID="NM_001305360.1"


# Use heredoc to create Primer3 parameters file
# PRIMER_TASK=generic is used with PRIMER_PICK settings to be PCR primers.
cat << EOF > ${output_top}/NM_001305360.1-primer3-params.txt
SEQUENCE_ID=${sequence_ID}
SEQUENCE_TEMPLATE=${sequence}
PRIMER_TASK=generic
PRIMER_MIN_TM=50
PRIMER_OPT_TM=60
SEQUENCE_PRIMER_PAIR_OK_REGION_LIST=1109,50,,
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=25
PRIMER_PRODUCT_SIZE_RANGE=75-150
PRIMER_MAX_NS_ACCEPTED=0
P3_FILE_FLAG=1
PRIMER_EXPLAIN_FLAG=1
PRIMER_THERMODYNAMIC_PARAMETERS_PATH=${primer3_config}
PRIMER_NUM_RETURN=3
PRIMER_PAIR_EXPLAIN=considered 0, ok 0
=
EOF


# Run Primer3
${primer3} \
--format_output \
--output="${output_top}/NM_001305360.1-primer3-primers.txt" \
"${output_top}/NM_001305360.1-primer3-params.txt"

# Run Primer3 with default output for parsable results
${primer3} \
--output="${output_top}/NM_001305360.1-primer3-primers-default-format.txt" \
"${output_top}/NM_001305360.1-primer3-params.txt"

5.1.1.1 Review primers

5.1.1.1.1 Easy-to-read
source .bashvars

cat "${output_top}/NM_001305360.1-primer3-primers.txt"
PRIMER PICKING RESULTS FOR NM_001305360.1

No mispriming library specified
Using 0-based sequence positions
OLIGO            start  len      tm     gc%  any_th  3'_th hairpin seq
LEFT PRIMER       1112   20   57.36   45.00    8.11   7.53    0.00 TCTCACTGTGGAAATGCACA
RIGHT PRIMER      1194   21   56.16   42.86    0.00   0.00    0.00 TGGACATTCATCTCTACTGCT
SEQUENCE SIZE: 1214
INCLUDED REGION SIZE: 1214

PRODUCT SIZE: 83, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00
PAIR_OK_REGIONS (left_start, left_len, right_start, right_len)*: 1109,50,1158,57

