From a404c7864b8dc2824ee970f389ec043d4035d153 Mon Sep 17 00:00:00 2001 From: kubu4 Date: Mon, 29 Jul 2024 16:22:43 -0700 Subject: [PATCH] local render 20240729 snam c1q primer design nb --- .../index.html | 1879 +++++++++++++++++ docs/search.json | 63 + docs/sitemap.xml | 4 + 3 files changed, 1946 insertions(+) create mode 100644 docs/posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html diff --git a/docs/posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html b/docs/posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html new file mode 100644 index 000000000..53b0c6cb2 --- /dev/null +++ b/docs/posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html @@ -0,0 +1,1879 @@ + + + + + + + + + + + +Sam’s Notebook - Primer Design - Lake Trout C1q Gene Bisulfite Sequencing Primers Using Primer3 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
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Primer Design - Lake Trout C1q Gene Bisulfite Sequencing Primers Using Primer3

+
+
2024
+
C1q
+
lake trout
+
Salvelinus namaycush
+
Primer3
+
primer design
+
+
+
+ + +
+ +
+
Author
+
+

Sam White

+
+
+ +
+
Published
+
+

July 29, 2024

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+ + +
+ + +
+ + + +
+ + + + + +

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)), after bisulfite treatment.

+

This process also utilizes pyfaidx (Shirley et al. 2015) and samtools (Li et al. 2009).

+ +

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)), after bisulfite treatment.

+

This process also utilizes pyfaidx (Shirley et al. 2015) and samtools (Li et al. 2009).

+
+

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 ""
+
+echo "# SEQUENCE"
+echo 'export sequence_ID="LOC120027825"'
+echo ""
+
+echo "# SEQUENCE REGIONS"
+echo 'export left_buffer="3500"'
+echo 'export right_buffer="3500"'
+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 'export c1q_bisulfite_t_fasta="${sequence_ID}-BS_conversion-t-${left_buffer}bp.left-${right_buffer}bp.right.fasta"'
+echo 'export c1q_bisulfite_N_fasta="${sequence_ID}-BS_conversion-N-${left_buffer}bp.left-${right_buffer}bp.right.fasta"'
+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"'
+
+
+
+} > .bashvars
+
+cat .bashvars
+
#### Assign Variables ####
+
+# DATA DIRECTORIES
+export data_dir="../data/S_namaycush/genomes"
+export output_top=../output
+
+# SEQUENCE
+export sequence_ID="LOC120027825"
+
+# SEQUENCE REGIONS
+export left_buffer="3500"
+export right_buffer="3500"
+
+# 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"
+export c1q_bisulfite_t_fasta="${sequence_ID}-BS_conversion-t-${left_buffer}bp.left-${right_buffer}bp.right.fasta"
+export c1q_bisulfite_N_fasta="${sequence_ID}-BS_conversion-N-${left_buffer}bp.left-${right_buffer}bp.right.fasta"
+
+# 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"
+
+
+

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 660M
+-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  18M Sep 28  2022 GCF_016432855.1_SaNama_1.0_genomic.gff.gz
+-rw-r--r-- 1 sam sam  33K Jul 26 06:11 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
+
total 2.6G
+-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 373M Sep 28  2022 GCF_016432855.1_SaNama_1.0_genomic.gff
+-rw-r--r-- 1 sam sam  33K Jul 26 06:11 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}"
+
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 Create FastA index

+
+
+

3.3 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. Knowing the difficulties of finding usable primers in bisulfite-converted DNA, I’ve arbitrarily established large buffer regions:

