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Instructions

This file describes the commands used to obtain the Targets, dDNAs, and Primers used in the manuscript.

In order to proceed with the strain engineering portion of the manuscript in a reasonable time frame, we used an incomplete version of AddTag at the time. We used AddTag r284 as the base version, which may have had uncommitted changes which could potentially alter the expected output. Please note that the commands listed here are for an outdated version of AddTag.

We recommend you use the latest version of AddTag that has the full complement of software features. The README.md file contains these same workflows but updated for the current AddTag version. Recent versions are less error-prone, are much more streamlined, and do not strictly require viewing of the log.txt file.

Setup AddTag

Download and install AddTag and its dependencies. For instructions, please refer to the README.md file located at the root of the Git repository.

Get genome data

Download the Candida albicans reference genome and annotations used for this study.

wget http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly22/archive/C_albicans_SC5314_version_A22-s07-m01-r19_chromosomes.fasta.gz
gunzip C_albicans_SC5314_version_A22-s07-m01-r19_chromosomes.fasta.gz
wget http://www.candidagenome.org/download/gff/C_albicans_SC5314/archive/C_albicans_SC5314_version_A22-s07-m01-r19_features.gff

Set convenience variables for referencing these two files.

GENOME_FASTA=C_albicans_SC5314_version_A22-s07-m01-r19_chromosomes.fasta
GENOME_GFF=C_albicans_SC5314_version_A22-s07-m01-r19_features.gff

Create the *.homologs file for the C. albicans genome.

python3 gff2homologs.py ${GENOME_FEATURES} > C_albicans_SC5314_A22_current_homologs.txt

Obtain the general *.homologs file included with the package, or create one with only the genes you are interested in manipulating.

cat << EOF > C_albicans_SC5314_A22_current_homologs.txt
ADE2	C3_04520C_A	C3_04520C_B
BRG1	C1_05140W_A	C1_05140W_B
CSR1	C4_04850C_A	C4_04850C_B
EFG1	CR_07890W_A	CR_07890W_B
ROB1	C1_13620W_A	C1_13620W_B
ZRT2	C2_02590W_A	C2_02590W_B
EOF

ADE2_CDS

For simplicity, we use a variable to hold the label for this computational experiment.

GENE=ADE2

Create and enter the directory for this experiment.

mkdir ${GENE}_CDS
cd ${GENE}_CDS

We create additional environmental variables for simplicity

DDNA_LEN=100
MIN_INS=0
MAX_INS=4
MIN_HOM=$(((DDNA_LEN-MAX_INS)/2)) # 48
MAX_HOM=$((DDNA_LEN/2)) # 50

Extract the annotations for ADE2 and put them in their own *.gff file.

addtag feature --gff ../${GENOME_FEATURES} --header --query ${GENE} > ${GENE}.gff

Identify the optimal Target sites and generate potential dDNAs.

addtag generate \
  --fasta ../${GENOME_FASTA} \
  --gff ${GENE}.gff \
  --homologs ../C_albicans_SC5314_A22_current_homologs.txt \
  --ko-gRNA \
  --ko-dDNA mintag \
  --ki-gRNA \
  --ki-dDNA \
  --features gene \
  --tag ID \
  --motifs 'N{17}|N{3}>NGG' \
  --off_target_motifs 'N{17}|N{3}>NAG' \
  --excise_upstream_homology ${MIN_HOM} ${MAX_HOM} \
  --excise_downstream_homology ${MIN_HOM} ${MAX_HOM} \
  --excise_donor_lengths ${DDNA_LEN} ${DDNA_LEN} \
  --excise_insert_lengths ${MIN_INS} ${MAX_INS} \
  --revert_amplification_primers \
  --max_homology_errors 0 \
  --revert_homology_length 100 200 \
  --primer_scan_limit 120 \
  --primer_pair_limit 120 \
  --folder ${GENE}g > ${GENE}g.out 2> ${GENE}g.err

We select the best +Target with no predicted off-targets (Hsu-Zhang = 100.0, and CFD = 100.0). This script can fail if no Targets meet this criteria.

TARGET=$(echo -e "import sys\nwith open(sys.argv[1], 'r') as flo:\n  for line in flo:\n    if not line.startswith('#'):\n      sline = line.split('\t')\n      if ((len(sline[1].split(',')) == 2) and sline[3].startswith('exTarget-') and (sline[5] == '100.0') and (sline[6] == '100.0')):\n        print(sline[3])\n        break\n" | python3 - ${GENE}g.out)
addtag extract \
  --fasta ${GENE}g/excision-spacers.fasta \
  --query "${TARGET}\b" > ${GENE}-ko-Target.fasta

Select the top-ranked ΔTarget and its associated ΔdDNA. When we ran it, exDonor-55 and reTarget-0 were optimal.

DONOR=exDonor-55
addtag extract --fasta ${GENE}g/excision-dDNAs.fasta --query "${DONOR}\b" > ${GENE}-ko-dDNA.fasta
TARGET=reTarget-0
addtag extract --fasta ${GENE}g/reversion-spacers.fasta --query "${TARGET}\b" > ${GENE}-ki-Target.fasta

Manually scan the output for the top-ranked AmpF/AmpR primers, and extract their associated AdDNA. We found reDonor-0.

reDonor-0       C3_04520C_A     0.005368097146465493    PrimerPair(names=('AmpF', 'AmpR'), seqs=('CTAAGAAGGGAAAAGCACCAC', 'CTCGGTACAATCTTGTCAATGAG'), amplicon_sizes=[('ADE2', 'C3_04520C_A', 0, 'Ca22chr3A_C_albicans_SC5314', 1980), ('ADE2', 'C3_04520C_B', 0, 'Ca22chr3B_C_albicans_SC5314', 1980)], Tms=(52.77, 53.05), GCs=(0.48, 0.43), min(dG)=-3.72)

DONOR=reDonor-0
addtag extract --fasta ${GENE}g/reversion-dDNAs.fasta --query "${DONOR}\b" > ${GENE}-ki-dDNA.fasta

Calculate a decent Primer Design for validating each genome engineering step. We killed the process after getting through the first cycle.

addtag confirm \
  --fasta ../${GENOME_FASTA} \
  --dDNAs ${GENE}-ko-dDNA.fasta ${GENE}-ki-dDNA.fasta \
  --primer_scan_limit 600 \
  --primer_pair_limit 300 \
  --i_primers_required y n y \
  --o_primers_required y n y \
  --specificity all \
  --max_number_designs_reported 1000 \ 
  --folder ${GENE}c > ${GENE}c.out 2> ${GENE}c.err \

After the first cycle, a usable set of primers is identified. Manually select the PrimerSet with the highest weight. You can extract the primer sets with the following command.