    0 GGAATTTTCAGATTTGAGAAGTGAAGAATATATCTGAGGTACTTTAAATATACGATGCGC
                                                                  

   60 TATGGGAAGTTTGCGACAAATAACGAAATTGAGTGAAAATGGTGTCCGGATTTTCTGTTC
                                                                  

  120 CCAACTAAAGAATCTAGAAAATAATTCAATTCTACTGAGGGTCAGTGGAATTCGCACCAG
                                                                  

  180 CAATGGGCTTCGCAATGCAGGAACAAAGGCTGACGTTGATGAAAATATAAAGAAATTTAA
                                                                  

  240 AGAGGAAAATTTTGAAAAAATTCCAGACCCCGAACAGCTCGATCACTTCAGGAAAACACA
                                                                  

  300 GAGCACAGATCAACTGGTGGAGTCAATGAAGAACCCTCCCCCAATGGGCACGCACACACT
                                                                  

  360 GCCCCACCCAATATGGTCTGAAGAAGAACTTCATAGTGTGAAGGTCACACATAAACCGCC
                                                                  

  420 CGAGGGATTCGTAGACAAGCTTGCCTTTCGATCAGTGAAGCTGCTACGTTCAACTTTTGA
                                                                  

  480 CCTTTTAACCGGGTTCAACTGGGGAGAAAGAACGGAGAAGAAATGGGTCTTACGGATTTG
                                                                  

  540 CTTCCTAGAGACTGTCGCTGGAGTTCCAGGAATGGTGGCAGCGATGACGCGACATTTACA
                                                                  

  600 CTCTTTGCGCCGACTCAAGCGGGATCACGGCTGGATTCATACCCTTTTAGAGGAAGCAGA
                                                                  

  660 AAACGAGAGAATGCATCTGATGACAGCATTGCAGCTTCGACAACCATCTTGGTTATTTAG
                                                                  

  720 AAGTGGCGTCATCGTGTCTCAGGGTGCTTTCGTCACGATGTTCAGCATTGCCTATATGCT
                                                                  

  780 AAGTCCTCGGTTTTGTCATCGTTTCGTTGGCTATCTAGAGGAGGAAGCAGTGTTTACTTA
                                                                  

  840 TTCAAAATGTTTGAAGGATATAGAGTCAGGGTCCCTTAAACACTGGCAAACAAAAGCAGC
                                                                  

  900 ACCCGACGTGGCCATCCGCTATTGGAAACTCCCAGAAACTGCATCCATGAAGGATGTAGT
                                                                  

  960 GTTGGCAATTCGAGCAGATGAGGCTCACCATAGGGTCGTGAACCATACTCTGGCTTCCAT
                                                                  

 1020 GAAAGAAGACGAATACAATCCTTATGAGCCAGGGAAGTGATATGGAAGCAGAGAAAACAG
                                                                  

 1080 TGTTAACATTCTGTAGTTATACTGTGAATACATCTCACTGTGGAAATGCACATTACTTGC
                                      >>>>>>>>>>>>>>>>>>>>        

 1140 CTTTGATACAATGTACATGTACATGTAGTAAGTAAGCAGTAGAGATGAATGTCCATTGTA
                                        <<<<<<<<<<<<<<<<<<<<<     

 1200 TAATTCAGGCCATG
                    

KEYS (in order of precedence):
>>>>>> left primer
<<<<<< right primer

ADDITIONAL OLIGOS
                    start  len      tm     gc%  any_th  3'_th hairpin seq

 1 LEFT PRIMER       1110   22   59.18   45.45    8.11   7.53    0.00 CATCTCACTGTGGAAATGCACA
   RIGHT PRIMER      1194   21   56.16   42.86    0.00   0.00    0.00 TGGACATTCATCTCTACTGCT
   PRODUCT SIZE: 85, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00

 2 LEFT PRIMER       1109   22   59.18   45.45    8.11   0.00    0.00 ACATCTCACTGTGGAAATGCAC
   RIGHT PRIMER      1194   21   56.16   42.86    0.00   0.00    0.00 TGGACATTCATCTCTACTGCT
   PRODUCT SIZE: 86, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00

Statistics
         con   too    in    in   not          no    tm    tm   high  high  high        high      
         sid  many   tar  excl    ok   bad    GC   too   too any_th 3'_th hair-  poly   end      
        ered    Ns   get   reg   reg   GC% clamp   low  high  compl compl   pin     X  stab    ok
Left    9120     0     0     0  8887     0     0    22     0      0     0     0     0     0   211
Right   9120     0     0     0  8836     0     0    90     0      0     0     0     0     0   194
Pair Stats:
considered 3, ok 3
libprimer3 release 2.6.1

5.1.2 XM_034462847.2

# Load bash variables into memory
source .bashvars

# Get sequence only, by skipping the first record
# Remove newlines so sequence is on a single line
sequence=$(awk 'NR > 1' "${data_dir}/${XM_034462847_fasta}" | tr -d '\n')

sequence_ID="XM_034462847.2"


# Use heredoc to create Primer3 parameters file
# PRIMER_TASK=generic is used with PRIMER_PICK settings to be PCR primers.
cat << EOF > ${output_top}/XM_034462847.2-primer3-params.txt
SEQUENCE_ID=${sequence_ID}
SEQUENCE_TEMPLATE=${sequence}
PRIMER_TASK=generic
PRIMER_MIN_TM=50
PRIMER_OPT_TM=60
SEQUENCE_PRIMER_PAIR_OK_REGION_LIST=90,25,,
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=25
PRIMER_PRODUCT_SIZE_RANGE=75-150
PRIMER_MAX_NS_ACCEPTED=0
P3_FILE_FLAG=1
PRIMER_EXPLAIN_FLAG=1
PRIMER_THERMODYNAMIC_PARAMETERS_PATH=${primer3_config}
PRIMER_NUM_RETURN=3
PRIMER_PAIR_EXPLAIN=considered 0, ok 0
=
EOF