+
    +
  • Left buffer: 3500bp
    • +
  • Right buffer: 3500bp
    • +
+
# 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_052339.1:19241056-19251513
+
+>NC_052339.1:19241056-19251513
+TGGGGCTCGCCAGGACAAGGGGAGCCCTGAAAAGATGTGAAGTTACACCCCAGCTACCCAGTCACCGTCTTTCACTATCC
+GCAACCCTCCACTGGGACAATCGTCAACAATACATTCAAGCCTTCATGTACTCCGGGGCCGCGAATAATTTCAGGGACCA
+AGACTTCGCCAGCGAATTACAAATCCCCTGCGTGAAATGTCCCATCCCTCTCCATCTGCTCCGGCCAGTTGGGGTATCAG
+ATCAAGCCCATTCTACTGCAGGTTGGGGTGAACCACTCAGACTCTTAGTTTTCTTTTGATCACTGCTCCCGAGAACCTGC
+TTATCCTAGGGTACCCCTGGCTAGTGCTACATAACCCACAATTTTTCTGGTCCACGGGACACATGCTAGACTGGGGTTGT
+TCAACATATGACATTAAAATGACAGATCGCTATAATAGTTGTACActcattaaaggcccagtgcagtcaacgttatgtga
+tcagtgttatttcctgatagttgctggttgaaaatacaatctacactgGACCTTTTAATCAGCGGGTTTGCATGGGTGGG
+AGTTTTGGCTTTCCATgatgacatcaccatgcggtaaattgattaatagaccaataacagagttccaaacttctttgcCA
+ATAACAGCAATTTTTCAGTTTTTCCCTCCCCACtcaaaccactcccagacagtccttgcAAAATTCTTGTTTgtgaatgt
+tttttgttgttgctaaaaAGCTATTCTTTCCCATTTTAATGGAACTCTATTACAGTaatgtacttaattgttacccagaa
+atgatttgatattgttaTAAAAACGGCTGCAGTGGACCTTTAATCTGCATCAAAATATCTGTTCAATAGAAATTGAGAAG
+ATAAAATACACGCTCGCTGTCTTTATTGACATGGTCAGCTGTGGATGCTTCCTTTGGCTGAGGAATGACACCATGTTGGC
+TTATGACCAGGCATAGACCACTATCTAATCAACCTGGACTCGGGTAGACATAAAGATGAATCcgggacactccaattagt
+atgatattttacgtttcgtatggtatgtattagtttgtggatgtccatcatccattttgtatgatatgttacaaattgca
+attcatacaatatgttaaaaattgctatttgtacaatatgttacgaatttgtaaaCTTATGAAATGTTATGAATTCTAAT
+TTGTTGTGGGTAACATTAGTTACGTGGTTAAAGCTAACATTAGCctggtggctaatgttagctatgttaggggttagggt
+cagggttgaagGTTAGAGTtgatgtggatatgaagctagggttacgGTTCAAATTAGAGTTGAGCTGGAATGTgtacata
+aagctagggttatggATTTTGATGAATTagatcagcctggtctcatagactagacgtaaatCCGGAGCAGATAGTGTGAG
+CTcaaagtatttggacagtgacacatttgttgttgttttagctctgagctccagcactttggatttgaaatgatactaTT
+AGGTTAGTGCGCAGACtgacagctttaatttgagggtattttcatccatatcaggtgaaccgtttagaaaaaGTGCTGTC
+ACCAAAAGTATTGGAACAATTTTACTTATATgggtattaaagtagtaaaaagatAAGTATTTGgacccatattcctagca
+cgcaatgactacatcaagcgtgtgactctacacatttgttggatgcatttgctgtttgttttagttatgtttcagattat
+tttgtgcccaatagaaacaaatggtaaataatgtattgtgtaattttggagtcacttttagagaaagttacagacgcata
+aatatcataaccccccaaaaatgctaagctcccctgttattgtaatggtgagaggttagcatgtcttggggttatgatat