grep -n 'cycle: 1' ${GENE}c/log.txt | cut -f 1 -d ':' | xargs -I {} head -n {} ${GENE}c/log.txt | grep optimal > optimal.txt

Then sort them by weight.

optimal = PrimerSet(weight=9.515753054508352e-05, [PrimerPair(names=('', ''), seqs=('GTGGTGGATTGGTATTTCTTTCTGTG', 'AAGACCCCAAACATTTTGACTCG'), amplicon_sizes=[('exDonor-55,reDonor-0', 1, 2, 'Ca22chr3B_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 2046), ('exDonor-55,reDonor-0', 0, 0, 'Ca22chr3A_C_albicans_SC5314', 2046), ('exDonor-55,reDonor-0', 1, 1, 'Ca22chr3B_C_albicans_SC5314-r1[exDonor-55]', 341), ('exDonor-55,reDonor-0', 1, 0, 'Ca22chr3B_C_albicans_SC5314', 2046), ('exDonor-55,reDonor-0', 0, 1, 'Ca22chr3A_C_albicans_SC5314-r1[exDonor-55]', 341), ('exDonor-55,reDonor-0', 0, 2, 'Ca22chr3A_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 2046)], Tms=(55.99, 55.4), GCs=(0.42, 0.43), min(dG)=-3.34), PrimerPair(names=('', ''), seqs=('GTGGTGGATTGGTATTTCTTTCTGTG', 'CATTGCCTGTCATTGGTGTTCC'), amplicon_sizes=[('exDonor-55,reDonor-0', 1, 2, 'Ca22chr3B_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 474), ('exDonor-55,reDonor-0', 0, 0, 'Ca22chr3A_C_albicans_SC5314', 474), ('exDonor-55,reDonor-0', 1, 0, 'Ca22chr3B_C_albicans_SC5314', 474), ('exDonor-55,reDonor-0', 0, 2, 'Ca22chr3A_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 474)], Tms=(55.99, 56.26), GCs=(0.42, 0.5), min(dG)=-2.53), PrimerPair(names=('', ''), seqs=('CCCCAATGTGTAACAAGTCATCG', 'AAGACCCCAAACATTTTGACTCG'), amplicon_sizes=[('exDonor-55,reDonor-0', 1, 2, 'Ca22chr3B_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 567), ('exDonor-55,reDonor-0', 0, 0, 'Ca22chr3A_C_albicans_SC5314', 567), ('exDonor-55,reDonor-0', 1, 0, 'Ca22chr3B_C_albicans_SC5314', 567), ('exDonor-55,reDonor-0', 0, 2, 'Ca22chr3A_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 567)], Tms=(55.68, 55.4), GCs=(0.48, 0.43), min(dG)=-3.58), PrimerPair(names=('', ''), seqs=('CAGAGTTGTGAGGTCTTGGTG', 'GGCGTATGATGGTAGAGGTAAC'), amplicon_sizes=[('exDonor-55,reDonor-0', 1, 0, 'Ca22chr3B_C_albicans_SC5314', 377), ('exDonor-55,reDonor-0', 0, 2, 'Ca22chr3A_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 377), ('exDonor-55,reDonor-0', 1, 2, 'Ca22chr3B_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 377), ('exDonor-55,reDonor-0', 0, 0, 'Ca22chr3A_C_albicans_SC5314', 377)], Tms=(54.42, 54.05), GCs=(0.52, 0.5), min(dG)=-2.0), None, None, None, PrimerPair(names=('', ''), seqs=('GTGGTGGATTGGTATTTCTTTCTGTG', 'CATTGCCTGTCATTGGTGTTCC'), amplicon_sizes=[('exDonor-55,reDonor-0', 1, 2, 'Ca22chr3B_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 474), ('exDonor-55,reDonor-0', 0, 0, 'Ca22chr3A_C_albicans_SC5314', 474), ('exDonor-55,reDonor-0', 1, 0, 'Ca22chr3B_C_albicans_SC5314', 474), ('exDonor-55,reDonor-0', 0, 2, 'Ca22chr3A_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 474)], Tms=(55.99, 56.26), GCs=(0.42, 0.5), min(dG)=-2.53), PrimerPair(names=('', ''), seqs=('CCCCAATGTGTAACAAGTCATCG', 'AAGACCCCAAACATTTTGACTCG'), amplicon_sizes=[('exDonor-55,reDonor-0', 1, 2, 'Ca22chr3B_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 567), ('exDonor-55,reDonor-0', 0, 0, 'Ca22chr3A_C_albicans_SC5314', 567), ('exDonor-55,reDonor-0', 1, 0, 'Ca22chr3B_C_albicans_SC5314', 567), ('exDonor-55,reDonor-0', 0, 2, 'Ca22chr3A_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 567)], Tms=(55.68, 55.4), GCs=(0.48, 0.43), min(dG)=-3.58), PrimerPair(names=('', ''), seqs=('CAGAGTTGTGAGGTCTTGGTG', 'GGCGTATGATGGTAGAGGTAAC'), amplicon_sizes=[('exDonor-55,reDonor-0', 1, 0, 'Ca22chr3B_C_albicans_SC5314', 377), ('exDonor-55,reDonor-0', 0, 2, 'Ca22chr3A_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 377), ('exDonor-55,reDonor-0', 1, 2, 'Ca22chr3B_C_albicans_SC5314-r1[exDonor-55]-r2[reDonor-0]', 377), ('exDonor-55,reDonor-0', 0, 0, 'Ca22chr3A_C_albicans_SC5314', 377)], Tms=(54.42, 54.05), GCs=(0.52, 0.5), min(dG)=-2.0)])

Finally change back to the parent folder

cd ..