# Run Primer3
${primer3} \
--format_output \
--output="${output_top}/XM_034462847.2-primer3-primers.txt" \
"${output_top}/XM_034462847.2-primer3-params.txt"

# Run Primer3 with default output for parsable results
${primer3} \
--output="${output_top}/XM_034462847.2-primer3-primers-default-format.txt" \
"${output_top}/XM_034462847.2-primer3-params.txt"

5.1.2.1 Review primers

5.1.2.1.1 Easy-to-read
source .bashvars

cat "${output_top}/XM_034462847.2-primer3-primers.txt"
PRIMER PICKING RESULTS FOR XM_034462847.2

No mispriming library specified
Using 0-based sequence positions
OLIGO            start  len      tm     gc%  any_th  3'_th hairpin seq
LEFT PRIMER         91   24   59.07   41.67    0.00   0.00    0.00 TCGATCACTTCAGAATTTCACAGG
RIGHT PRIMER       169   20   60.03   60.00    0.00   0.00    0.00 CCATTGGGGGAGGGTTCTTC
SEQUENCE SIZE: 931
INCLUDED REGION SIZE: 931

PRODUCT SIZE: 79, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00
PAIR_OK_REGIONS (left_start, left_len, right_start, right_len)*: 90,25,139,109

    0 CTTCGCAATGCAGGAACAAAGGCTGACGTTGATGAAAATATAAAGAAATTTAAAGAGGAA
                                                                  

   60 AATTTTGAAAAAATTCCAGACCCCGAACAGCTCGATCACTTCAGAATTTCACAGGAAAAC
                                     >>>>>>>>>>>>>>>>>>>>>>>>     

  120 ACAGAGCACAGATCAACTGGTGGAGTCAATGAAGAACCCTCCCCCAATGGGCACGCACAC
                                    <<<<<<<<<<<<<<<<<<<<          

  180 ACTGCCCCACCCAATATGGTCTGAAGAAGAACTTCATAGTGTGAAGGTCACACATAAACC
                                                                  

  240 GCCCGAGGGATTCGTAGACAAGCTTGCCTTTCGATCAGTGAAGCTGCTACGTTCAACTTT
                                                                  

  300 TGACCTTTTAACCGGGTTCAACTGGGGAGAAAGAACGGAGAAGAAATGGGTGTTACGGAT
                                                                  

  360 TTGCTTCCTAGAGACTGTCGCTGGAGTTCCAGGGATGGTGGCAGCGATGACGCGACATTT
                                                                  

  420 ACACTCTTTGCGCCGACTCAAGCGGGATCACGGCTGGATTCATACCCTTTTAGAGGAAGC
                                                                  

  480 AGAAAACGAAAGAATGCATCTGATGACAGCATTGCAGCTTCGACAACCATCTTGGTTATT
                                                                  

  540 TAGAAGTGGCGTCATCGTGTCTCAGGGTGCTTTCGTCACGATGTTCAGCATTGCCTATAT
                                                                  

  600 GCTAAGCCCTCGGTTTTGTCATCGGTTCGTTGGCTATCTAGAGGAGGAAGCAGTGTTTAC
                                                                  

  660 TTACTCAAAATGTTTGAAGGATATAGAGTCAGGGCCCCTTAAACACTGGCAAACAAAAGC
                                                                  

  720 AGCACCCGACGTGGCTATCCGCTATTGGAAACTCCCAGAAACTGCATCCATGAAGGATGT
                                                                  

  780 AGTGTTGGCAATTCGAGCAGATGAGGCTCACCATAGGGTCGTGAACCATACTCTGGCTTC
                                                                  

  840 CATGAAAGAAGACGAATACAATCCTTATGAGCCAGGGAAGTGATATGGAAGCAGAGAAAA
                                                                  

  900 CAGCGTTAACATTCTGTAGTTATACTGTGAA
                                     

KEYS (in order of precedence):
>>>>>> left primer
<<<<<< right primer

ADDITIONAL OLIGOS
                    start  len      tm     gc%  any_th  3'_th hairpin seq

