1 CREATE BASH VARIABLES FILE
2 DOWNLOAD NCBI GENOME FILES
3 EXTRACT C1Q GENE SEQUENCE
- 3.1 Peek at GFF
- 3.2 Format region and extract C1q sequence as FastA.
4 EXTRACT C1Q WITH 5’/3’ BUFFER REGIONS
5 PRIMER DESIGN USING PRIMER3
- 5.1 Design primers
- 5.2 Amplicon length
6 SPLIT GENOME
- 6.1 Split mulit-FastA file in to individual FastA files with PyFaidx
7 PRIMER SEARCH WITH EMBOSS PRIMERSEARCH
8 SUMMARY
9 CITATIONS

This notebook describes using Primer3 (Untergasser et al. 2012; Koressaar and Remm 2007) to reproducibly design primers for sequencing of the lake trout (S.namaycush) (Wikipedia) C1q gene (LOC120027825 (GitHub Issue)). Additionally, this is primarily to confirm that we can successfully amplify the C1q gene, since the bisulfite PCR was unsuccessful (Notebook).

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.

The notebook entry was rendered directly from the R Markdown file, 20240916-snam-c1q-primer-design.Rmd (GitHub - commit ea893a0e9607662b85278bb620f042994efbf374).

1 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/S_namaycush/genomes"'
echo 'export output_top="../output"'
echo 'export genome_fasta_splits_dir="${data_dir}/fasta_splits"'
echo ""

echo "# SEQUENCE"
echo 'export sequence_ID="LOC120027825"'
echo ""

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

echo "# INPUT FILES"
echo 'export genome_fasta="GCF_016432855.1_SaNama_1.0_genomic.fna"'
echo 'export genome_gff="GCF_016432855.1_SaNama_1.0_genomic.gff"'
echo 'export ncbi_gff_gz="GCF_016432855.1_SaNama_1.0_genomic.gff.gz"'
echo 'export ncbi_fasta_gz="GCF_016432855.1_SaNama_1.0_genomic.fna.gz"'
echo 'export ncbi_md5sums="md5checksums.txt"'
echo 'export ncbi_url="https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/016/432/855/GCF_016432855.1_SaNama_1.0/"'
echo ""

echo "# OUTPUT FILES"
echo 'export c1q_fasta="${sequence_ID}.fasta"'
echo 'export c1q_buffer_fasta="${sequence_ID}-${left_buffer}bp.left-${right_buffer}bp.right.fasta"'
echo 'export c1q_faidx_region_file="${sequence_ID}"-region.txt'
echo 'export c1q_buffer_faidx_region_file="${sequence_ID}-${left_buffer}bp.left-${right_buffer}bp.right-region.txt"'
echo ""

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

echo "# PROGRAMS"
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/S_namaycush/genomes"
export output_top="../output"
export genome_fasta_splits_dir="${data_dir}/fasta_splits"

# SEQUENCE
export sequence_ID="LOC120027825"

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

# INPUT FILES
export genome_fasta="GCF_016432855.1_SaNama_1.0_genomic.fna"
export genome_gff="GCF_016432855.1_SaNama_1.0_genomic.gff"
export ncbi_gff_gz="GCF_016432855.1_SaNama_1.0_genomic.gff.gz"
export ncbi_fasta_gz="GCF_016432855.1_SaNama_1.0_genomic.fna.gz"
export ncbi_md5sums="md5checksums.txt"
export ncbi_url="https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/016/432/855/GCF_016432855.1_SaNama_1.0/"

# OUTPUT FILES
export c1q_fasta="${sequence_ID}.fasta"
export c1q_buffer_fasta="${sequence_ID}-${left_buffer}bp.left-${right_buffer}bp.right.fasta"
export c1q_faidx_region_file="${sequence_ID}"-region.txt
export c1q_buffer_faidx_region_file="${sequence_ID}-${left_buffer}bp.left-${right_buffer}bp.right-region.txt"

# SET CPUS
export threads=40

# PROGRAMS
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"

2 DOWNLOAD NCBI GENOME FILES

2.1 Download the actual 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 3.3G
drwxr-xr-x 2 sam sam 164K Sep 16 16:44 fasta_splits
-rw-r--r-- 1 sam sam 2.3G Jan 13  2021 GCF_016432855.1_SaNama_1.0_genomic.fna
-rw-r--r-- 1 sam sam 154K Sep 16 13:22 GCF_016432855.1_SaNama_1.0_genomic.fna.fai
-rw-r--r-- 1 sam sam 642M Jan 13  2021 GCF_016432855.1_SaNama_1.0_genomic.fna.gz
-rw-r--r-- 1 sam sam 373M Sep 28  2022 GCF_016432855.1_SaNama_1.0_genomic.gff
-rw-r--r-- 1 sam sam  18M Sep 28  2022 GCF_016432855.1_SaNama_1.0_genomic.gff.gz
-rw-r--r-- 1 sam sam  33K Sep 16 01:22 md5checksums.txt

2.2 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_016432855.1_SaNama_1.0_genomic.fna.gz: OK
./GCF_016432855.1_SaNama_1.0_genomic.gff.gz: OK

2.3 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_016432855.1_SaNama_1.0_genomic.fna already exists;    not overwritten
gzip: GCF_016432855.1_SaNama_1.0_genomic.gff already exists;    not overwritten
total 3.3G
drwxr-xr-x 2 sam sam 164K Sep 16 16:44 fasta_splits
-rw-r--r-- 1 sam sam 2.3G Jan 13  2021 GCF_016432855.1_SaNama_1.0_genomic.fna
-rw-r--r-- 1 sam sam 154K Sep 16 13:22 GCF_016432855.1_SaNama_1.0_genomic.fna.fai
-rw-r--r-- 1 sam sam 642M Jan 13  2021 GCF_016432855.1_SaNama_1.0_genomic.fna.gz
-rw-r--r-- 1 sam sam 373M Sep 28  2022 GCF_016432855.1_SaNama_1.0_genomic.gff
-rw-r--r-- 1 sam sam  18M Sep 28  2022 GCF_016432855.1_SaNama_1.0_genomic.gff.gz
-rw-r--r-- 1 sam sam  33K Sep 16 01:22 md5checksums.txt

3 EXTRACT C1Q GENE SEQUENCE

3.1 Peek at GFF

# Load bash variables into memory
source .bashvars

awk '$3=="gene"' "${data_dir}/${genome_gff}" | grep "${sequence_ID}" | column -t

NC_052339.1  Gnomon  gene  19244556  19248013  .  -  .  ID=gene-LOC120027825;Dbxref=GeneID:120027825;Name=LOC120027825;gbkey=Gene;gene=LOC120027825;gene_biotype=protein_coding

3.2 Format region and extract C1q sequence as FastA.

# Load bash variables into memory
source .bashvars

# Format region for use with pyfaidx
region=$(awk '$3=="gene"' "${data_dir}/${genome_gff}" | grep "${sequence_ID}" | awk '{print $1":"$4"-"$5}')

echo "${region}"
echo ""