EFG1 CDS

GENE=EFG1
cd ${GENE}_CDS

DDNA_LEN=100
MIN_INS=0
MAX_INS=4
MIN_HOM=$(((DDNA_LEN-MAX_INS)/2)) # 48
MAX_HOM=$((DDNA_LEN/2)) # 50
addtag feature --gff ../${GENOME_FEATURES} --header --query CR_07890W --linked_tags ID > EFG1.gff
addtag generate \
  --fasta ../${GENOME_FASTA} \
  --gff ${GENE}.gff \
  --homologs ../C_albicans_SC5314_A22_current_homologs.txt \
  --ko-gRNA \
  --ko-dDNA mintag \
  --ki-gRNA \
  --ki-dDNA \
  --features gene \
  --tag ID \
  --motifs 'N{17}|N{3}>NGG' \
  --off_target_motifs 'N{17}|N{3}>NAG' \
  --excise_upstream_homology ${MIN_HOM} ${MAX_HOM} \
  --excise_downstream_homology ${MIN_HOM} ${MAX_HOM} \
  --excise_donor_lengths ${DDNA_LEN} ${DDNA_LEN} \
  --excise_insert_lengths ${MIN_INS} ${MAX_INS} \
  --revert_amplification_primers \
  --excise_upstream_feature_trim 0 3 \
  --excise_downstream_feature_trim 7 10 \
  --max_homology_errors 0 \
  --revert_homology_length 100 200 \
  --primer_scan_limit 120 \
  --primer_pair_limit 120 \
  --folder ${GENE}g > ${GENE}g.out 2> ${GENE}g.err
addtag extract --fasta ${GENE}g/excision-spacers.fasta --query 'exTarget-97\b' > ${GENE}-ko-spacer.fasta
addtag extract --fasta ${GENE}g/reversion-spacers.fasta --query 'reTarget-118\b' > ${GENE}-ki-spacer.fasta
addtag extract --fasta ${GENE}g/excision-dDNAs.fasta --query 'exDonor-7183\b' > ${GENE}-ko-dDNA.fasta
addtag extract --fasta ${GENE}g/reversion-dDNAs.fasta --query 'reDonor-0\b' > ${GENE}-ki-dDNA.fasta

AmpF/AmpR

reDonor-0       CR_07890W_A     0.002814545952972088    PrimerPair(names=('AmpF', 'AmpR'), seqs=('CACATTAGTTGCTCAGGTCAC', 'GTCAATGGATTTGGGAGAAGA'), amplicon_sizes=[('EFG1', 'CR_07890W_A', 0, 'Ca22chrRA_C_albicans_SC5314', 1972), ('EFG1', 'CR_07890W_B', 0, 'Ca22chrRB_C_albicans_SC5314', 1985)], Tms=(52.89, 52.04), GCs=(0.48, 0.43), min(dG)=-3.46)
reDonor-349     CR_07890W_B     0.002814545952972088    PrimerPair(names=('AmpF', 'AmpR'), seqs=('CACATTAGTTGCTCAGGTCAC', 'GTCAATGGATTTGGGAGAAGA'), amplicon_sizes=[('EFG1', 'CR_07890W_A', 0, 'Ca22chrRA_C_albicans_SC5314', 1972), ('EFG1', 'CR_07890W_B', 0, 'Ca22chrRB_C_albicans_SC5314', 1985)], Tms=(52.89, 52.04), GCs=(0.48, 0.43), min(dG)=-3.46)

addtag confirm \
--fasta ../${GENOME_FASTA} \
--dDNAs ${GENE}-ko-dDNA.fasta ${GENE}-ki-dDNA.fasta \
--primer_scan_limit 120 \
--primer_pair_limit 120 \
--i_primers_required y n y \
--o_primers_required y n y \
--specificity all \
--max_number_designs_reported 1000 \
--cycle_start 10 \
--folder ${GENE}c > ${GENE}c.out 2> ${GENE}c.err
grep -n 'cycle: 11' ${GENE}c/log.txt | cut -f 1 -d ':' | xargs -I {} head -n {} ${GENE}c/log.txt | grep optimal > optimal.txt

cPCR primer set identified

optimal = PrimerSet(weight=2.8889235120047e-11, [PrimerPair(names=('', ''), seqs=('TTAACCCCTTTGTGTCCCTT',    'CCCAAATAGTATAAATTCGTTCATGTC'), amplicon_sizes=[('exDonor-7183,reDonor-0', 1, 1, 'Ca22chrRB_C_albicans_SC5314-r1[exDonor-7183]', 575), ('exDonor-7183,reDonor-0', 1, 2, 'Ca22chrRB_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 2236), ('exDonor-7183,reDonor-0', 0, 2, 'Ca22chrRA_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 2236), ('exDonor-7183,reDonor-0', 0, 0, 'Ca22chrRA_C_albicans_SC5314', 2236), ('exDonor-7183,reDonor-0', 0, 1, 'Ca22chrRA_C_albicans_SC5314-r1[exDonor-7183]', 574), ('exDonor-7183,reDonor-0', 1, 0, 'Ca22chrRB_C_albicans_SC5314', 2249)], Tms=(52.59, 52.55), GCs=(0.45, 0.33), min(dG)=-3.06), PrimerPair(names=('', ''), seqs=('TTAACCCCTTTGTGTCCCTT',   'GCTGTTGTTGTTGTTGTCCT'), amplicon_sizes=[('exDonor-7183,reDonor-0', 1, 2, 'Ca22chrRB_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 657), ('exDonor-7183,reDonor-0', 0, 2, 'Ca22chrRA_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 657), ('exDonor-7183,reDonor-0', 0, 0, 'Ca22chrRA_C_albicans_SC5314', 657), ('exDonor-7183,reDonor-0', 1, 0, 'Ca22chrRB_C_albicans_SC5314', 658)], Tms=(52.59, 52.48), GCs=(0.45, 0.45), min(dG)=-2.3), PrimerPair(names=('', ''), seqs=('ACCAATCACCCCAAGTTCAG',     'CCCAAATAGTATAAATTCGTTCATGTC'), amplicon_sizes=[('exDonor-7183,reDonor-0', 0, 0, 'Ca22chrRA_C_albicans_SC5314', 301), ('exDonor-7183,reDonor-0', 1, 0, 'Ca22chrRB_C_albicans_SC5314', 301), ('exDonor-7183,reDonor-0', 1, 2, 'Ca22chrRB_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 301), ('exDonor-7183,reDonor-0', 0, 2, 'Ca22chrRA_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 301)], Tms=(53.12, 52.55), GCs=(0.5, 0.33), min(dG)=-3.06), PrimerPair(names=('', ''), seqs=('CCCCCATACCTTCCAATTCTAC',   'GACACATTACTGCCACCACTG'), amplicon_sizes=[('exDonor-7183,reDonor-0', 0, 0, 'Ca22chrRA_C_albicans_SC5314', 682), ('exDonor-7183,reDonor-0', 1, 0, 'Ca22chrRB_C_albicans_SC5314', 682), ('exDonor-7183,reDonor-0', 1, 2, 'Ca22chrRB_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 682), ('exDonor-7183,reDonor-0', 0, 2, 'Ca22chrRA_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 682)], Tms=(53.82, 55.19), GCs=(0.5, 0.52), min(dG)=-1.59), None, None, None, PrimerPair(names=('', ''), seqs=('TTAACCCCTTTGTGTCCCTT', 'GCTGTTGTTGTTGTTGTCCT'), amplicon_sizes=[('exDonor-7183,reDonor-0', 1, 2, 'Ca22chrRB_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 657), ('exDonor-7183,reDonor-0', 0, 2, 'Ca22chrRA_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 657), ('exDonor-7183,reDonor-0', 0, 0, 'Ca22chrRA_C_albicans_SC5314', 657), ('exDonor-7183,reDonor-0', 1, 0, 'Ca22chrRB_C_albicans_SC5314', 658)], Tms=(52.59, 52.48), GCs=(0.45, 0.45), min(dG)=-2.3), PrimerPair(names=('', ''), seqs=('ACCAATCACCCCAAGTTCAG', 'CCCAAATAGTATAAATTCGTTCATGTC'), amplicon_sizes=[('exDonor-7183,reDonor-0', 0, 0, 'Ca22chrRA_C_albicans_SC5314', 301), ('exDonor-7183,reDonor-0', 1, 0, 'Ca22chrRB_C_albicans_SC5314', 301), ('exDonor-7183,reDonor-0', 1, 2, 'Ca22chrRB_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 301), ('exDonor-7183,reDonor-0', 0, 2, 'Ca22chrRA_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 301)], Tms=(53.12, 52.55), GCs=(0.5, 0.33), min(dG)=-3.06), PrimerPair(names=('', ''), seqs=('CCCCCATACCTTCCAATTCTAC', 'GACACATTACTGCCACCACTG'), amplicon_sizes=[('exDonor-7183,reDonor-0', 0, 0, 'Ca22chrRA_C_albicans_SC5314', 682), ('exDonor-7183,reDonor-0', 1, 0, 'Ca22chrRB_C_albicans_SC5314', 682), ('exDonor-7183,reDonor-0', 1, 2, 'Ca22chrRB_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 682), ('exDonor-7183,reDonor-0', 0, 2, 'Ca22chrRA_C_albicans_SC5314-r1[exDonor-7183]-r2[reDonor-0]', 682)], Tms=(53.82, 55.19), GCs=(0.5, 0.52), min(dG)=-1.59)])