 1 LEFT PRIMER         91   24   59.07   41.67    0.00   0.00    0.00 TCGATCACTTCAGAATTTCACAGG
   RIGHT PRIMER       196   20   60.03   55.00    0.00   0.00    0.00 ATATTGGGTGGGGCAGTGTG
   PRODUCT SIZE: 106, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00

 2 LEFT PRIMER         91   24   59.07   41.67    0.00   0.00    0.00 TCGATCACTTCAGAATTTCACAGG
   RIGHT PRIMER       197   20   60.03   55.00    0.00   0.00    0.00 CATATTGGGTGGGGCAGTGT
   PRODUCT SIZE: 107, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00

Statistics
         con   too    in    in   not          no    tm    tm   high  high  high        high      
         sid  many   tar  excl    ok   bad    GC   too   too any_th 3'_th hair-  poly   end      
        ered    Ns   get   reg   reg   GC% clamp   low  high  compl compl   pin     X  stab    ok
Left    6856     0     0     0  6820     0     0    10     0      0     0     0     0     0    26
Right   6856     0     0     0  6148     0     0    42   255      0     0   185     0     0   226
Pair Stats:
considered 4, ok 4
libprimer3 release 2.6.1

5.1.3 XM_066083499.1

# Load bash variables into memory
source .bashvars

# Get sequence only, by skipping the first record
# Remove newlines so sequence is on a single line
sequence=$(awk 'NR > 1' "${data_dir}/${XM_066083499_fasta}" | tr -d '\n')

sequence_ID="XM_066083499.1"


# Use heredoc to create Primer3 parameters file
# PRIMER_TASK=generic is used with PRIMER_PICK settings to be PCR primers.
cat << EOF > ${output_top}/XM_066083499.1-primer3-params.txt
SEQUENCE_ID=${sequence_ID}
SEQUENCE_TEMPLATE=${sequence}
PRIMER_TASK=generic
PRIMER_MIN_TM=50
PRIMER_OPT_TM=60
SEQUENCE_PRIMER_PAIR_OK_REGION_LIST=,,1100,50
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=25
PRIMER_PRODUCT_SIZE_RANGE=75-150
PRIMER_MAX_NS_ACCEPTED=0
P3_FILE_FLAG=1
PRIMER_EXPLAIN_FLAG=1
PRIMER_THERMODYNAMIC_PARAMETERS_PATH=${primer3_config}
PRIMER_NUM_RETURN=3
PRIMER_PAIR_EXPLAIN=considered 0, ok 0
=
EOF


# Run Primer3
${primer3} \
--format_output \
--output="${output_top}/XM_066083499.1-primer3-primers.txt" \
"${output_top}/XM_066083499.1-primer3-params.txt"

# Run Primer3 with default output for parsable results
${primer3} \
--output="${output_top}/XM_066083499.1-primer3-primers-default-format.txt" \
"${output_top}/XM_066083499.1-primer3-params.txt"

5.1.3.1 Review primers

5.1.3.1.1 Easy-to-read
source .bashvars

cat "${output_top}/XM_066083499.1-primer3-primers.txt"
PRIMER PICKING RESULTS FOR XM_066083499.1

No mispriming library specified
Using 0-based sequence positions
OLIGO            start  len      tm     gc%  any_th  3'_th hairpin seq
LEFT PRIMER       1028   20   60.25   60.00    0.00   0.00    0.00 CAGACGAGGCTAATCACCGG
RIGHT PRIMER      1141   21   58.73   47.62    0.00   0.00   34.07 TGAGGCACATTAAGGGTCTGA
SEQUENCE SIZE: 1329
INCLUDED REGION SIZE: 1329

PRODUCT SIZE: 114, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00
PAIR_OK_REGIONS (left_start, left_len, right_start, right_len)*: 967,134,1100,50