# Extract region
${pyfaidx} "${data_dir}/${genome_fasta}" "${region}" | tee "${output_top}/${c1q_fasta}"

NC_052339.1:19244556-19248013

>NC_052339.1:19244556-19248013
TGGGATGTGTAATGGTCATATTTATTACCATACCTCTCCACTTCTTCCTCTTTCCctttacaatttttattttttttaaa
taataaagtGATGATTTATTTATTGGTTAAAGAGGAAAATCCTCACATCACAGAATGGTGCTGTCCTCACACTGGGAAGA
GCAGGAAGCCACTGAAGGTGTTGTGGTTATCATGGCTATCATGGAGACCCTGGTCTTCAGGGAGACGAAGGTAGACCACA
TCCTCCTTCTCCAGCTCTAGTGTCAACGCATTAGATATGTACTGCCAACCCCCAACTGTATTTCTTTCTACATTATATAG
AACTCTTTGATTATTGTGAAACATCATGATGCCCATGAGTTGTGGGTGGCGGGATGACATGGCCGTGAATCTGAAGAAGT
AGACTCCTTTCACTGATGCTGTGAAGATGCCTGCATCAGAGGAGAAATAGAGTGATATCAGCACTGTCTGTCTCATGTTG
CTGTAGCTGGAATGTTTTtaacaaataaactcagcaaaaaaagaaatgtcccttttcaggaccctgtctttcaaagataa
ttcgtaaaaatccaaataacttcacagatcttcattttaaagggtttaaacactgtttcccatgcttcttcaatgaacca
taaacaattaatgaacatgcacctgtggaacggtcgttaagacactaacagcttacagacggtaggcagttAGGCcaaag
ttatgaaaacttaggacactaaagaggcctttctactgacactgaaaaacaccaaaagaaagatgtccagggtccctgct
catctgcaggaacgtgccttagacatgctgcaaggaggcatgaggactacagatgtggccagggcaataaattgcaatgt
ccgtactgtgagacacctaacacagcgctacagggagacaggacggacagctgatcgtcctcgcagtgacagaccacgtg
taacaacacctgcacaggatcggtacatccgaacatcacacctgcgggacaggtaccggatggcaacaacaactgcccga
gttacaccaggaacgcacaatccctccatcagtgctcagactgtctgcaataggctaagagaggttggactgagggcttg
taggcctgttgtcaGGCAGGTCATCACCGGCAActacgtcgcctatgggcacaaacccaccgtcactggaccagacagcc
gcggttttgtctcaccaggggtgatggtcggaattgcgtttatcgttgaaggaatgagcgttacactgaggcctgtactc
tggagcgggatcgatttggaggtggagggtccgtcatggtctggggcggtgtatcacagcatcatcggactgagcttgtt
gtctttgcaggaaatctcaacgctgtgcgttacagggaagacatcctcctccctcatatggtacccttcctgcaggctca
tcctgacatgaccctccagcatgacaatgccaccagccatactgctcatgttgtgcgtgatttcctgcaagacaggaatg
tcagtgttctgccatggccagcgaagagcccagatctcaatcccattgagcacgtctgggaccttttggatcggagggtg
agagctagggccattcgccacagaaatgtctgggaacttgcaggtgccttggtggaagagtggggtaacatctcacagca
agaactggcaaatctggtgcagtccatgaggaggagatgcactgcagtacttaatgcagctggtggccacaccagatact
gactgttacttttgattttgaccccacctttgttcagggacacattattccatttctgttagtcacatgtctgtggaact
tgttcagtttatgtctcagttgttgaatattgttatgttcatacaaatatttacacatgttaagttgactgaaaataaac
gcagttgacagtgagaggacgtttcttttaaCAGTGAGAGGAGTTTAGTATAATTAAGTCAGGGACTAGGTAACACAAAT
TACCACCAAACACAATAAGTATCAAACTAAACATTGTTATAACTAAAATAAAATCAGGGTGACAGATTAAGTAACATAAA
ATATGTCATTAAAAAAAATGACCGTAgacatacagtactagtcaaaagtttggacatacttagaggtgtgggtttttctt
tatttttactattttctacattgtagcataatagtgaagacataaactatgaaataacaaatatggaatcatgtagtaac
caaaatagtatttaaaaaatctaaatatatttcagatttttcaaagtagccaccctttgcctcgatgacagctttgcact
ctcttggcattctctcttcacctggaatgcttttccaagagtgtgcaaaactgtcatcaaggcaaagggtgactacttta
aagaatgtcaaatataaaatatattttgatttgttttatactttttgggttactacatgattccatatgtgttatttaat
agttttgaagtcttcactattattctacaatgtagaaaatagtaaaataaagaatccctggaataagtaggtgtgtccaa
acttttgactggtactgtacgtgttTGGTATCATATAGCTAGTAGTTACAAAAGTAAACTCATTTAAAAGCTTGAAACGT
AGAATTTGTCCCTCAGAATTATGTCCACTCTGAAAATCTAGTGTCTTATCATGATTTTCAAAGTAAAATATAAAACAAGT
TAGTGAAGTAAAATTATATACAGAGATTACTGAGAAACATGCAGCAAAATCATCCCAGAAGAATTACATTAAATTGCATT
CATAAAAAATTATGATGAGATGACTGACTCAATATTTGGCATTGTTGGATGAATGTTATGCAACATTCAAATGGATTTCC
CTCTTGTTAGTTAGTACCTGTAATTGGGTTGTAGGCCTTGCCGATGTTGGTGATGACTCTACTGAAGATCAGTGTAGTAG
CAGTACTGAATGGACCTACGTTTCCAGAGTCAGTCAAACCAGCAGAGAAGGCCACAGTTGGTCTGCCTGTGAAAAGAAGT
TGATTATTTAAAAGGACAATCTGAGATTGAAATAACAACAAGGCAAATGCTTACTTATCCACCTGTATCAATccaattta
caaatgcctgatATTTCTGACATGACCTGCTTTCTCTTTCTCCAGCTCTTCCACCTTGCTCTTCTGGAGCTGCAGTTCAG
TCTTGGTGACGCTCAGCTCCTGTCTCTGTTCCACCATCATGTCTCTCAGCTCCTCCAGCTTAGACCAGATGTCAGGGGTC
TTGTGGGCCGTCTCTGTAGCTTCAGCCCATGCCCCAAACAGAGAAAACAGCAGCAGAGCTACAGCACCTCTCATTCTGAA
ACGACACATCTTCTGAAATGATCTAGTCTTTGAGGCTAGAAATTTACAGGATTAAAGTAGGAGCTATATCTGTGTGAAAT
ATATTGTGTCTCTGTGGA

4 EXTRACT C1Q WITH 5’/3’ BUFFER REGIONS

Since we want to sequence the entirety of the C1q gene, we need to add some buffer sequence outside of the 5’/3’ ends of the gene. I’ve arbitrarily established large buffer regions:

Left buffer: 500bp
Right buffer: 500bp

# Load bash variables into memory
source .bashvars

# Format region for use with pyfaidx
# Use awk to add/subtract desired buffer regions
region=$(awk '$3=="gene"' "${data_dir}/${genome_gff}" \
| grep "${sequence_ID}" \
| awk -v left_buffer="$left_buffer" -v right_buffer="$right_buffer" '{print $1":"$4 - left_buffer"-"$5 + right_buffer}')

echo "Region formatting: ${region}"
echo ""