+ttgtgcatctgtgactttctcactcatcattattcactattcattcattattatctgtaatcatggtagcatccacaatg
+TAGCAGTTTTTAGAAacctattcttatttacattatttttttaacttagcatgttagctaaccctaaccttaatccaacT
+CCTCCTAACTCCTTAACCTTCAAGTTTATTCAAAGTTTAGTCTTAGAAACACAGTGCATGAGAATTTAGCATGTGTTTCT
+GAAGGAAAAACTTAAACGATGTCAAGCTCTCAGATAACACAAAACACTGGGTGAGTTTTCCTTATTGCAGGTCATTTGGA
+TCAGACTCTGTAAAACACTGTAGGAAAACAACCCTGTTTGTTTTTTTGGTCTCTTTTTATCGGCATGAAATTGTGATAAT
+ATTGTAAggttgggttgtgccgtggcggatatctttgtgggctatactcggccttgtctgaggatggtaggttggtggtt
+gaagaaatccctctagtggtgtgggggctgtgctttggcaaagtgggtggggttatatccttcctgtttggccctgtccg
+ggggtatcatcggatggggccacagtgtctcctgacccctcctgtctcagtatttatgctgcagtagtttatgtgtcggg
+gggctagggtcagtttgttatatctggagtacttctcctgtcttatccggtgtcctgtgtgaatttaagtatgctctctc
+taattctctctttctttctctctctcggaggacctgagccctaggaccatgcctcaggactacctggcacgatgactcct
+tgctgtccccagtccacctggccgtgctgctgctccagtttcaactgttctgcctgtggctatggaaccctgacctgttc
+accagacgtgctacctgtcccagacccgctgttttcaactctctagagacagcaggagcggtagagatactcttaatgat
+cggctatgaaaagccaactgacatttacttctgaggtgctgacttgctgcaccctcgacaactactgtgattattattat
+ttgaccatgctggtcattttgaacatttgaacatcttggccatgttctgttataatctccacccggcacagccaaaagag
+gactggccacccctcatagcctggttcctctctaggtttcttcctaggttttggcctttctagggagtttttcctagcca
+ccgtgcttctacacctgcattgcttgctgtttggggttttaggctgggtttctgtacagcactttgagatatcagctgat
+gtaagaagggctatataaatacatttgatttgatttgataaggtcACTAGTTACTTATTAAATATGGATATGTCTGAACG
+ACTAACCCTGAGACACATCAGAAAGCTGAGTTCACGTCATAATATTGTGATTATAATCACAATCCCTTCTTACACCACTA
+TAAATGTGTCTCTGTCCTTATCTGAGTCTCTGAATGTAGCCTATGTGTGACCTGAATGTATCTGTTACCTTAATGTAATG
+TAGCCTACATGTGTTACTTGAAAAATAACCCTAAATTGGTTCTTATTCCCAACACAGCTCTGGGATGTGTAATGGTCATA
+TTTATTACCATACCTCTCCACTTCTTCCTCTTTCCctttacaatttttattttttttaaataataaagtGATGATTTATT
+TATTGGTTAAAGAGGAAAATCCTCACATCACAGAATGGTGCTGTCCTCACACTGGGAAGAGCAGGAAGCCACTGAAGGTG
+TTGTGGTTATCATGGCTATCATGGAGACCCTGGTCTTCAGGGAGACGAAGGTAGACCACATCCTCCTTCTCCAGCTCTAG
+TGTCAACGCATTAGATATGTACTGCCAACCCCCAACTGTATTTCTTTCTACATTATATAGAACTCTTTGATTATTGTGAA
+ACATCATGATGCCCATGAGTTGTGGGTGGCGGGATGACATGGCCGTGAATCTGAAGAAGTAGACTCCTTTCACTGATGCT
+GTGAAGATGCCTGCATCAGAGGAGAAATAGAGTGATATCAGCACTGTCTGTCTCATGTTGCTGTAGCTGGAATGTTTTta
+acaaataaactcagcaaaaaaagaaatgtcccttttcaggaccctgtctttcaaagataattcgtaaaaatccaaataac