    0 ATATATTGAGACCGTCTTGCATATTTCAGAAAATAAGAAAAAAAGTCAGTGTAAGGTATT
                                                                  

   60 TTCAGATTTGAGAAGTGAAGAATATATCTGAGGTATTTTAAATATACGAATCGCTATGGG
                                                                  

  120 AAGTTTACGACAAATAACGAAATTGAGTGAAAATGGTGTCCGGATTTTCTGTTCCCAACT
                                                                  

  180 AAAGAATCTAGAAAATAATTCAATTCTACTGAGGGTCAGTGGAATTCGCACCAGCAATGG
                                                                  

  240 GCTTCGCAATGCAGGAACAAAGGCTGACGTTGATGAAAATATAAAGAAATTTAAAGAGGA
                                                                  

  300 AAATTTTGAAAAAATTCCAGACCCCGAACAGCTCGATCACTTCAGGAAAACACAGAGCAC
                                                                  

  360 AGATCAACTGGTGGAGTCAATGAAGAACCCTCCCCCAATGGGCACGCACACACTGCCCCA
                                                                  

  420 CCCAATATGGTCTGAAGAAGAACTTCATAGTGTGAAGGTCACACATAAACCGCCCGAGGG
                                                                  

  480 ATTCGTAGACAAGCTTGCCTTTCGATCAGTGAAGCTGCTACGTTCAACTTTTGACCTTTT
                                                                  

  540 AACCGGGTTCAACTGGGGAGAAAGAACGGAGAAGAAATGGGTGTTACGGATTTGCTTCCT
                                                                  

  600 AGAGACTGTCGCTGGAGTTCCAGGGATGGTGGCAGCGATGACGCGACATTTACACTCTTT
                                                                  

  660 GCGCCGACTCAAGCGGGATCACGGCTGGATTCATACCCTTTTAGAGGAAGCAGAAAACGA
                                                                  

  720 AAGAATGCATCTGATGACAGCATTGCAGCTTCGACAACCATCTTGGTTATTTAGAAGTGG
                                                                  

  780 CGTCATCGTGTCTCAGGGTGCTTTCGTCACGATGTTCAGCATTGCCTATATGCTAAGCCC
                                                                  

  840 TCGGTTTTGTCATCGGTTCGTTGGCTATCTAGAGGAGGAAGCAGTGTTTACTTACTCAAA
                                                                  

  900 ATGTTTGAAGGATATAGAGTCAGGGCCCCTTAAACACTGGCAAACAAAAGCAGCACCCGA
                                                                  

  960 CGTGGCTATCCGCTATTGGAAACTCCCAAATAACGGAACAATGAAAGACGTCATATTTAA
                                                                  

 1020 CATCCGAGCAGACGAGGCTAATCACCGGAAAGTGAACCATGTTTTGGCATCCATGAAAAC
              >>>>>>>>>>>>>>>>>>>>                                

 1080 CGACCAGTACAACCCATTTCTACCCGGTCAGTAGTTTTAAATCAGACCCTTAATGTGCCT
                                               <<<<<<<<<<<<<<<<<<<

 1140 CATCTCTGTTTACAGGAAACTGCATCCATGAAGGATGTAGTGTTGGCAATTCGAGCAGAT
      <<                                                          

 1200 GAGGCTCACCATAGGGTCGTGAACCATACTCTGGCTTCCATGAAAGAAGACGAATACAAT
                                                                  

 1260 CCTTATGAGCCAGGGAAGTGATATGGAAGCAGAGAAAACAGCGTTAACATTCTGTAGTTA
                                                                  

 1320 TACTGTGAA
               

KEYS (in order of precedence):
>>>>>> left primer
<<<<<< right primer

ADDITIONAL OLIGOS
                    start  len      tm     gc%  any_th  3'_th hairpin seq

 1 LEFT PRIMER       1023   20   60.32   60.00    0.00   0.00   42.78 CCGAGCAGACGAGGCTAATC
   RIGHT PRIMER      1141   21   58.73   47.62    0.00   0.00   34.07 TGAGGCACATTAAGGGTCTGA
   PRODUCT SIZE: 119, PAIR ANY_TH COMPL: 0.07, PAIR 3'_TH COMPL: 0.00

 2 LEFT PRIMER       1024   20   59.62   55.00    0.00   0.00   42.78 CGAGCAGACGAGGCTAATCA
   RIGHT PRIMER      1141   21   58.73   47.62    0.00   0.00   34.07 TGAGGCACATTAAGGGTCTGA
   PRODUCT SIZE: 118, PAIR ANY_TH COMPL: 0.07, PAIR 3'_TH COMPL: 0.00