# Extract region
${pyfaidx} "${data_dir}/${genome_fasta}" "${region}" | tee "${output_top}/${c1q_buffer_fasta}"

Region formatting: NC_052339.1:19244056-19248513

>NC_052339.1:19244056-19248513
catgttctgttataatctccacccggcacagccaaaagaggactggccacccctcatagcctggttcctctctaggtttc
ttcctaggttttggcctttctagggagtttttcctagccaccgtgcttctacacctgcattgcttgctgtttggggtttt
aggctgggtttctgtacagcactttgagatatcagctgatgtaagaagggctatataaatacatttgatttgatttgata
aggtcACTAGTTACTTATTAAATATGGATATGTCTGAACGACTAACCCTGAGACACATCAGAAAGCTGAGTTCACGTCAT
AATATTGTGATTATAATCACAATCCCTTCTTACACCACTATAAATGTGTCTCTGTCCTTATCTGAGTCTCTGAATGTAGC
CTATGTGTGACCTGAATGTATCTGTTACCTTAATGTAATGTAGCCTACATGTGTTACTTGAAAAATAACCCTAAATTGGT
TCTTATTCCCAACACAGCTCTGGGATGTGTAATGGTCATATTTATTACCATACCTCTCCACTTCTTCCTCTTTCCcttta
caatttttattttttttaaataataaagtGATGATTTATTTATTGGTTAAAGAGGAAAATCCTCACATCACAGAATGGTG
CTGTCCTCACACTGGGAAGAGCAGGAAGCCACTGAAGGTGTTGTGGTTATCATGGCTATCATGGAGACCCTGGTCTTCAG
GGAGACGAAGGTAGACCACATCCTCCTTCTCCAGCTCTAGTGTCAACGCATTAGATATGTACTGCCAACCCCCAACTGTA
TTTCTTTCTACATTATATAGAACTCTTTGATTATTGTGAAACATCATGATGCCCATGAGTTGTGGGTGGCGGGATGACAT
GGCCGTGAATCTGAAGAAGTAGACTCCTTTCACTGATGCTGTGAAGATGCCTGCATCAGAGGAGAAATAGAGTGATATCA
GCACTGTCTGTCTCATGTTGCTGTAGCTGGAATGTTTTtaacaaataaactcagcaaaaaaagaaatgtcccttttcagg
accctgtctttcaaagataattcgtaaaaatccaaataacttcacagatcttcattttaaagggtttaaacactgtttcc
catgcttcttcaatgaaccataaacaattaatgaacatgcacctgtggaacggtcgttaagacactaacagcttacagac
ggtaggcagttAGGCcaaagttatgaaaacttaggacactaaagaggcctttctactgacactgaaaaacaccaaaagaa
agatgtccagggtccctgctcatctgcaggaacgtgccttagacatgctgcaaggaggcatgaggactacagatgtggcc
agggcaataaattgcaatgtccgtactgtgagacacctaacacagcgctacagggagacaggacggacagctgatcgtcc
tcgcagtgacagaccacgtgtaacaacacctgcacaggatcggtacatccgaacatcacacctgcgggacaggtaccgga
tggcaacaacaactgcccgagttacaccaggaacgcacaatccctccatcagtgctcagactgtctgcaataggctaaga
gaggttggactgagggcttgtaggcctgttgtcaGGCAGGTCATCACCGGCAActacgtcgcctatgggcacaaacccac
cgtcactggaccagacagccgcggttttgtctcaccaggggtgatggtcggaattgcgtttatcgttgaaggaatgagcg
ttacactgaggcctgtactctggagcgggatcgatttggaggtggagggtccgtcatggtctggggcggtgtatcacagc
atcatcggactgagcttgttgtctttgcaggaaatctcaacgctgtgcgttacagggaagacatcctcctccctcatatg
gtacccttcctgcaggctcatcctgacatgaccctccagcatgacaatgccaccagccatactgctcatgttgtgcgtga
tttcctgcaagacaggaatgtcagtgttctgccatggccagcgaagagcccagatctcaatcccattgagcacgtctggg
accttttggatcggagggtgagagctagggccattcgccacagaaatgtctgggaacttgcaggtgccttggtggaagag
tggggtaacatctcacagcaagaactggcaaatctggtgcagtccatgaggaggagatgcactgcagtacttaatgcagc
tggtggccacaccagatactgactgttacttttgattttgaccccacctttgttcagggacacattattccatttctgtt
agtcacatgtctgtggaacttgttcagtttatgtctcagttgttgaatattgttatgttcatacaaatatttacacatgt
taagttgactgaaaataaacgcagttgacagtgagaggacgtttcttttaaCAGTGAGAGGAGTTTAGTATAATTAAGTC
AGGGACTAGGTAACACAAATTACCACCAAACACAATAAGTATCAAACTAAACATTGTTATAACTAAAATAAAATCAGGGT
GACAGATTAAGTAACATAAAATATGTCATTAAAAAAAATGACCGTAgacatacagtactagtcaaaagtttggacatact
tagaggtgtgggtttttctttatttttactattttctacattgtagcataatagtgaagacataaactatgaaataacaa
atatggaatcatgtagtaaccaaaatagtatttaaaaaatctaaatatatttcagatttttcaaagtagccaccctttgc
ctcgatgacagctttgcactctcttggcattctctcttcacctggaatgcttttccaagagtgtgcaaaactgtcatcaa
ggcaaagggtgactactttaaagaatgtcaaatataaaatatattttgatttgttttatactttttgggttactacatga
ttccatatgtgttatttaatagttttgaagtcttcactattattctacaatgtagaaaatagtaaaataaagaatccctg
gaataagtaggtgtgtccaaacttttgactggtactgtacgtgttTGGTATCATATAGCTAGTAGTTACAAAAGTAAACT
CATTTAAAAGCTTGAAACGTAGAATTTGTCCCTCAGAATTATGTCCACTCTGAAAATCTAGTGTCTTATCATGATTTTCA
AAGTAAAATATAAAACAAGTTAGTGAAGTAAAATTATATACAGAGATTACTGAGAAACATGCAGCAAAATCATCCCAGAA
GAATTACATTAAATTGCATTCATAAAAAATTATGATGAGATGACTGACTCAATATTTGGCATTGTTGGATGAATGTTATG
CAACATTCAAATGGATTTCCCTCTTGTTAGTTAGTACCTGTAATTGGGTTGTAGGCCTTGCCGATGTTGGTGATGACTCT
ACTGAAGATCAGTGTAGTAGCAGTACTGAATGGACCTACGTTTCCAGAGTCAGTCAAACCAGCAGAGAAGGCCACAGTTG
GTCTGCCTGTGAAAAGAAGTTGATTATTTAAAAGGACAATCTGAGATTGAAATAACAACAAGGCAAATGCTTACTTATCC
ACCTGTATCAATccaatttacaaatgcctgatATTTCTGACATGACCTGCTTTCTCTTTCTCCAGCTCTTCCACCTTGCT
CTTCTGGAGCTGCAGTTCAGTCTTGGTGACGCTCAGCTCCTGTCTCTGTTCCACCATCATGTCTCTCAGCTCCTCCAGCT
TAGACCAGATGTCAGGGGTCTTGTGGGCCGTCTCTGTAGCTTCAGCCCATGCCCCAAACAGAGAAAACAGCAGCAGAGCT
ACAGCACCTCTCATTCTGAAACGACACATCTTCTGAAATGATCTAGTCTTTGAGGCTAGAAATTTACAGGATTAAAGTAG
GAGCTATATCTGTGTGAAATATATTGTGTCTCTGTGGAAAAAAGAGGGTTCTTAAATATCAGGGAGTATTATTTGATTAG
GATTACTGGTAGGATTCTTTATTTGTTTTTACTGGCAATGAGGTGAAGGTTTCTTGCGTCATTCACCAGTTGTTGTTACT
GTATTTCGTCATACTTAATTTTGATAGTCCCCTAGATAGGGAgtgggcgtacacatcacagacaaactaaaatggtccac
acagacagtgtggtaaagaaggaaggccaaaaagatcatcaaggacaacaaccacccgagccactgcctgttcacactgc
tatcatccagaaggtgaggtcagtacaggtacatcaagctgggaccgagagattgagaaacagcttctatctcaaggcca
tcagactgctaaacagcaatcattaACTGAGAGAGTCTGCTGCCCACATTGAGAACCAATCAcaggacactttaataaat
ggatcactagtcactttaaacaatgccactttaaataatggcactttaataatgctta

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 nucletoide 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
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.