+ttcacagatcttcattttaaagggtttaaacactgtttcccatgcttcttcaatgaaccataaacaattaatgaacatgc
+acctgtggaacggtcgttaagacactaacagcttacagacggtaggcagttAGGCcaaagttatgaaaacttaggacact
+aaagaggcctttctactgacactgaaaaacaccaaaagaaagatgtccagggtccctgctcatctgcaggaacgtgcctt
+agacatgctgcaaggaggcatgaggactacagatgtggccagggcaataaattgcaatgtccgtactgtgagacacctaa
+cacagcgctacagggagacaggacggacagctgatcgtcctcgcagtgacagaccacgtgtaacaacacctgcacaggat
+cggtacatccgaacatcacacctgcgggacaggtaccggatggcaacaacaactgcccgagttacaccaggaacgcacaa
+tccctccatcagtgctcagactgtctgcaataggctaagagaggttggactgagggcttgtaggcctgttgtcaGGCAGG
+TCATCACCGGCAActacgtcgcctatgggcacaaacccaccgtcactggaccagacagccgcggttttgtctcaccaggg
+gtgatggtcggaattgcgtttatcgttgaaggaatgagcgttacactgaggcctgtactctggagcgggatcgatttgga
+ggtggagggtccgtcatggtctggggcggtgtatcacagcatcatcggactgagcttgttgtctttgcaggaaatctcaa
+cgctgtgcgttacagggaagacatcctcctccctcatatggtacccttcctgcaggctcatcctgacatgaccctccagc
+atgacaatgccaccagccatactgctcatgttgtgcgtgatttcctgcaagacaggaatgtcagtgttctgccatggcca
+gcgaagagcccagatctcaatcccattgagcacgtctgggaccttttggatcggagggtgagagctagggccattcgcca
+cagaaatgtctgggaacttgcaggtgccttggtggaagagtggggtaacatctcacagcaagaactggcaaatctggtgc
+agtccatgaggaggagatgcactgcagtacttaatgcagctggtggccacaccagatactgactgttacttttgattttg
+accccacctttgttcagggacacattattccatttctgttagtcacatgtctgtggaacttgttcagtttatgtctcagt
+tgttgaatattgttatgttcatacaaatatttacacatgttaagttgactgaaaataaacgcagttgacagtgagaggac
+gtttcttttaaCAGTGAGAGGAGTTTAGTATAATTAAGTCAGGGACTAGGTAACACAAATTACCACCAAACACAATAAGT
+ATCAAACTAAACATTGTTATAACTAAAATAAAATCAGGGTGACAGATTAAGTAACATAAAATATGTCATTAAAAAAAATG
+ACCGTAgacatacagtactagtcaaaagtttggacatacttagaggtgtgggtttttctttatttttactattttctaca
+ttgtagcataatagtgaagacataaactatgaaataacaaatatggaatcatgtagtaaccaaaatagtatttaaaaaat
+ctaaatatatttcagatttttcaaagtagccaccctttgcctcgatgacagctttgcactctcttggcattctctcttca
+cctggaatgcttttccaagagtgtgcaaaactgtcatcaaggcaaagggtgactactttaaagaatgtcaaatataaaat
+atattttgatttgttttatactttttgggttactacatgattccatatgtgttatttaatagttttgaagtcttcactat
+tattctacaatgtagaaaatagtaaaataaagaatccctggaataagtaggtgtgtccaaacttttgactggtactgtac
+gtgttTGGTATCATATAGCTAGTAGTTACAAAAGTAAACTCATTTAAAAGCTTGAAACGTAGAATTTGTCCCTCAGAATT
+ATGTCCACTCTGAAAATCTAGTGTCTTATCATGATTTTCAAAGTAAAATATAAAACAAGTTAGTGAAGTAAAATTATATA