Statistics
         con   too    in    in   not          no    tm    tm   high  high  high        high      
         sid  many   tar  excl    ok   bad    GC   too   too any_th 3'_th hair-  poly   end      
        ered    Ns   get   reg   reg   GC% clamp   low  high  compl compl   pin     X  stab    ok
Left   10040     0     0     0  9132     0     0    66    87      0     0   179     0     0   576
Right  10040     0     0     0  9804     0     0    49     0      0     0     0     0     0   187
Pair Stats:
considered 3, ok 3
libprimer3 release 2.6.1

6 SPLIT GENOME FASTA

For some reason, I feel like the EMBOSS PrimerSearch tool will not work on mulit-FastA files (or, it makes the results more difficult to decipher?), but I’m just going off of memory and repeating a process I’ve done previously.

This will split the genome multi-FastA file into separate FastA files.

6.1 Split mulit-FastA file in to individual FastA files with PyFaidx

# Load bash variables into memory
source .bashvars

# Make directory if it doesn't exist
mkdir --parents ${genome_fasta_splits_dir}

cd ${genome_fasta_splits_dir}

# Count sequences in FastA
echo "-------------------------------------------------------------------"
echo "NUMBER OF SEQUENCES IN ORIGINAL FASTA"
grep -c "^>" ../${genome_fasta}
echo "-------------------------------------------------------------------"
echo ""
echo ""



# Split FastA
${pyfaidx} \
--split-files \
../${genome_fasta}

# Count number of individual FastA files
echo "-------------------------------------------------------------------"
echo "NUMBER OF INDIVIDUAL FASTA FILES"
ls -1 *.fna | wc -l
echo "-------------------------------------------------------------------"
-------------------------------------------------------------------
NUMBER OF SEQUENCES IN ORIGINAL FASTA
29
-------------------------------------------------------------------


-------------------------------------------------------------------
NUMBER OF INDIVIDUAL FASTA FILES
29
-------------------------------------------------------------------

7 PRIMER SEARCH WITH EMBOSS PRIMERSEARCH

This will run EMBOSS PrimerSearch against the genome to assess primer specificity.

7.1 Create EMBOSS PrimerSearch Primers File

Create a tab-delimited file to use with EMBOSS PrimerSearch.

# Load bash variables into memory
source .bashvars

# Remove if exists.
# Prevents appending to output file repeatedly
if [ "${output_top}/emboss-primers.txt" ]; then
  rm "${output_top}/emboss-primers.txt"
fi

for primer_file in ${output_top}/*primer3-primers-default-format.txt
do
  seq_id=$(grep "SEQUENCE_ID=" "${primer_file}" \
  | sed 's/SEQUENCE_ID=//')
  
  left_primer=$(grep "PRIMER_LEFT_0_SEQUENCE=" "${primer_file}" \
  | sed 's/PRIMER_LEFT_0_SEQUENCE=//' \
  | tr '[:lower:]' '[:upper:]')

  right_primer=$(grep "PRIMER_RIGHT_0_SEQUENCE=" "${primer_file}" \
  | sed 's/PRIMER_RIGHT_0_SEQUENCE=//' \
  | tr '[:lower:]' '[:upper:]')
  
  # Create EMBOSS primer file
  printf "%s\t%s\t%s\t\n" "${seq_id}" "${left_primer}" "${right_primer}" \
  | tee --append "${output_top}/emboss-primers.txt"
  
done
NM_001305360.1  TCTCACTGTGGAAATGCACA    TGGACATTCATCTCTACTGCT   
XM_034462847.2  TCGATCACTTCAGAATTTCACAGG    CCATTGGGGGAGGGTTCTTC    
XM_066083499.1  CAGACGAGGCTAATCACCGG    TGAGGCACATTAAGGGTCTGA

7.2 Run EMBOSS PrimerSearch

This will run EMBOSS PrimerSearch and allow for a 10% mismatch in primer annealing sites (${primersearch} -auto ${fasta} ${primers} 10).