# Load bash variables into memory
source .bashvars

# Get sequence only, by skipping the first record
# Remove newlines so sequence is on a single line
c1q_sequence=$(awk 'NR > 1' "${output_top}/${c1q_fasta}" | tr -d '\n')

# Determine length of C1q
c1q_length=${#c1q_sequence}

# Get sequence only, by skipping the first record
# Remove newlines so sequence is on a single line
c1q_buffer_sequence=$(awk 'NR > 1' "${output_top}/${c1q_buffer_fasta}" | tr -d '\n')

# Determine length of C1q with the 5'/3' buffer sequences included
c1q_buffer_length=${#c1q_buffer_sequence}

# Calculate combined length of 5'buffer region and C1q length
c1q_buffer_and_seq_length=$((left_buffer + c1q_length))


# Use heredoc to create Primer3 parameters file
cat << EOF > ${output_top}/primer3-params.txt
SEQUENCE_ID=${sequence_ID}
SEQUENCE_TEMPLATE=${c1q_buffer_sequence}
PRIMER_TASK=pick_sequencing_primers
SEQUENCE_TARGET=${left_buffer},${c1q_length}
PRIMER_MIN_TM=50
PRIMER_OPT_TM=60
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=25
PRIMER_MAX_NS_ACCEPTED=0
P3_FILE_FLAG=1
PRIMER_EXPLAIN_FLAG=1
PRIMER_THERMODYNAMIC_PARAMETERS_PATH=${primer3_config}
PRIMER_NUM_RETURN=10
PRIMER_PAIR_EXPLAIN=considered 0, ok 0
=
EOF


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

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


# Pull out any primers falling outside of target
tail -n 30 "${output_top}/primer3-primers.txt" \
| head -n -12 \
| awk -v left_buffer="$left_buffer" -v target="$c1q_buffer_and_seq_length" '$3 <= left_buffer || $3 >= target {print $0}'


### Write the left and right primer start positions to files for later use in R chunk(s).
# Pull out left primer start position
left_primer_start=$(tail -n 30 "${output_top}/primer3-primers.txt" \
| head -n -12 \
| awk -v left_buffer="$left_buffer" -v target="$c1q_buffer_and_seq_length" '$3 <= left_buffer || $3 >= target {print $0}' \
| grep "PRIMER" \
| awk 'NR == 1 {print $3}')

# Pull out right primer start position
right_primer_start=$(tail -n 30 "${output_top}/primer3-primers.txt" \
| head -n -12 \
| awk -v left_buffer="$left_buffer" -v target="$c1q_buffer_and_seq_length" '$3 <= left_buffer || $3 >= target {print $0}' \
| grep "PRIMER" \
| awk 'NR == 2 {print $3}')

echo "${left_primer_start}" > "${output_top}/left-primer-start.txt"
echo "${right_primer_start}" > "${output_top}/right-primer-start.txt"

echo ""
echo ""
echo "----------------------------------------------------------------------------------"
echo ""
echo "Primer results file:"
echo ""
echo ""
# Print the full output file
cat "${output_top}/primer3-primers.txt"

echo ""
echo ""
echo "----------------------------------------------------------------------------------"
echo ""
echo "Primer default (i.e. computer-friendly) results file:"
echo ""
echo ""

# Print the full output file
cat "${output_top}/primer3-primers-default-format.txt"

 1 LEFT_PRIMER        277   20   58.01   50.00    0.00   0.00    0.00 ACGACTAACCCTGAGACACA

                     start  len      tm     gc%  any_th  3'_th hairpin seq
 8 RIGHT_PRIMER      4188   21   59.22   47.62    3.32   0.00    0.00 tggccttccttctttaccaca


----------------------------------------------------------------------------------

Primer results file:


PRIMER PICKING RESULTS FOR LOC120027825

No mispriming library specified
Using 0-based sequence positions
SEQUENCE SIZE: 4458
INCLUDED REGION SIZE: 4458

TARGETS (start, len)*: 500,3458
    0 catgttctgttataatctccacccggcacagccaaaagaggactggccacccctcatagc
                                                                  

   60 ctggttcctctctaggtttcttcctaggttttggcctttctagggagtttttcctagcca
                                                                  

  120 ccgtgcttctacacctgcattgcttgctgtttggggttttaggctgggtttctgtacagc
                                                                  

  180 actttgagatatcagctgatgtaagaagggctatataaatacatttgatttgatttgata
                                                                  

  240 aggtcACTAGTTACTTATTAAATATGGATATGTCTGAACGACTAACCCTGAGACACATCA
                                           >>>>>>>>>>>>>>>>>>>>   

  300 GAAAGCTGAGTTCACGTCATAATATTGTGATTATAATCACAATCCCTTCTTACACCACTA
                                                                  

  360 TAAATGTGTCTCTGTCCTTATCTGAGTCTCTGAATGTAGCCTATGTGTGACCTGAATGTA
                                                                  

  420 TCTGTTACCTTAATGTAATGTAGCCTACATGTGTTACTTGAAAAATAACCCTAAATTGGT
                                                                  

  480 TCTTATTCCCAACACAGCTCTGGGATGTGTAATGGTCATATTTATTACCATACCTCTCCA
                          ****************************************

  540 CTTCTTCCTCTTTCCctttacaatttttattttttttaaataataaagtGATGATTTATT
      ************************************************************

  600 TATTGGTTAAAGAGGAAAATCCTCACATCACAGAATGGTGCTGTCCTCACACTGGGAAGA
      **********************************<<<<<<<<<<<<<<<<<<<<******

  660 GCAGGAAGCCACTGAAGGTGTTGTGGTTATCATGGCTATCATGGAGACCCTGGTCTTCAG
      ************************************************************

  720 GGAGACGAAGGTAGACCACATCCTCCTTCTCCAGCTCTAGTGTCAACGCATTAGATATGT
      ************************************************************

  780 ACTGCCAACCCCCAACTGTATTTCTTTCTACATTATATAGAACTCTTTGATTATTGTGAA
      *>>>>>>>>>>>>>>>>>>>>***************************************

  840 ACATCATGATGCCCATGAGTTGTGGGTGGCGGGATGACATGGCCGTGAATCTGAAGAAGT
      ************************************************************

  900 AGACTCCTTTCACTGATGCTGTGAAGATGCCTGCATCAGAGGAGAAATAGAGTGATATCA
      ************************************************************

  960 GCACTGTCTGTCTCATGTTGCTGTAGCTGGAATGTTTTtaacaaataaactcagcaaaaa
      ************************************************************

 1020 aagaaatgtcccttttcaggaccctgtctttcaaagataattcgtaaaaatccaaataac
      ************************************************************

 1080 ttcacagatcttcattttaaagggtttaaacactgtttcccatgcttcttcaatgaacca
      ************************************************************

 1140 taaacaattaatgaacatgcacctgtggaacggtcgttaagacactaacagcttacagac
      *******************<<<<<<<<<<<<<<<<<<<<*********************

 1200 ggtaggcagttAGGCcaaagttatgaaaacttaggacactaaagaggcctttctactgac
      ************************************************************

 1260 actgaaaaacaccaaaagaaagatgtccagggtccctgctcatctgcaggaacgtgcctt
      *******************************************>>>>>>>>>>>>>>>>>

 1320 agacatgctgcaaggaggcatgaggactacagatgtggccagggcaataaattgcaatgt
      >>>*********************************************************

 1380 ccgtactgtgagacacctaacacagcgctacagggagacaggacggacagctgatcgtcc
      ************************************************************

 1440 tcgcagtgacagaccacgtgtaacaacacctgcacaggatcggtacatccgaacatcaca
      ************************************************************

 1500 cctgcgggacaggtaccggatggcaacaacaactgcccgagttacaccaggaacgcacaa
      ************************************************************

 1560 tccctccatcagtgctcagactgtctgcaataggctaagagaggttggactgagggcttg
      ************************************************************

 1620 taggcctgttgtcaGGCAGGTCATCACCGGCAActacgtcgcctatgggcacaaacccac
      ****************<<<<<<<<<<<<<<<<<<<<************************