BRG1_CDS

GENE=BRG1
cd ${GENE}_CDS

DDNA_LEN=100
MIN_INS=0
MAX_INS=4
MIN_HOM=$(((DDNA_LEN-MAX_INS)/2)) # 48
MAX_HOM=$((DDNA_LEN/2)) # 50

Include only the BRG1 features.

addtag feature --gff ../${GENOME_FEATURES} --header --query ${GENE} > ${GENE}.gff

With this version of AddTag, there was a bug in the feature expansion, so it is corrected here.

awk '{$4-=42}1' FS='\t' OFS='\t' ${GENE}.gff > ${GENE}ex.gff

Run AddTag to find Targets, make dDNAs, and find AmpF/AmpR primers.

addtag generate \
  --fasta ../${GENOME_FASTA} \
  --gff ${GENE}ex.gff \
  --homologs ../C_albicans_SC5314_A22_current_homologs.txt \
  --ko-gRNA \
  --ko-dDNA mintag \
  --ki-gRNA \
  --ki-dDNA \
  --features gene \
  --tag ID \
  --motifs 'N{17}|N{3}>NGG' \
  --off_target_motifs 'N{17}|N{3}>NAG' \
  --excise_upstream_homology ${MIN_HOM} ${MAX_HOM} \
  --excise_downstream_homology ${MIN_HOM} ${MAX_HOM} \
  --excise_donor_lengths ${DDNA_LEN} ${DDNA_LEN} \
  --excise_insert_lengths ${MIN_INS} ${MAX_INS} \
  --revert_amplification_primers \
  --max_homology_errors 0 \
  --revert_homology_length 100 200 \
  --primer_scan_limit 120 \
  --primer_pair_limit 120 \
  --folder ${GENE}g > ${GENE}g.out 2> ${GENE}g.err
TARGET=$(echo -e "import sys\nwith open(sys.argv[1], 'r') as flo:\n  for line in flo:\n    if not line.startswith('#'):\n      sline = line.split('\t')\n      if ((len(sline[1].split(',')) == 2) and sline[3].startswith('exTarget-') and (sline[5] == '100.0') and (sline[6] == '100.0')):\n        print(sline[3])\n        break\n" | python3 - ${GENE}g.out)
addtag extract \
  --fasta ${GENE}g/excision-spacers.fasta \
  --query "${TARGET}\b" > ${GENE}-ko-Target.fasta
addtag extract --fasta ${GENE}g/excision-dDNAs.fasta --query 'exDonor-440\b' > ${GENE}-ko-dDNA.fasta
addtag extract --fasta ${GENE}g/reversion-spacers.fasta --query 'reTarget-628\b' > ${GENE}-ki-spacer.fasta
addtag extract --fasta ${GENE}g/reversion-dDNAs.fasta --query 'reDonor-80\b' > ${GENE}-ki-dDNA.fasta

A single optimal primer pair was predicted to create one amplicon for each homologous chromosome.

reDonor-0       C1_05140W_B     4.45941697919701e-05    PrimerPair(names=('AmpF', 'AmpR'), seqs=('TATAAATATCAGGTCATAGATCCCTG', 'GTTCAAACAACAATACTGTAGCAG'), amplicon_sizes=[('BRG1', 'C1_05140W_B', 0, 'Ca22chr1B_C_albicans_SC5314', 1608), ('BRG1', 'C1_05140W_A', 0, 'Ca22chr1A_C_albicans_SC5314', 1605)], Tms=(52.03, 52.39), GCs=(0.35, 0.38), min(dG)=-3.44)
reDonor-80      C1_05140W_A     4.45941697919701e-05    PrimerPair(names=('AmpF', 'AmpR'), seqs=('TATAAATATCAGGTCATAGATCCCTG', 'GTTCAAACAACAATACTGTAGCAG'), amplicon_sizes=[('BRG1', 'C1_05140W_B', 0, 'Ca22chr1B_C_albicans_SC5314', 1608), ('BRG1', 'C1_05140W_A', 0, 'Ca22chr1A_C_albicans_SC5314', 1605)], Tms=(52.03, 52.39), GCs=(0.35, 0.38), min(dG)=-3.44)

Identify the set of primers to use for cPCR validation

addtag confirm \
  --fasta ../${GENOME_FASTA} \
  --dDNAs ${GENE}-ko-dDNA.fasta ${GENE}-ki-dDNA.fasta \
  --primer_scan_limit 3600 \
  --primer_pair_limit 600 \
  --i_primers_required y n y \
  --o_primers_required y n y \
  --specificity all \
  --max_number_designs_reported 1000 \
  --cycle_start 6 
  --folder ${GENE}c > ${GENE}c.out 2> ${GENE}c.err

After 6 cycles, a usable set of primers was identified.