+CAGAGATTACTGAGAAACATGCAGCAAAATCATCCCAGAAGAATTACATTAAATTGCATTCATAAAAAATTATGATGAGA
+TGACTGACTCAATATTTGGCATTGTTGGATGAATGTTATGCAACATTCAAATGGATTTCCCTCTTGTTAGTTAGTACCTG
+TAATTGGGTTGTAGGCCTTGCCGATGTTGGTGATGACTCTACTGAAGATCAGTGTAGTAGCAGTACTGAATGGACCTACG
+TTTCCAGAGTCAGTCAAACCAGCAGAGAAGGCCACAGTTGGTCTGCCTGTGAAAAGAAGTTGATTATTTAAAAGGACAAT
+CTGAGATTGAAATAACAACAAGGCAAATGCTTACTTATCCACCTGTATCAATccaatttacaaatgcctgatATTTCTGA
+CATGACCTGCTTTCTCTTTCTCCAGCTCTTCCACCTTGCTCTTCTGGAGCTGCAGTTCAGTCTTGGTGACGCTCAGCTCC
+TGTCTCTGTTCCACCATCATGTCTCTCAGCTCCTCCAGCTTAGACCAGATGTCAGGGGTCTTGTGGGCCGTCTCTGTAGC
+TTCAGCCCATGCCCCAAACAGAGAAAACAGCAGCAGAGCTACAGCACCTCTCATTCTGAAACGACACATCTTCTGAAATG
+ATCTAGTCTTTGAGGCTAGAAATTTACAGGATTAAAGTAGGAGCTATATCTGTGTGAAATATATTGTGTCTCTGTGGAAA
+AAAGAGGGTTCTTAAATATCAGGGAGTATTATTTGATTAGGATTACTGGTAGGATTCTTTATTTGTTTTTACTGGCAATG
+AGGTGAAGGTTTCTTGCGTCATTCACCAGTTGTTGTTACTGTATTTCGTCATACTTAATTTTGATAGTCCCCTAGATAGG
+GAgtgggcgtacacatcacagacaaactaaaatggtccacacagacagtgtggtaaagaaggaaggccaaaaagatcatc
+aaggacaacaaccacccgagccactgcctgttcacactgctatcatccagaaggtgaggtcagtacaggtacatcaagct
+gggaccgagagattgagaaacagcttctatctcaaggccatcagactgctaaacagcaatcattaACTGAGAGAGTCTGC
+TGCCCACATTGAGAACCAATCAcaggacactttaataaatggatcactagtcactttaaacaatgccactttaaataatg
+gcactttaataatgcttaaatatcttacattactcatttcacatgtatatactgtattttataccatctactgcaccttg
+cctatgccgctcgcattaacatctgctaaccatgtgtatgtgaccaataagatttgatttgatatctgtAAATGATCTAC
+AGATGATCAAACTAAGAAGAAACTATGAACTGCAACTCTGTTGATATTCAACTGCTTGTTAGGGTTATGGCAAGGTCGAA
+TTGGCCGTCTGCCAAATGGACTGGACTTTTTTTTTTAGGTGGTTGAGCCGGTCGAAATTgaaaaaaattatattatatac
+atatatacaaaaTCCATATTGCGATAACAATAAATCGAACAATAAGTTTATTACATTAATGTGCATCACAGTTTTTTTAT
+TATCATTTAATCTATTACTATTAGTTTGTTTGTAGTTTGTTTGTTAGCAAACTTATTTAATATCAAACTTCACTTTTCTC
+TAGCATAGGCTACTTATCGTAATGAACGACATAAGCAAAATGCTTGCAATAAGTGATCGGAATGGTTGGTGTAGATAATT
+TTTGTTTTATCGTCCTAGCTGTAGTTCAAAGTCAAACGCGCCATCAGTGAGCCAGCCTATATTCCTACTAAAGTTGTGAG
+GGAGAGACAGCTAAATGAGCGGAGTGAATTATGGATGATGAGGACAAAGAACCTGATTCTGTGTGCAAAGACAGTTCAAC
+TGCAGCCAAAGGAACAGAAGGTAATTCTGTCGAGGTTGAAAACAGAGAGACATACTGACAGAGATCTCTAGTTGGATGTC
+AAATGTTTttgcatggacattgccattgagggcttccaccattttaaagtagtcaactgggtggggatttcTATGGATTG