 1680 cgtcactggaccagacagccgcggttttgtctcaccaggggtgatggtcggaattgcgtt
      ************************************************************

 1740 tatcgttgaaggaatgagcgttacactgaggcctgtactctggagcgggatcgatttgga
      **************************************>>>>>>>>>>>>>>>>>>>>**

 1800 ggtggagggtccgtcatggtctggggcggtgtatcacagcatcatcggactgagcttgtt
      ************************************************************

 1860 gtctttgcaggaaatctcaacgctgtgcgttacagggaagacatcctcctccctcatatg
      ************************************************************

 1920 gtacccttcctgcaggctcatcctgacatgaccctccagcatgacaatgccaccagccat
      ************************************************************

 1980 actgctcatgttgtgcgtgatttcctgcaagacaggaatgtcagtgttctgccatggcca
      ************************************************************

 2040 gcgaagagcccagatctcaatcccattgagcacgtctgggaccttttggatcggagggtg
      ************************************************************

 2100 agagctagggccattcgccacagaaatgtctgggaacttgcaggtgccttggtggaagag
      ************************************************************

 2160 tggggtaacatctcacagcaagaactggcaaatctggtgcagtccatgaggaggagatgc
      **********<<<<<<<<<<<<<<<<<<<<******************************

 2220 actgcagtacttaatgcagctggtggccacaccagatactgactgttacttttgattttg
      ************************************************************

 2280 accccacctttgttcagggacacattattccatttctgttagtcacatgtctgtggaact
      **>>>>>>>>>>>>>>>>>>>>**************************************

 2340 tgttcagtttatgtctcagttgttgaatattgttatgttcatacaaatatttacacatgt
      ************************************************************

 2400 taagttgactgaaaataaacgcagttgacagtgagaggacgtttcttttaaCAGTGAGAG
      ************************************************************

 2460 GAGTTTAGTATAATTAAGTCAGGGACTAGGTAACACAAATTACCACCAAACACAATAAGT
      ************************************************************

 2520 ATCAAACTAAACATTGTTATAACTAAAATAAAATCAGGGTGACAGATTAAGTAACATAAA
      ************************************************************

 2580 ATATGTCATTAAAAAAAATGACCGTAgacatacagtactagtcaaaagtttggacatact
      *********************************************************<<<

 2640 tagaggtgtgggtttttctttatttttactattttctacattgtagcataatagtgaaga
      <<<<<<<<<<<<<<<<<<<*****************************************

 2700 cataaactatgaaataacaaatatggaatcatgtagtaaccaaaatagtatttaaaaaat
      ************************************************************

 2760 ctaaatatatttcagatttttcaaagtagccaccctttgcctcgatgacagctttgcact
      *******************************>>>>>>>>>>>>>>>>>>>>*********

 2820 ctcttggcattctctcttcacctggaatgcttttccaagagtgtgcaaaactgtcatcaa
      ************************************************************

 2880 ggcaaagggtgactactttaaagaatgtcaaatataaaatatattttgatttgttttata
      ************************************************************

 2940 ctttttgggttactacatgattccatatgtgttatttaatagttttgaagtcttcactat
      ************************************************************

 3000 tattctacaatgtagaaaatagtaaaataaagaatccctggaataagtaggtgtgtccaa
      ************************************************************

 3060 acttttgactggtactgtacgtgttTGGTATCATATAGCTAGTAGTTACAAAAGTAAACT
      ************************************************************

 3120 CATTTAAAAGCTTGAAACGTAGAATTTGTCCCTCAGAATTATGTCCACTCTGAAAATCTA
      **************************<<<<<<<<<<<<<<<<<<<<<<<***********

 3180 GTGTCTTATCATGATTTTCAAAGTAAAATATAAAACAAGTTAGTGAAGTAAAATTATATA
      ************************************************************

 3240 CAGAGATTACTGAGAAACATGCAGCAAAATCATCCCAGAAGAATTACATTAAATTGCATT
      ********************>>>>>>>>>>>>>>>>>>>>>>>>>***************

 3300 CATAAAAAATTATGATGAGATGACTGACTCAATATTTGGCATTGTTGGATGAATGTTATG
      ************************************************************

 3360 CAACATTCAAATGGATTTCCCTCTTGTTAGTTAGTACCTGTAATTGGGTTGTAGGCCTTG
      ************************************************************

 3420 CCGATGTTGGTGATGACTCTACTGAAGATCAGTGTAGTAGCAGTACTGAATGGACCTACG
      ************************************************************

 3480 TTTCCAGAGTCAGTCAAACCAGCAGAGAAGGCCACAGTTGGTCTGCCTGTGAAAAGAAGT
      ************************************************************

 3540 TGATTATTTAAAAGGACAATCTGAGATTGAAATAACAACAAGGCAAATGCTTACTTATCC
      ************************************************************

 3600 ACCTGTATCAATccaatttacaaatgcctgatATTTCTGACATGACCTGCTTTCTCTTTC
      ************************************************************

 3660 TCCAGCTCTTCCACCTTGCTCTTCTGGAGCTGCAGTTCAGTCTTGGTGACGCTCAGCTCC
      **<<<<<<<<<<<<<<<<<<<<**************************************

 3720 TGTCTCTGTTCCACCATCATGTCTCTCAGCTCCTCCAGCTTAGACCAGATGTCAGGGGTC
      ***********************************************>>>>>>>>>>>>>

 3780 TTGTGGGCCGTCTCTGTAGCTTCAGCCCATGCCCCAAACAGAGAAAACAGCAGCAGAGCT
      >>>>>>>*****************************************************

 3840 ACAGCACCTCTCATTCTGAAACGACACATCTTCTGAAATGATCTAGTCTTTGAGGCTAGA
      ************************************************************

 3900 AATTTACAGGATTAAAGTAGGAGCTATATCTGTGTGAAATATATTGTGTCTCTGTGGAAA
      **********************************************************  

 3960 AAAGAGGGTTCTTAAATATCAGGGAGTATTATTTGATTAGGATTACTGGTAGGATTCTTT
                                                                  

 4020 ATTTGTTTTTACTGGCAATGAGGTGAAGGTTTCTTGCGTCATTCACCAGTTGTTGTTACT
                                                                  

 4080 GTATTTCGTCATACTTAATTTTGATAGTCCCCTAGATAGGGAgtgggcgtacacatcaca
                                                                  

 4140 gacaaactaaaatggtccacacagacagtgtggtaaagaaggaaggccaaaaagatcatc
                                  <<<<<<<<<<<<<<<<<<<<<           

 4200 aaggacaacaaccacccgagccactgcctgttcacactgctatcatccagaaggtgaggt
                                                                  

 4260 cagtacaggtacatcaagctgggaccgagagattgagaaacagcttctatctcaaggcca
                                                                  

 4320 tcagactgctaaacagcaatcattaACTGAGAGAGTCTGCTGCCCACATTGAGAACCAAT
                                                                  

 4380 CAcaggacactttaataaatggatcactagtcactttaaacaatgccactttaaataatg
                                                                  

 4440 gcactttaataatgctta
                        

KEYS (in order of precedence):
****** target
>>>>>> left primer
<<<<<< right primer
^^^^^^ left primer / right primer overlap