grep -n 'cycle: 7' ${GENE}c/log.txt | cut -f 1 -d ':' | xargs -I {} head -n {} ${GENE}c/log.txt | grep -c optimal > optimal.txt

optimal = PrimerSet(weight=6.792474801162299e-10, [PrimerPair(names=('', ''), seqs=('TGCAGCTTTTGTACTACATTTGG', 'CCAGCTCAGGATATAATTTACAGC'), amplicon_sizes=[('exDonor-440,reDonor-80', 0, 1, 'Ca22chr1B_C_albicans_SC5314-r1[exDonor-440]', 636), ('exDonor-440,reDonor-80', 1, 0, 'Ca22chr1A_C_albicans_SC5314', 1950), ('exDonor-440,reDonor-80', 1, 2, 'Ca22chr1A_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 1950), ('exDonor-440,reDonor-80', 0, 2, 'Ca22chr1B_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 1947), ('exDonor-440,reDonor-80', 0, 0, 'Ca22chr1B_C_albicans_SC5314', 1950), ('exDonor-440,reDonor-80', 1, 1, 'Ca22chr1A_C_albicans_SC5314-r1[exDonor-440]', 639)], Tms=(53.24, 52.99), GCs=(0.39, 0.42), min(dG)=-3.26), PrimerPair(names=('', ''), seqs=('TGCAGCTTTTGTACTACATTTGG', 'ACCTCCACTAATGGTTGATCG'), amplicon_sizes=[('exDonor-440,reDonor-80', 1, 0, 'Ca22chr1A_C_albicans_SC5314', 521), ('exDonor-440,reDonor-80', 1, 2, 'Ca22chr1A_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 521), ('exDonor-440,reDonor-80', 0, 2, 'Ca22chr1B_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 518), ('exDonor-440,reDonor-80', 0, 0, 'Ca22chr1B_C_albicans_SC5314', 521)], Tms=(53.24, 52.84), GCs=(0.39, 0.48), min(dG)=-3.3), PrimerPair(names=('', ''), seqs=('GTCATTCATCAACCACCACCA', 'CCAGCTCAGGATATAATTTACAGC'), amplicon_sizes=[('exDonor-440,reDonor-80', 1, 0, 'Ca22chr1A_C_albicans_SC5314', 337), ('exDonor-440,reDonor-80', 1, 2, 'Ca22chr1A_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 337), ('exDonor-440,reDonor-80', 0, 2, 'Ca22chr1B_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 337), ('exDonor-440,reDonor-80', 0, 0, 'Ca22chr1B_C_albicans_SC5314', 337)], Tms=(54.01, 52.99), GCs=(0.48, 0.42), min(dG)=-3.05), PrimerPair(names=('', ''), seqs=('CCACCACAACAACCACAATCAG', 'CGACCGTTCTTCCCTTTTGTC'), amplicon_sizes=[('exDonor-440,reDonor-80', 1, 2, 'Ca22chr1A_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 569), ('exDonor-440,reDonor-80', 1, 0, 'Ca22chr1A_C_albicans_SC5314', 569), ('exDonor-440,reDonor-80', 0, 2, 'Ca22chr1B_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 569), ('exDonor-440,reDonor-80', 0, 0, 'Ca22chr1B_C_albicans_SC5314', 569)], Tms=(56.07, 55.5), GCs=(0.5, 0.52), min(dG)=-1.87), None, None, None, PrimerPair(names=('', ''), seqs=('TGCAGCTTTTGTACTACATTTGG', 'ACCTCCACTAATGGTTGATCG'), amplicon_sizes=[('exDonor-440,reDonor-80', 1, 0, 'Ca22chr1A_C_albicans_SC5314', 521), ('exDonor-440,reDonor-80', 1, 2, 'Ca22chr1A_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 521), ('exDonor-440,reDonor-80', 0, 2, 'Ca22chr1B_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 518), ('exDonor-440,reDonor-80', 0, 0, 'Ca22chr1B_C_albicans_SC5314', 521)], Tms=(53.24, 52.84), GCs=(0.39, 0.48), min(dG)=-3.3), PrimerPair(names=('', ''), seqs=('GTCATTCATCAACCACCACCA', 'CCAGCTCAGGATATAATTTACAGC'), amplicon_sizes=[('exDonor-440,reDonor-80', 1, 0, 'Ca22chr1A_C_albicans_SC5314', 337), ('exDonor-440,reDonor-80', 1, 2, 'Ca22chr1A_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 337), ('exDonor-440,reDonor-80', 0, 2, 'Ca22chr1B_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 337), ('exDonor-440,reDonor-80', 0, 0, 'Ca22chr1B_C_albicans_SC5314', 337)], Tms=(54.01, 52.99), GCs=(0.48, 0.42), min(dG)=-3.05), PrimerPair(names=('', ''), seqs=('CCACCACAACAACCACAATCAG', 'CGACCGTTCTTCCCTTTTGTC'), amplicon_sizes=[('exDonor-440,reDonor-80', 1, 2, 'Ca22chr1A_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 569), ('exDonor-440,reDonor-80', 1, 0, 'Ca22chr1A_C_albicans_SC5314', 569), ('exDonor-440,reDonor-80', 0, 2, 'Ca22chr1B_C_albicans_SC5314-r1[exDonor-440]-r2[reDonor-80]', 569), ('exDonor-440,reDonor-80', 0, 0, 'Ca22chr1B_C_albicans_SC5314', 569)], Tms=(56.07, 55.5), GCs=(0.5, 0.52), min(dG)=-1.87)])

Change back to the parent folder

cd ..

CSR1_US (ZAP1_US)

Obtain or create the *.gff annotation for the CSR1 self-regulator.

cat << EOF > ZRT2_US.gff
# seqid	source	feature	start	end	score	strand	frame	attribute
Ca22chr4A_C_albicans_SC5314	Seher	protein_bind	1044578	1044588	.	-	.	ID=CSR1_US_A
Ca22chr4B_C_albicans_SC5314	Seher	protein_bind	1044606	1044616	.	-	.	ID=CSR1_US_B
EOF

Obtain or create the *.homologs file for this new annotation.

cat << EOF > CSR1_US_homologs.txt
CSR1_US	CSR1_US_A	CSR1_US_B
EOF

Mask the Csr1 transcription factor binding site by changing the characters to lower case, and save as C_albicans_SC5314_A22_current_chromosomes_CSR1_US.fasta

Make a FASTA file with the modified binding site

cat << EOF > CSR1_US_MOD.fasta
>CSR1_US
CAGCTGTTCTA
EOF

Make a FASTA file with the addtag sequence

cat << EOF > CSR1_US_UNITAG.fasta
>CSR1_US
CGTACGCTGCAGGTCGACAGTGG
EOF

The generated ΔdDNA are longer to accomodate the 23 nt addtag sequence.