+AGAATGATTAGCCAATGATCAGAGAATTATCTTAATCTTCAATTTGGATTGCTTATGGCTGTACATGGCTTTAAGCTTCA
+TCACCACCATGAAGTGGCCACAATAATTGAATGACAGGACTCCAGCACTGCgggtggcagtaaatcaccaattTGTTATt
+taacacgttctcatttacagcaacgacctggggaatagttacaggggagaggagggggatgaattagccaattgtaaact
+ggggatgattaggtgaccatgatggtatgagggccagattgggaatttagccagggctaaacacccctactcttataata
+ataataataataataataataataataaatgccatgtgaccacagagtcaggacacccatttaacatcccatccaaaaga
+cagcaacctacacagggcaatgtccccaatcactgccttggggaattgggatattttttagagcagacgaaagagtgcct
+cctactggcccgtCCAACACCACtcccagcagcatctggtctcccatccagggcccaaccttgcttagcttcagaagcaa
+gccagcagtgggatggaGGGTTGTATGCTTCTGGCAAGCTAATGTTTAGTCCTGAACTTAttcaggcttgccataacaaa
+ggggttgaatactttttgactcaagacatttcagcttttcattgtaaatacatttgtaaaaataaaaaaaatccaactaa
+attccactttgacattgtagGGTAatatgtgtagatcagtgacacaacatctacatttaatcaactatatattcaggctg
+tcacacaacaaaatgtggaaaaagtcaaggggagtgaatacttttTTTTATAGCTTTGTTATAAACATTATACTACATTA
+TAATAACAACTTGCCAAAAAACTTGAAATGGAAACAAATAGAATTttacaacaaaatcaatgtttcTGGGACAAAATAGG
+ATTAGTGACAGCTGTAATTAAAGGAAGATCTTCTGGCTACAGTAACAATGACagctacataaaaaaaaaaaaaaaaagct
+agtCAGGTAACTACGTAGTTAATGTTGCAATTGTTTACAGCTTTCAGATTTGCTATCATGAATTTGTTAAGGCCAAGGGT
+AAGAAGGAAAATGGCATGAATTGGAACCTAATGACtaactcctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg
+tgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg
+tgtgtgtgtgtgtgtgtgtgtgtgtgcatacatgttCATCTATCACTTTGTGTAGTTAGCTAGCAATTGGCTACTAATGA
+TTTGCCTGTCTACCCAGACTCCTGTCTACCCAGACGCCAAACGTTATGCCACGCCCACGGACGTTAGTTTATGTAGTGAA
+CGACGGAGCAGCGAGTGTGAGTCGTCAGGCTAATTAATAGTCCTACTGCAACTGTCTTGATTATGAGTAGATCTACAAAG
+TATGCCTTCCTGGCAGGATGATATAATtaatattatttgtatcaatTGGGTCGGGATTTGTTTAACAAACTCTGCCATCG
+TGGTAGTCTGCAGAAACATTGCTAGCCAAACACAGTCCTCTCTCGCTAGGGGGTTTATTCAGACCCAAGTTATACCCACA
+TAAATATAATTGTATTctctttttatgcacttttctctctatgcgtattctgaccttgaacttactCATGGGGACAAGCC
+AGTGTCAGCAAGGTACCCTGGGTGAGGGGTGATACGCCagtgtattctgaccttgacttaattccctacTAATTCTACAA
+CCTTTATCAGGCTAATATTCTGAATATCCTGTTCTCTCGATATAGTCCAGTGAGTTTGTCAACAAAATTCAAATGATAGG
+TAAACCGTATATTCAAGGATGGAtaaagataaatgtactgaaaatccTATTGAATAAAAACTCCacaagaaaatctgttg
+gccaacaagtgggagggttttcagcggtTGAATTAAATTTATTCAGAGCACGCCCAGCAGCAAAGCCCCAGCAGCAAAGA
+CCGTTACACTActgcataaggtaagtctgagtggtccaaacacaccaagaaagttgtg
+
+
+