                    start  len      tm     gc%  any_th  3'_th hairpin seq
 1 LEFT_PRIMER        277   20   58.01   50.00    0.00   0.00    0.00 ACGACTAACCCTGAGACACA
 2 LEFT_PRIMER        781   20   59.96   55.00    0.00   0.00    0.00 CTGCCAACCCCCAACTGTAT
 3 LEFT_PRIMER       1303   20   60.04   55.00   14.25   0.00   43.28 ctgcaggaacgtgccttaga
 4 LEFT_PRIMER       1778   20   59.90   55.00    0.00   0.00   39.00 tctggagcgggatcgatttg
 5 LEFT_PRIMER       2282   20   59.82   55.00    0.00   0.00   42.25 cccacctttgttcagggaca
 6 LEFT_PRIMER       2791   20   60.04   55.00    0.00   0.00    0.00 accctttgcctcgatgacag
 7 LEFT_PRIMER       3260   25   60.63   40.00    0.00   0.00    0.00 GCAGCAAAATCATCCCAGAAGAATT
 8 LEFT_PRIMER       3767   20   60.04   60.00    0.00   0.00    0.00 GATGTCAGGGGTCTTGTGGG

                     start  len      tm     gc%  any_th  3'_th hairpin seq
 1 RIGHT_PRIMER       653   20   59.96   55.00    0.00   0.00   35.43 CAGTGTGAGGACAGCACCAT
 2 RIGHT_PRIMER      1178   20   59.97   55.00   11.27   0.00   41.51 taacgaccgttccacaggtg
 3 RIGHT_PRIMER      1655   20   59.75   55.00    1.90   0.00   40.58 tagTTGCCGGTGATGACCTG
 4 RIGHT_PRIMER      2189   20   59.53   50.00    0.00   0.00   34.84 tgccagttcttgctgtgaga
 5 RIGHT_PRIMER      2658   22   57.21   40.91    0.00   0.00    0.00 agaaaaacccacacctctaagt
 6 RIGHT_PRIMER      3168   23   58.90   43.48    0.00   0.00    0.00 AGTGGACATAATTCTGAGGGACA
 7 RIGHT_PRIMER      3681   20   59.68   55.00    0.00   0.00   35.36 AGAGCAAGGTGGAAGAGCTG
 8 RIGHT_PRIMER      4188   21   59.22   47.62    3.32   0.00    0.00 tggccttccttctttaccaca

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    2518     0     0     0     0   162     0   221   612      0     0   176    10     0     8
Right   2560     0     0     0     0   108     0   205   530      0     0   221     0     0     8
Pair Stats:
considered 0, ok 0
libprimer3 release 2.6.1




----------------------------------------------------------------------------------

Primer default (i.e. computer-friendly) results file:


SEQUENCE_ID=LOC120027825
SEQUENCE_TEMPLATE=catgttctgttataatctccacccggcacagccaaaagaggactggccacccctcatagcctggttcctctctaggtttcttcctaggttttggcctttctagggagtttttcctagccaccgtgcttctacacctgcattgcttgctgtttggggttttaggctgggtttctgtacagcactttgagatatcagctgatgtaagaagggctatataaatacatttgatttgatttgataaggtcACTAGTTACTTATTAAATATGGATATGTCTGAACGACTAACCCTGAGACACATCAGAAAGCTGAGTTCACGTCATAATATTGTGATTATAATCACAATCCCTTCTTACACCACTATAAATGTGTCTCTGTCCTTATCTGAGTCTCTGAATGTAGCCTATGTGTGACCTGAATGTATCTGTTACCTTAATGTAATGTAGCCTACATGTGTTACTTGAAAAATAACCCTAAATTGGTTCTTATTCCCAACACAGCTCTGGGATGTGTAATGGTCATATTTATTACCATACCTCTCCACTTCTTCCTCTTTCCctttacaatttttattttttttaaataataaagtGATGATTTATTTATTGGTTAAAGAGGAAAATCCTCACATCACAGAATGGTGCTGTCCTCACACTGGGAAGAGCAGGAAGCCACTGAAGGTGTTGTGGTTATCATGGCTATCATGGAGACCCTGGTCTTCAGGGAGACGAAGGTAGACCACATCCTCCTTCTCCAGCTCTAGTGTCAACGCATTAGATATGTACTGCCAACCCCCAACTGTATTTCTTTCTACATTATATAGAACTCTTTGATTATTGTGAAACATCATGATGCCCATGAGTTGTGGGTGGCGGGATGACATGGCCGTGAATCTGAAGAAGTAGACTCCTTTCACTGATGCTGTGAAGATGCCTGCATCAGAGGAGAAATAGAGTGATATCAGCACTGTCTGTCTCATGTTGCTGTAGCTGGAATGTTTTtaacaaataaactcagcaaaaaaagaaatgtcccttttcaggaccctgtctttcaaagataattcgtaaaaatccaaataacttcacagatcttcattttaaagggtttaaacactgtttcccatgcttcttcaatgaaccataaacaattaatgaacatgcacctgtggaacggtcgttaagacactaacagcttacagacggtaggcagttAGGCcaaagttatgaaaacttaggacactaaagaggcctttctactgacactgaaaaacaccaaaagaaagatgtccagggtccctgctcatctgcaggaacgtgccttagacatgctgcaaggaggcatgaggactacagatgtggccagggcaataaattgcaatgtccgtactgtgagacacctaacacagcgctacagggagacaggacggacagctgatcgtcctcgcagtgacagaccacgtgtaacaacacctgcacaggatcggtacatccgaacatcacacctgcgggacaggtaccggatggcaacaacaactgcccgagttacaccaggaacgcacaatccctccatcagtgctcagactgtctgcaataggctaagagaggttggactgagggcttgtaggcctgttgtcaGGCAGGTCATCACCGGCAActacgtcgcctatgggcacaaacccaccgtcactggaccagacagccgcggttttgtctcaccaggggtgatggtcggaattgcgtttatcgttgaaggaatgagcgttacactgaggcctgtactctggagcgggatcgatttggaggtggagggtccgtcatggtctggggcggtgtatcacagcatcatcggactgagcttgttgtctttgcaggaaatctcaacgctgtgcgttacagggaagacatcctcctccctcatatggtacccttcctgcaggctcatcctgacatgaccctccagcatgacaatgccaccagccatactgctcatgttgtgcgtgatttcctgcaagacaggaatgtcagtgttctgccatggccagcgaagagcccagatctcaatcccattgagcacgtctgggaccttttggatcggagggtgagagctagggccattcgccacagaaatgtctgggaacttgcaggtgccttggtggaagagtggggtaacatctcacagcaagaactggcaaatctggtgcagtccatgaggaggagatgcactgcagtacttaatgcagctggtggccacaccagatactgactgttacttttgattttgaccccacctttgttcagggacacattattccatttctgttagtcacatgtctgtggaacttgttcagtttatgtctcagttgttgaatattgttatgttcatacaaatatttacacatgttaagttgactgaaaataaacgcagttgacagtgagaggacgtttcttttaaCAGTGAGAGGAGTTTAGTATAATTAAGTCAGGGACTAGGTAACACAAATTACCACCAAACACAATAAGTATCAAACTAAACATTGTTATAACTAAAATAAAATCAGGGTGACAGATTAAGTAACATAAAATATGTCATTAAAAAAAATGACCGTAgacatacagtactagtcaaaagtttggacatacttagaggtgtgggtttttctttatttttactattttctacattgtagcataatagtgaagacataaactatgaaataacaaatatggaatcatgtagtaaccaaaatagtatttaaaaaatctaaatatatttcagatttttcaaagtagccaccctttgcctcgatgacagctttgcactctcttggcattctctcttcacctggaatgcttttccaagagtgtgcaaaactgtcatcaaggcaaagggtgactactttaaagaatgtcaaatataaaatatattttgatttgttttatactttttgggttactacatgattccatatgtgttatttaatagttttgaagtcttcactattattctacaatgtagaaaatagtaaaataaagaatccctggaataagtaggtgtgtccaaacttttgactggtactgtacgtgttTGGTATCATATAGCTAGTAGTTACAAAAGTAAACTCATTTAAAAGCTTGAAACGTAGAATTTGTCCCTCAGAATTATGTCCACTCTGAAAATCTAGTGTCTTATCATGATTTTCAAAGTAAAATATAAAACAAGTTAGTGAAGTAAAATTATATACAGAGATTACTGAGAAACATGCAGCAAAATCATCCCAGAAGAATTACATTAAATTGCATTCATAAAAAATTATGATGAGATGACTGACTCAATATTTGGCATTGTTGGATGAATGTTATGCAACATTCAAATGGATTTCCCTCTTGTTAGTTAGTACCTGTAATTGGGTTGTAGGCCTTGCCGATGTTGGTGATGACTCTACTGAAGATCAGTGTAGTAGCAGTACTGAATGGACCTACGTTTCCAGAGTCAGTCAAACCAGCAGAGAAGGCCACAGTTGGTCTGCCTGTGAAAAGAAGTTGATTATTTAAAAGGACAATCTGAGATTGAAATAACAACAAGGCAAATGCTTACTTATCCACCTGTATCAATccaatttacaaatgcctgatATTTCTGACATGACCTGCTTTCTCTTTCTCCAGCTCTTCCACCTTGCTCTTCTGGAGCTGCAGTTCAGTCTTGGTGACGCTCAGCTCCTGTCTCTGTTCCACCATCATGTCTCTCAGCTCCTCCAGCTTAGACCAGATGTCAGGGGTCTTGTGGGCCGTCTCTGTAGCTTCAGCCCATGCCCCAAACAGAGAAAACAGCAGCAGAGCTACAGCACCTCTCATTCTGAAACGACACATCTTCTGAAATGATCTAGTCTTTGAGGCTAGAAATTTACAGGATTAAAGTAGGAGCTATATCTGTGTGAAATATATTGTGTCTCTGTGGAAAAAAGAGGGTTCTTAAATATCAGGGAGTATTATTTGATTAGGATTACTGGTAGGATTCTTTATTTGTTTTTACTGGCAATGAGGTGAAGGTTTCTTGCGTCATTCACCAGTTGTTGTTACTGTATTTCGTCATACTTAATTTTGATAGTCCCCTAGATAGGGAgtgggcgtacacatcacagacaaactaaaatggtccacacagacagtgtggtaaagaaggaaggccaaaaagatcatcaaggacaacaaccacccgagccactgcctgttcacactgctatcatccagaaggtgaggtcagtacaggtacatcaagctgggaccgagagattgagaaacagcttctatctcaaggccatcagactgctaaacagcaatcattaACTGAGAGAGTCTGCTGCCCACATTGAGAACCAATCAcaggacactttaataaatggatcactagtcactttaaacaatgccactttaaataatggcactttaataatgctta
PRIMER_TASK=pick_sequencing_primers
SEQUENCE_TARGET=500,3458
PRIMER_MIN_TM=50
PRIMER_OPT_TM=60
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=25
PRIMER_MAX_NS_ACCEPTED=0
P3_FILE_FLAG=1
PRIMER_EXPLAIN_FLAG=1
PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/home/shared/primer3-2.6.1/src/primer3_config
PRIMER_NUM_RETURN=10
PRIMER_PAIR_EXPLAIN=considered 0, ok 0
PRIMER_LEFT_EXPLAIN=sequencing locations 8, considered 2518, GC content failed 162, low tm 221, high tm 612, high hairpin stability 176, long poly-x seq 10, ok 8
PRIMER_RIGHT_EXPLAIN=sequencing locations 8, considered 2560, GC content failed 108, low tm 205, high tm 530, high hairpin stability 221, ok 8
PRIMER_PAIR_EXPLAIN=considered 0, ok 0
PRIMER_LEFT_NUM_RETURNED=8
PRIMER_RIGHT_NUM_RETURNED=8
PRIMER_INTERNAL_NUM_RETURNED=0
PRIMER_PAIR_NUM_RETURNED=0
PRIMER_LEFT_0_PENALTY=1.991121
PRIMER_RIGHT_0_PENALTY=0.035881
PRIMER_LEFT_0_SEQUENCE=ACGACTAACCCTGAGACACA
PRIMER_RIGHT_0_SEQUENCE=CAGTGTGAGGACAGCACCAT
PRIMER_LEFT_0=277,20
PRIMER_RIGHT_0=653,20
PRIMER_LEFT_0_TM=58.009
PRIMER_RIGHT_0_TM=59.964
PRIMER_LEFT_0_GC_PERCENT=50.000
PRIMER_RIGHT_0_GC_PERCENT=55.000
PRIMER_LEFT_0_SELF_ANY_TH=0.00
PRIMER_RIGHT_0_SELF_ANY_TH=0.00
PRIMER_LEFT_0_SELF_END_TH=0.00
PRIMER_RIGHT_0_SELF_END_TH=0.00
PRIMER_LEFT_0_HAIRPIN_TH=0.00
PRIMER_RIGHT_0_HAIRPIN_TH=35.43
PRIMER_LEFT_0_END_STABILITY=3.7200
PRIMER_RIGHT_0_END_STABILITY=3.5500
PRIMER_LEFT_1_PENALTY=0.039618
PRIMER_RIGHT_1_PENALTY=0.032403
PRIMER_LEFT_1_SEQUENCE=CTGCCAACCCCCAACTGTAT
PRIMER_RIGHT_1_SEQUENCE=taacgaccgttccacaggtg
PRIMER_LEFT_1=781,20
PRIMER_RIGHT_1=1178,20
PRIMER_LEFT_1_TM=59.960
PRIMER_RIGHT_1_TM=59.968
PRIMER_LEFT_1_GC_PERCENT=55.000
PRIMER_RIGHT_1_GC_PERCENT=55.000
PRIMER_LEFT_1_SELF_ANY_TH=0.00
PRIMER_RIGHT_1_SELF_ANY_TH=11.27
PRIMER_LEFT_1_SELF_END_TH=0.00
PRIMER_RIGHT_1_SELF_END_TH=0.00
PRIMER_LEFT_1_HAIRPIN_TH=0.00
PRIMER_RIGHT_1_HAIRPIN_TH=41.51
PRIMER_LEFT_1_END_STABILITY=2.2900
PRIMER_RIGHT_1_END_STABILITY=4.0000
PRIMER_LEFT_2_PENALTY=0.037326
PRIMER_RIGHT_2_PENALTY=0.249120
PRIMER_LEFT_2_SEQUENCE=ctgcaggaacgtgccttaga
PRIMER_RIGHT_2_SEQUENCE=tagTTGCCGGTGATGACCTG
PRIMER_LEFT_2=1303,20
PRIMER_RIGHT_2=1655,20
PRIMER_LEFT_2_TM=60.037
PRIMER_RIGHT_2_TM=59.751
PRIMER_LEFT_2_GC_PERCENT=55.000
PRIMER_RIGHT_2_GC_PERCENT=55.000
PRIMER_LEFT_2_SELF_ANY_TH=14.25
PRIMER_RIGHT_2_SELF_ANY_TH=1.90
PRIMER_LEFT_2_SELF_END_TH=0.00
PRIMER_RIGHT_2_SELF_END_TH=0.00
PRIMER_LEFT_2_HAIRPIN_TH=43.28
PRIMER_RIGHT_2_HAIRPIN_TH=40.58
PRIMER_LEFT_2_END_STABILITY=2.1000
PRIMER_RIGHT_2_END_STABILITY=4.0000
PRIMER_LEFT_3_PENALTY=0.104855
PRIMER_RIGHT_3_PENALTY=0.469494
PRIMER_LEFT_3_SEQUENCE=tctggagcgggatcgatttg
PRIMER_RIGHT_3_SEQUENCE=tgccagttcttgctgtgaga
PRIMER_LEFT_3=1778,20
PRIMER_RIGHT_3=2189,20
PRIMER_LEFT_3_TM=59.895
PRIMER_RIGHT_3_TM=59.531
PRIMER_LEFT_3_GC_PERCENT=55.000
PRIMER_RIGHT_3_GC_PERCENT=50.000
PRIMER_LEFT_3_SELF_ANY_TH=0.00
PRIMER_RIGHT_3_SELF_ANY_TH=0.00
PRIMER_LEFT_3_SELF_END_TH=0.00
PRIMER_RIGHT_3_SELF_END_TH=0.00
PRIMER_LEFT_3_HAIRPIN_TH=39.00
PRIMER_RIGHT_3_HAIRPIN_TH=34.84
PRIMER_LEFT_3_END_STABILITY=2.3200
PRIMER_RIGHT_3_END_STABILITY=3.2700
PRIMER_LEFT_4_PENALTY=0.184933
PRIMER_RIGHT_4_PENALTY=4.790718
PRIMER_LEFT_4_SEQUENCE=cccacctttgttcagggaca
PRIMER_RIGHT_4_SEQUENCE=agaaaaacccacacctctaagt
PRIMER_LEFT_4=2282,20
PRIMER_RIGHT_4=2658,22
PRIMER_LEFT_4_TM=59.815
PRIMER_RIGHT_4_TM=57.209
PRIMER_LEFT_4_GC_PERCENT=55.000
PRIMER_RIGHT_4_GC_PERCENT=40.909
PRIMER_LEFT_4_SELF_ANY_TH=0.00
PRIMER_RIGHT_4_SELF_ANY_TH=0.00
PRIMER_LEFT_4_SELF_END_TH=0.00
PRIMER_RIGHT_4_SELF_END_TH=0.00
PRIMER_LEFT_4_HAIRPIN_TH=42.25
PRIMER_RIGHT_4_HAIRPIN_TH=0.00
PRIMER_LEFT_4_END_STABILITY=4.0200
PRIMER_RIGHT_4_END_STABILITY=2.2400
PRIMER_LEFT_5_PENALTY=0.036010
PRIMER_RIGHT_5_PENALTY=4.104086
PRIMER_LEFT_5_SEQUENCE=accctttgcctcgatgacag
PRIMER_RIGHT_5_SEQUENCE=AGTGGACATAATTCTGAGGGACA
PRIMER_LEFT_5=2791,20
PRIMER_RIGHT_5=3168,23
PRIMER_LEFT_5_TM=60.036
PRIMER_RIGHT_5_TM=58.896
PRIMER_LEFT_5_GC_PERCENT=55.000
PRIMER_RIGHT_5_GC_PERCENT=43.478
PRIMER_LEFT_5_SELF_ANY_TH=0.00
PRIMER_RIGHT_5_SELF_ANY_TH=0.00
PRIMER_LEFT_5_SELF_END_TH=0.00
PRIMER_RIGHT_5_SELF_END_TH=0.00
PRIMER_LEFT_5_HAIRPIN_TH=0.00
PRIMER_RIGHT_5_HAIRPIN_TH=0.00
PRIMER_LEFT_5_END_STABILITY=3.5100
PRIMER_RIGHT_5_END_STABILITY=4.0200
PRIMER_LEFT_6_PENALTY=5.626116
PRIMER_RIGHT_6_PENALTY=0.324285
PRIMER_LEFT_6_SEQUENCE=GCAGCAAAATCATCCCAGAAGAATT
PRIMER_RIGHT_6_SEQUENCE=AGAGCAAGGTGGAAGAGCTG
PRIMER_LEFT_6=3260,25
PRIMER_RIGHT_6=3681,20
PRIMER_LEFT_6_TM=60.626
PRIMER_RIGHT_6_TM=59.676
PRIMER_LEFT_6_GC_PERCENT=40.000
PRIMER_RIGHT_6_GC_PERCENT=55.000
PRIMER_LEFT_6_SELF_ANY_TH=0.00
PRIMER_RIGHT_6_SELF_ANY_TH=0.00
PRIMER_LEFT_6_SELF_END_TH=0.00
PRIMER_RIGHT_6_SELF_END_TH=0.00
PRIMER_LEFT_6_HAIRPIN_TH=0.00
PRIMER_RIGHT_6_HAIRPIN_TH=35.36
PRIMER_LEFT_6_END_STABILITY=2.1700
PRIMER_RIGHT_6_END_STABILITY=4.2400
PRIMER_LEFT_7_PENALTY=0.035056
PRIMER_RIGHT_7_PENALTY=1.777878
PRIMER_LEFT_7_SEQUENCE=GATGTCAGGGGTCTTGTGGG
PRIMER_RIGHT_7_SEQUENCE=tggccttccttctttaccaca
PRIMER_LEFT_7=3767,20
PRIMER_RIGHT_7=4188,21
PRIMER_LEFT_7_TM=60.035
PRIMER_RIGHT_7_TM=59.222
PRIMER_LEFT_7_GC_PERCENT=60.000
PRIMER_RIGHT_7_GC_PERCENT=47.619
PRIMER_LEFT_7_SELF_ANY_TH=0.00
PRIMER_RIGHT_7_SELF_ANY_TH=3.32
PRIMER_LEFT_7_SELF_END_TH=0.00
PRIMER_RIGHT_7_SELF_END_TH=0.00
PRIMER_LEFT_7_HAIRPIN_TH=0.00
PRIMER_RIGHT_7_HAIRPIN_TH=0.00
PRIMER_LEFT_7_END_STABILITY=4.6100
PRIMER_RIGHT_7_END_STABILITY=4.1700
=