GENE=CSR1_US
cd ${GENE}

DDNA_LEN=130
addtag generate \
  --fasta ../C_albicans_SC5314_A22_current_chromosomes_CSR1_US.fasta \
  --gff ${GENE}.gff ../${GENOME_FEATURES} \
  --homologs ${GENE}_homologs.txt \
  --ko-gRNA \
  --ko-dDNA ${GENE}_UNITAG.fasta \
  --ki-gRNA \
  --ki-dDNA ${GENE}_MOD.fasta \
  --features protein_bind \
  --excluded_features gene \
  --tag ID \
  --motifs 'N{17}|N{3}>NGG' \
  --off_target_motifs 'N{17}|N{3}>NAG' \
  --excise_donor_lengths ${DDNA_LEN} ${DDNA_LEN} \
  --feature_expansion_method center_both \
  --feature_expansion_pad 200 \
  --feature_expansion_lengths 300 500 \
  --revert_amplification_primers \
  --max_homology_errors 0 \
  --revert_homology_length 100 200 \
  --primer_scan_limit 120 \
  --primer_pair_limit 120 \
  --folder ${GENE}g > ${GENE}g.out 2> ${GENE}g.err
reDonor-0       CSR1_US_B_derived-0     0.03497651705045208     PrimerPair(names=('AmpF', 'AmpR'), seqs=('GTGAACCACTCATCATCATTGG', 'ACGACTAATGCTATGACTGCTC'), amplicon_sizes=[('CSR1_US', 'CSR1_US_A', 0, 'Ca22chr4A_C_albicans_SC5314', 641), ('CSR1_US', 'CSR1_US_B', 0, 'Ca22chr4B_C_albicans_SC5314', 642)], Tms=(53.25, 53.32), GCs=(0.45, 0.45), min(dG)=-3.47)
reDonor-809     CSR1_US_A_derived-0     0.03497651705045208     PrimerPair(names=('AmpF', 'AmpR'), seqs=('GTGAACCACTCATCATCATTGG', 'ACGACTAATGCTATGACTGCTC'), amplicon_sizes=[('CSR1_US', 'CSR1_US_A', 0, 'Ca22chr4A_C_albicans_SC5314', 641), ('CSR1_US', 'CSR1_US_B', 0, 'Ca22chr4B_C_albicans_SC5314', 642)], Tms=(53.25, 53.32), GCs=(0.45, 0.45), min(dG)=-3.47)

addtag extract --fasta ${GENE}g/reversion-spacers.fasta --query 'reTarget-0\b' > ${GENE}-ki-spacer.fasta
addtag extract --fasta ${GENE}g/excision-spacers.fasta --query 'exTarget-5\b' > ${GENE}-ko-spacer.fasta
addtag extract --fasta ${GENE}g/reversion-dDNAs.fasta --query 'reDonor-809\b' > ${GENE}-ki-dDNA.fasta
addtag extract --fasta ${GENE}g/excision-dDNAs.fasta --query 'exDonor-0\b' > ${GENE}-ko-dDNA.fasta
addtag confirm \
  --fasta ../${GENOME_FASTA} \
  --dDNAs ${GENE}-ko-dDNA.fasta ${GENE}-ki-dDNA.fasta \
  --primer_scan_limit 120 \
  --primer_pair_limit 120 \
  --i_primers_required y n y \
  --o_primers_required y y y \
  --specificity all \
  --max_number_designs_reported 1000 \
  --cycle_start 13 \
  --folder ${GENE}c > ${GENE}c.out 2> ${GENE}c.err

The {$GENE}c/log.txt contains the optimal primer set.