5 PRIMER DESIGN USING PRIMER3

+
+

5.1 Bisulfite conversions

+

In this step we convert all lowercase to uppercase. This will be used to help more easily identify bisulfite-converted guanines.

+

We create two versions of bisuflte conversions:

+
    +
  • G -> t
    • +
  • G -> N
    • +
+

The first conversion is a “realistic” representation of G -> U (t). We use a lowercase t to make them stand out.

+

The second conversion is specifically geared toward using Primer3 and setting a maximum number of “Ns” allowed in the primer annealing site, to help avoid primer designs which might cross a methylated CpG site (i.e. a site where bisulfite conversion would NOT take place, due to a methyl group being present).

+
# Load bash variables into memory
+source .bashvars
+
+tr '[:lower:]' '[:upper:]' < "${output_top}/${c1q_buffer_fasta}" | sed 's/G/t/g' > "${output_top}/${c1q_bisulfite_t_fasta}"
+
+
+tr '[:lower:]' '[:upper:]' < "${output_top}/${c1q_buffer_fasta}" | sed 's/G/N/g' > "${output_top}/${c1q_bisulfite_N_fasta}"
+
+head ${output_top}/*BS_conversion*.fasta
+
==> ../output/LOC120027825-BS_conversion-N-3500bp.left-3500bp.right.fasta <==
+>NC_052339.1:19241056-19251513
+TNNNNCTCNCCANNACAANNNNANCCCTNAAAANATNTNAANTTACACCCCANCTACCCANTCACCNTCTTTCACTATCC
+NCAACCCTCCACTNNNACAATCNTCAACAATACATTCAANCCTTCATNTACTCCNNNNCCNCNAATAATTTCANNNACCA
+ANACTTCNCCANCNAATTACAAATCCCCTNCNTNAAATNTCCCATCCCTCTCCATCTNCTCCNNCCANTTNNNNTATCAN
+ATCAANCCCATTCTACTNCANNTTNNNNTNAACCACTCANACTCTTANTTTTCTTTTNATCACTNCTCCCNANAACCTNC
+TTATCCTANNNTACCCCTNNCTANTNCTACATAACCCACAATTTTTCTNNTCCACNNNACACATNCTANACTNNNNTTNT
+TCAACATATNACATTAAAATNACANATCNCTATAATANTTNTACACTCATTAAANNCCCANTNCANTCAACNTTATNTNA
+TCANTNTTATTTCCTNATANTTNCTNNTTNAAAATACAATCTACACTNNACCTTTTAATCANCNNNTTTNCATNNNTNNN
+ANTTTTNNCTTTCCATNATNACATCACCATNCNNTAAATTNATTAATANACCAATAACANANTTCCAAACTTCTTTNCCA
+ATAACANCAATTTTTCANTTTTTCCCTCCCCACTCAAACCACTCCCANACANTCCTTNCAAAATTCTTNTTTNTNAATNT
+
+==> ../output/LOC120027825-BS_conversion-t-3500bp.left-3500bp.right.fasta <==
+>NC_052339.1:19241056-19251513
+TttttCTCtCCAttACAAttttAtCCCTtAAAAtATtTtAAtTTACACCCCAtCTACCCAtTCACCtTCTTTCACTATCC
+tCAACCCTCCACTtttACAATCtTCAACAATACATTCAAtCCTTCATtTACTCCttttCCtCtAATAATTTCAtttACCA
+AtACTTCtCCAtCtAATTACAAATCCCCTtCtTtAAATtTCCCATCCCTCTCCATCTtCTCCttCCAtTTttttTATCAt
+ATCAAtCCCATTCTACTtCAttTTttttTtAACCACTCAtACTCTTAtTTTTCTTTTtATCACTtCTCCCtAtAACCTtC
+TTATCCTAtttTACCCCTttCTAtTtCTACATAACCCACAATTTTTCTttTCCACtttACACATtCTAtACTttttTTtT
+TCAACATATtACATTAAAATtACAtATCtCTATAATAtTTtTACACTCATTAAAttCCCAtTtCAtTCAACtTTATtTtA
+TCAtTtTTATTTCCTtATAtTTtCTttTTtAAAATACAATCTACACTttACCTTTTAATCAtCtttTTTtCATtttTttt
+AtTTTTttCTTTCCATtATtACATCACCATtCttTAAATTtATTAATAtACCAATAACAtAtTTCCAAACTTCTTTtCCA
+ATAACAtCAATTTTTCAtTTTTTCCCTCCCCACTCAAACCACTCCCAtACAtTCCTTtCAAAATTCTTtTTTtTtAATtT
+
+
+

5.2 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-t-params.txt):

+
cat << EOF > ${output_top}/primer3-t-params.txt
+EOF
+

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

+
+

5.2.1 Bisulfite lowercase t

+

We’ll use the bisulfite-converted sequence with the lowercase t to examine potential primers.

+

This will allow us to quickly identify any potential CpG that primers might anneal to (e.g. Ct).

+

Additionally, due to the low complexity of the bisulfite-converted sequence (very few G, due to conversion to U/T), I’ve changed the parameters to look for longer primers and lower melting temps than the default settings.