Looks like we have one primer pair where both are outside of the C1q gene sequence.

5.2 Amplicon length

The primer pair will produced an amplicon length of 3911bp.

Next, we need to test them against the entire genome to assess specificity.

6 SPLIT GENOME

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 | wc -l
echo "-------------------------------------------------------------------"

-------------------------------------------------------------------
NUMBER OF SEQUENCES IN ORIGINAL FASTA
4121
-------------------------------------------------------------------


-------------------------------------------------------------------
NUMBER OF INDIVIDUAL FASTA FILES
4122
-------------------------------------------------------------------

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

# Get primer info from Primer3 default format output
seq_id=$(grep "SEQUENCE_ID=" "${output_top}/primer3-primers-default-format.txt" | sed 's/SEQUENCE_ID=//')

left_primer=$(grep "PRIMER_LEFT_0_SEQUENCE=" "${output_top}/primer3-primers-default-format.txt" | sed 's/PRIMER_LEFT_0_SEQUENCE=//' | tr '[:lower:]' '[:upper:]')

right_primer=$(grep "PRIMER_RIGHT_7_SEQUENCE=" "${output_top}/primer3-primers-default-format.txt" | sed 's/PRIMER_RIGHT_7_SEQUENCE=//' | tr '[:lower:]' '[:upper:]')

# Create EMBOSS primer file
printf "%s\t%s\t%s\t\n" "${seq_id}" "${left_primer}" "${right_primer}" | tee "${output_top}/emboss-primers.txt"

LOC120027825    ACGACTAACCCTGAGACACA    TGGCCTTCCTTCTTTACCACA

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    5m28.518s
user    4m14.155s
sys 1m45.833s

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_052339.primersearch


Primer name LOC120027825
Amplimer 1
    Sequence: NC_052339.1  
    
    ACGACTAACCCTGAGACACA hits forward strand at 19244333 with 0 mismatches
    TGGCCTTCCTTCTTTACCACA hits reverse strand at [18091675] with 0 mismatches
    Amplimer length: 3912 bp
----------------------------------

8 SUMMARY

EMBOSS PrimerSearch has identified only a single location in the genome where the forward and reverse primers match, as well as in the expected location on chromosome NC_052339. This means our primers are specific to our desired target in the genome.

9 CITATIONS

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.