Afterwards, the resulting output files (*.primersearch) will be searched for the term Amplimer, indicating a PCR product would be produced. If no amplimer is identified in a *.primersearch file, then that file is deleted. This should leave just the results in which primer matches were identified.

# Load bash variables into memory
source .bashvars

cd ${genome_fasta_splits_dir}

primers="../../output/emboss-primers.txt"


time \
for fasta in *.fna
  do
  # Remove path from FastA filename
  fasta_no_path=$(echo ${fasta##*/})
  
  # Remove file extension from FastA filename
  fasta_no_ext=$(echo ${fasta_no_path%%.*})
  
  # Convert filename to lowercase
  # Will be used for output from EMBOSS PrimerSearch
  fasta_no_ext_lower=$(echo ${fasta_no_ext} | tr '[:upper:]' '[:lower:]')
  
  ###### Run EMBOSS PrimerSearch on all FastA files ########
  # Allows for a 10% mismatch
  ${primersearch} -auto ${fasta} ${primers} 10
  ##### END EMBOSS ##########
  
  # Find EMBOSS PrimerSearch output files with primer matches
  # Remove those without a match
  if ! grep --quiet "Amplimer" "${fasta_no_ext_lower}.primersearch"
    then rm ${fasta_no_ext_lower}.primersearch
  fi
done
real    1m8.469s
user    1m5.269s
sys 0m3.411s

7.3 Check primer matches

This will print the contents of any remaining *.primersearch output files (i.e. sequences with primer matches)

# Load bash variables into memory
source .bashvars

cd ${genome_fasta_splits_dir}

# Check contents of files with matches
for file in *.primersearch
  do
  echo "FILE: ${file}"
  echo ""
  cat ${file}
  echo "----------------------------------"
  echo ""
done
FILE: nc_088857.primersearch


Primer name NM_001305360.1
Amplimer 1
    Sequence: NC_088857.1  
    
    TGGACATTCATCTCTACTGCT hits forward strand at 40310307 with 0 mismatches
    TCTCACTGTGGAAATGCACA hits reverse strand at [16964538] with 0 mismatches
    Amplimer length: 83 bp

Primer name XM_034462847.2

Primer name XM_066083499.1
Amplimer 1
    Sequence: NC_088857.1  
    
    TGAGGCACATTAAGGGTCTGA hits forward strand at 40310581 with 0 mismatches
    CAGACGAGGCTAATCACCGG hits reverse strand at [16964233] with 0 mismatches
    Amplimer length: 114 bp
----------------------------------

8 SUMMARY

EMBOSS PrimerSearch has identified a single amplicon on chromosome NC_088857.1 for NM_001305360.1 and XM_066083499.1, and each predicted amplicon matches the lengths generated by Primer3. However, no amplicon is predicted for XM_034462847.2. This could possibly be explained by what appears to be an insertion of ~10bp in this variant.

Anyway, I’ll get these ordered and tested out!

9 CITATIONS

Kans, James. 2013. Entrez Direct: E-Utilities on the Unix Command Line. Bethesda (MD): National Center for Biotechnology Information (US). https://www.ncbi.nlm.nih.gov/books/NBK179288/.
Koressaar, Triinu, and Maido Remm. 2007. “Enhancements and Modifications of Primer Design Program Primer3.” Bioinformatics 23 (10): 1289–91. https://doi.org/10.1093/bioinformatics/btm091.
Rice, Peter, Ian Longden, and Alan Bleasby. 2000. “EMBOSS: The European Molecular Biology Open Software Suite.” Trends in Genetics 16 (6): 276–77. https://doi.org/10.1016/s0168-9525(00)02024-2.
Shirley, Matthew D, Zhaorong Ma, Brent S Pedersen, and Sarah J Wheelan. 2015. “Efficient”Pythonic” Access to FASTA Files Using Pyfaidx.” http://dx.doi.org/10.7287/peerj.preprints.970v1.
Untergasser, Andreas, Ioana Cutcutache, Triinu Koressaar, Jian Ye, Brant C. Faircloth, Maido Remm, and Steven G. Rozen. 2012. “Primer3new Capabilities and Interfaces.” Nucleic Acids Research 40 (15): e115–15. https://doi.org/10.1093/nar/gks596.