optimal = PrimerSet(weight=1.1089802250429206e-12, [PrimerPair(names=('', ''), seqs=('CTGTGATCGTGATTATGAATGTGGC', 'ACGTTGTTCGTCTCAAGCTGG'), amplicon_sizes=[('exDonor-0,reDonor-809', 0, 2, 'Ca22chr4B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 978), ('exDonor-0,reDonor-809', 0, 1, 'Ca22chr4B_C_albicans_SC5314-r1[exDonor-0]', 578), ('exDonor-0,reDonor-809', 1, 2, 'Ca22chr4A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 977), ('exDonor-0,reDonor-809', 1, 0, 'Ca22chr4A_C_albicans_SC5314', 974), ('exDonor-0,reDonor-809', 0, 0, 'Ca22chr4B_C_albicans_SC5314', 977), ('exDonor-0,reDonor-809', 1, 1, 'Ca22chr4A_C_albicans_SC5314-r1[exDonor-0]', 577)], Tms=(56.19, 56.41), GCs=(0.44, 0.52), min(dG)=-3.44), PrimerPair(names=('', ''), seqs=('CTGTGATCGTGATTATGAATGTGGC', 'TACCCAGCATCATCATCATCG'), amplicon_sizes=[('exDonor-0,reDonor-809', 0, 2, 'Ca22chr4B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 442), ('exDonor-0,reDonor-809', 1, 2, 'Ca22chr4A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 442), ('exDonor-0,reDonor-809', 1, 0, 'Ca22chr4A_C_albicans_SC5314', 439), ('exDonor-0,reDonor-809', 0, 0, 'Ca22chr4B_C_albicans_SC5314', 442)], Tms=(56.19, 55.05), GCs=(0.44, 0.48), min(dG)=-2.94), PrimerPair(names=('', ''), seqs=('GTTGTCGATGATGATGATGCTGG', 'ACGTTGTTCGTCTCAAGCTGG'), amplicon_sizes=[('exDonor-0,reDonor-809', 1, 0, 'Ca22chr4A_C_albicans_SC5314', 561), ('exDonor-0,reDonor-809', 0, 0, 'Ca22chr4B_C_albicans_SC5314', 561), ('exDonor-0,reDonor-809', 0, 2, 'Ca22chr4B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 562), ('exDonor-0,reDonor-809', 1, 2, 'Ca22chr4A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 561)], Tms=(55.92, 56.41), GCs=(0.48, 0.52), min(dG)=-3.44), PrimerPair(names=('', ''), seqs=('CGATGATGATGATGCTGGGTA', 'GTGTTACTTGGTAGCACTTTGATC'), amplicon_sizes=[('exDonor-0,reDonor-809', 0, 0, 'Ca22chr4B_C_albicans_SC5314', 319), ('exDonor-0,reDonor-809', 0, 2, 'Ca22chr4B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 320), ('exDonor-0,reDonor-809', 1, 2, 'Ca22chr4A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 320), ('exDonor-0,reDonor-809', 1, 0, 'Ca22chr4A_C_albicans_SC5314', 320)], Tms=(55.05, 53.68), GCs=(0.48, 0.42), min(dG)=-4.24), PrimerPair(names=('', ''), seqs=('CTGTGATCGTGATTATGAATGTGGC', 'TGGTGTTACTTGGTAGCCCAC'), amplicon_sizes=[('exDonor-0,reDonor-809', 0, 1, 'Ca22chr4B_C_albicans_SC5314-r1[exDonor-0]', 343), ('exDonor-0,reDonor-809', 1, 1, 'Ca22chr4A_C_albicans_SC5314-r1[exDonor-0]', 343)], Tms=(56.19, 55.41), GCs=(0.44, 0.52), min(dG)=-3.82), PrimerPair(names=('', ''), seqs=('AGAAAGTGGCGTTTAATAAATACTACGTAC', 'ACGTTGTTCGTCTCAAGCTGG'), amplicon_sizes=[('exDonor-0,reDonor-809', 0, 1, 'Ca22chr4B_C_albicans_SC5314-r1[exDonor-0]', 300), ('exDonor-0,reDonor-809', 1, 1, 'Ca22chr4A_C_albicans_SC5314-r1[exDonor-0]', 299)], Tms=(55.13, 56.41), GCs=(0.33, 0.52), min(dG)=-4.58), None, PrimerPair(names=('', ''), seqs=('CTGTGATCGTGATTATGAATGTGGC', 'TACCCAGCATCATCATCATCG'), amplicon_sizes=[('exDonor-0,reDonor-809', 0, 2, 'Ca22chr4B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 442), ('exDonor-0,reDonor-809', 1, 2, 'Ca22chr4A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 442), ('exDonor-0,reDonor-809', 1, 0, 'Ca22chr4A_C_albicans_SC5314', 439), ('exDonor-0,reDonor-809', 0, 0, 'Ca22chr4B_C_albicans_SC5314', 442)], Tms=(56.19, 55.05), GCs=(0.44, 0.48), min(dG)=-2.94), PrimerPair(names=('', ''), seqs=('GTTGTCGATGATGATGATGCTGG', 'ACGTTGTTCGTCTCAAGCTGG'), amplicon_sizes=[('exDonor-0,reDonor-809', 1, 0, 'Ca22chr4A_C_albicans_SC5314', 561), ('exDonor-0,reDonor-809', 0, 0, 'Ca22chr4B_C_albicans_SC5314', 561), ('exDonor-0,reDonor-809', 0, 2, 'Ca22chr4B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 562), ('exDonor-0,reDonor-809', 1, 2, 'Ca22chr4A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 561)], Tms=(55.92, 56.41), GCs=(0.48, 0.52), min(dG)=-3.44), PrimerPair(names=('', ''), seqs=('CGATGATGATGATGCTGGGTA', 'GTGTTACTTGGTAGCACTTTGATC'), amplicon_sizes=[('exDonor-0,reDonor-809', 0, 0, 'Ca22chr4B_C_albicans_SC5314', 319), ('exDonor-0,reDonor-809', 0, 2, 'Ca22chr4B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 320), ('exDonor-0,reDonor-809', 1, 2, 'Ca22chr4A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-809]', 320), ('exDonor-0,reDonor-809', 1, 0, 'Ca22chr4A_C_albicans_SC5314', 320)], Tms=(55.05, 53.68), GCs=(0.48, 0.42), min(dG)=-4.24)])

ZRT2 US

Obtain or create the *.gff annotations for the region that includes both CSR1 binding sites, and everything in between.

cat << EOF > ZRT2_US.gff
# seqid	source	feature	start	end	score	strand	frame	attribute
Ca22chr2A_C_albicans_SC5314	Seher	protein_bind	523078	523744	.	+	.	ID=ZRT2_US_A
Ca22chr2B_C_albicans_SC5314	Seher	protein_bind	523078	523743	.	+	.	ID=ZRT2_US_B

Obtain or create the *.homologs file for this new annotation.

cat << EOF > ZRT2_US_homologs.txt
ZRT2_US	ZRT2_US_A	ZRT2_US_B
EOF

Make a FASTA file with the addtag sequence

cat << EOF > ZRT2_US_UNITAG.fasta
>ZRT2_US
CGTACGCTGCAGGTCGACAGTGG
EOF
GENE=ZRT2_US
cd ${GENE}

DDNA_LEN=130
addtag generate \
  --fasta ../${GENOME_FASTA} \
  --gff ${GENE}.gff ../${GENOME_FEATURES} \
  --homologs ${GENE}_homologs.txt \
  --ko-gRNA \
  --ko-dDNA ${GENE}_UNITAG.fasta \
  --ki-gRNA \
  --ki-dDNA ${GENE}_MOD-00-A.fasta \
  --features protein_bind \
  --excluded_features gene \
  --tag ID \
  --motifs 'N{17}|N{3}>NGG' \
  --off_target_motifs 'N{17}|N{3}>NAG' \
  --excise_donor_lengths ${DDNA_LEN} ${DDNA_LEN} \
  --revert_amplification_primers \
  --max_homology_errors 0 \
  --case upper-only \
  --revert_homology_length 100 200 \
  --primer_scan_limit 120 \
  --primer_pair_limit 120 \
  --folder ${GENE}g > ${GENE}g.out 2> ${GENE}g.err
reDonor-0	ZRT2_US_B	0.0021891124190610164	PrimerPair(names=('AmpF', 'AmpR'), seqs=('GCATATTTACTTGCTTGCCTG', 'TTGACAGGAATATGGAGGGTA'), amplicon_sizes=[('ZRT2_US', 'ZRT2_US_B', 0, 'Ca22chr2B_C_albicans_SC5314', 953), ('ZRT2_US', 'ZRT2_US_A', 0, 'Ca22chr2A_C_albicans_SC5314', 955)], Tms=(52.02, 52.71), GCs=(0.43, 0.43), min(dG)=-4.99)
reDonor-407	ZRT2_US_A	0.0021891124190610164	PrimerPair(names=('AmpF', 'AmpR'), seqs=('GCATATTTACTTGCTTGCCTG', 'TTGACAGGAATATGGAGGGTA'), amplicon_sizes=[('ZRT2_US', 'ZRT2_US_B', 0, 'Ca22chr2B_C_albicans_SC5314', 953), ('ZRT2_US', 'ZRT2_US_A', 0, 'Ca22chr2A_C_albicans_SC5314', 955)], Tms=(52.02, 52.71), GCs=(0.43, 0.43), min(dG)=-4.99)

addtag extract --fasta ${GENE}g/reversion-spacers.fasta --query 'reTarget-0\b' > ${GENE}-ki-spacer.fasta
addtag extract --fasta ${GENE}g/excision-spacers.fasta --query 'exTarget-11\b' > ${GENE}-ko-spacer.fasta
addtag extract --fasta ${GENE}g/reversion-dDNAs.fasta --query 'reDonor-407\b' > ${GENE}-ki-dDNA.fasta
addtag extract --fasta ${GENE}g/excision-dDNAs.fasta --query 'exDonor-0\b' > ${GENE}-ko-dDNA.fasta
addtag confirm \
  --fasta ../${GENOME_FASTA} \
  --dDNAs ${GENE}-ko-dDNA.fasta ${GENE}-ki-dDNA.fasta \
  --case upper-only \
  --primer_scan_limit 120 \
  --primer_pair_limit 120 \
  --i_primers_required y n y \
  --o_primers_required y n y \
  --specificity all \
  --max_number_designs_reported 1000 \
  --folder ${GENE}c > ${GENE}c.out 2> ${GENE}c.err

The {$GENE}c/log.txt contains the optimal primer set.

optimal = PrimerSet(weight=9.355670319407343e-06, [PrimerPair(names=('', ''), seqs=('GAACCAATCCTTCCACATAGC', 'GCTGGGAATTGATAATGAAAGC'), amplicon_sizes=[('exDonor-0,reDonor-407', 1, 0, 'Ca22chr2B_C_albicans_SC5314', 1059), ('exDonor-0,reDonor-407', 1, 2, 'Ca22chr2B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 1061), ('exDonor-0,reDonor-407', 0, 0, 'Ca22chr2A_C_albicans_SC5314', 1061), ('exDonor-0,reDonor-407', 0, 2, 'Ca22chr2A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 1061), ('exDonor-0,reDonor-407', 1, 1, 'Ca22chr2B_C_albicans_SC5314-r1[exDonor-0]', 416), ('exDonor-0,reDonor-407', 0, 1, 'Ca22chr2A_C_albicans_SC5314-r1[exDonor-0]', 417)], Tms=(52.7, 52.04), GCs=(0.48, 0.41), min(dG)=-3.45), PrimerPair(names=('', ''), seqs=('GAACCAATCCTTCCACATAGC', 'TTGCGTTTCGGGTATAATCAC'), amplicon_sizes=[('exDonor-0,reDonor-407', 1, 0, 'Ca22chr2B_C_albicans_SC5314', 654), ('exDonor-0,reDonor-407', 1, 2, 'Ca22chr2B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 655), ('exDonor-0,reDonor-407', 0, 0, 'Ca22chr2A_C_albicans_SC5314', 655), ('exDonor-0,reDonor-407', 0, 2, 'Ca22chr2A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 655)], Tms=(52.7, 52.81), GCs=(0.48, 0.43), min(dG)=-3.13), PrimerPair(names=('', ''), seqs=('TATTGGTCGGATTGGGTTAC', 'GCTGGGAATTGATAATGAAAGC'), amplicon_sizes=[('exDonor-0,reDonor-407', 0, 0, 'Ca22chr2A_C_albicans_SC5314', 369), ('exDonor-0,reDonor-407', 0, 2, 'Ca22chr2A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 369), ('exDonor-0,reDonor-407', 1, 0, 'Ca22chr2B_C_albicans_SC5314', 368), ('exDonor-0,reDonor-407', 1, 2, 'Ca22chr2B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 369)], Tms=(52.36, 52.04), GCs=(0.45, 0.41), min(dG)=-1.9), PrimerPair(names=('', ''), seqs=('GAGAAGAACCATAAAGTCCAAGC', 'CACCTCAAACCACACACTAC'), amplicon_sizes=[('exDonor-0,reDonor-407', 1, 2, 'Ca22chr2B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 337), ('exDonor-0,reDonor-407', 1, 0, 'Ca22chr2B_C_albicans_SC5314', 337), ('exDonor-0,reDonor-407', 0, 2, 'Ca22chr2A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 337), ('exDonor-0,reDonor-407', 0, 0, 'Ca22chr2A_C_albicans_SC5314', 337)], Tms=(53.16, 52.36), GCs=(0.43, 0.5), min(dG)=-2.71), None, None, None, PrimerPair(names=('', ''), seqs=('GAACCAATCCTTCCACATAGC', 'TTGCGTTTCGGGTATAATCAC'), amplicon_sizes=[('exDonor-0,reDonor-407', 1, 0, 'Ca22chr2B_C_albicans_SC5314', 654), ('exDonor-0,reDonor-407', 1, 2, 'Ca22chr2B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 655), ('exDonor-0,reDonor-407', 0, 0, 'Ca22chr2A_C_albicans_SC5314', 655), ('exDonor-0,reDonor-407', 0, 2, 'Ca22chr2A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 655)], Tms=(52.7, 52.81), GCs=(0.48, 0.43), min(dG)=-3.13), PrimerPair(names=('', ''), seqs=('TATTGGTCGGATTGGGTTAC', 'GCTGGGAATTGATAATGAAAGC'), amplicon_sizes=[('exDonor-0,reDonor-407', 0, 0, 'Ca22chr2A_C_albicans_SC5314', 369), ('exDonor-0,reDonor-407', 0, 2, 'Ca22chr2A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 369), ('exDonor-0,reDonor-407', 1, 0, 'Ca22chr2B_C_albicans_SC5314', 368), ('exDonor-0,reDonor-407', 1, 2, 'Ca22chr2B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 369)], Tms=(52.36, 52.04), GCs=(0.45, 0.41), min(dG)=-1.9), PrimerPair(names=('', ''), seqs=('GAGAAGAACCATAAAGTCCAAGC', 'CACCTCAAACCACACACTAC'), amplicon_sizes=[('exDonor-0,reDonor-407', 1, 2, 'Ca22chr2B_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 337), ('exDonor-0,reDonor-407', 1, 0, 'Ca22chr2B_C_albicans_SC5314', 337), ('exDonor-0,reDonor-407', 0, 2, 'Ca22chr2A_C_albicans_SC5314-r1[exDonor-0]-r2[reDonor-407]', 337), ('exDonor-0,reDonor-407', 0, 0, 'Ca22chr2A_C_albicans_SC5314', 337)], Tms=(53.16, 52.36), GCs=(0.43, 0.5), min(dG)=-2.71)])

FAQ

Why do overall PCR primer stringency levels differ for each gene?

The AddTag software tried all primers at all higher stringencies (lower numbers), and didn’t find a viable design until it reached the listed number. To save computation time, we had the program skip up to 6, 10, and 13 for BRG1, EFG1, and CSR1.

Why are you filtering the whole-genome GFF?

Early versions of AddTag needed abridged GFF as input, otherwise they would attempt to design Targets and dDNAs for all Features of the selected type. Later versions of AddTag introduced the functionality to list the specific feature IDs you wish to include.