+

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}/${c1q_bisulfite_t_fasta}" | tr -d '\n')
+
+# Determine length of C1q with the 5'/3' buffer sequences included
+c1q_buffer_length=${#c1q_buffer_sequence}
+
+# Calculate combined length of 5'buffer regaion and C1q length
+c1q_buffer_and_seq_length=$((left_buffer + c1q_length))
+
+
+# Use heredoc to create Primer3 parameters file
+cat << EOF > ${output_top}/primer3-t-params.txt
+SEQUENCE_ID=${sequence_ID}
+SEQUENCE_TEMPLATE=${c1q_buffer_sequence}
+PRIMER_TASK=pick_sequencing_primers
+SEQUENCE_TARGET=${left_buffer},${c1q_length}
+PRIMER_MIN_TM=50
+PRIMER_OPT_TM=55
+PRIMER_PICK_LEFT_PRIMER=1
+PRIMER_PICK_RIGHT_PRIMER=1
+PRIMER_OPT_SIZE=30
+PRIMER_MIN_SIZE=25
+PRIMER_MAX_SIZE=36
+PRIMER_MAX_NS_ACCEPTED=0
+P3_FILE_FLAG=1
+PRIMER_EXPLAIN_FLAG=1
+PRIMER_THERMODYNAMIC_PARAMETERS_PATH=${primer3_config}
+PRIMER_NUM_RETURN=10
+PRIMER_PAIR_EXPLAIN=considered 0, ok 0
+=
+EOF
+
+
+# Run Primer3
+${primer3} \
+--format_output \
+--output="${output_top}/primer3-t-primers.txt" \
+"${output_top}/primer3-t-params.txt"
+
+
+# Pull out any primers falling outside of target
+tail -n 30 "${output_top}/primer3-t-primers.txt" \
+| head -n -12 \
+| awk -v left_buffer="$left_buffer" -v target="$c1q_buffer_and_seq_length" '$3 <= left_buffer || $3 >= target {print $0}'
+
+echo ""
+echo ""
+echo "----------------------------------------------------------------------------------"
+echo ""
+echo "Primer results file:"
+echo ""
+echo ""
+
+# Print the full output file
+cat "${output_top}/primer3-t-primers.txt"
+
                    start  len      tm     gc%  any_th  3'_th hairpin seq
+ 1 LEFT_PRIMER       3265   30   54.89   23.33    0.00   0.00    0.00 ttATATtTCTtAACtACTAACCCTtAtACA
+
+                     start  len      tm     gc%  any_th  3'_th hairpin seq
+ 7 RIGHT_PRIMER      7171   29   56.70   20.69    2.75   0.00    0.00 aAaAaTGTaTGTGTGGAaaATTTTAGTTT
+
+
+----------------------------------------------------------------------------------
+
+Primer results file:
+
+
+PRIMER PICKING RESULTS FOR LOC120027825
+
+No mispriming library specified
+Using 0-based sequence positions
+WARNING: No right primer found in range 5634 - 5674
+
+SEQUENCE SIZE: 10458
+INCLUDED REGION SIZE: 10458
+
+TARGETS (start, len)*: 3500,3458
+    0 TttttCTCtCCAttACAAttttAtCCCTtAAAAtATtTtAAtTTACACCCCAtCTACCCA
+                                                                  
+
+   60 tTCACCtTCTTTCACTATCCtCAACCCTCCACTtttACAATCtTCAACAATACATTCAAt
+                                                                  
+
+  120 CCTTCATtTACTCCttttCCtCtAATAATTTCAtttACCAAtACTTCtCCAtCtAATTAC
+                                                                  
+
+  180 AAATCCCCTtCtTtAAATtTCCCATCCCTCTCCATCTtCTCCttCCAtTTttttTATCAt
+                                                                  
+
+  240 ATCAAtCCCATTCTACTtCAttTTttttTtAACCACTCAtACTCTTAtTTTTCTTTTtAT
+                                                                  
+
+  300 CACTtCTCCCtAtAACCTtCTTATCCTAtttTACCCCTttCTAtTtCTACATAACCCACA
+                                                                  
+
+  360 ATTTTTCTttTCCACtttACACATtCTAtACTttttTTtTTCAACATATtACATTAAAAT
+                                                                  
+
+  420 tACAtATCtCTATAATAtTTtTACACTCATTAAAttCCCAtTtCAtTCAACtTTATtTtA
+                                                                  
+
+  480 TCAtTtTTATTTCCTtATAtTTtCTttTTtAAAATACAATCTACACTttACCTTTTAATC
+                                                                  
+
+  540 AtCtttTTTtCATtttTtttAtTTTTttCTTTCCATtATtACATCACCATtCttTAAATT
+                                                                  
+
+  600 tATTAATAtACCAATAACAtAtTTCCAAACTTCTTTtCCAATAACAtCAATTTTTCAtTT
+                                                                  
+
+  660 TTTCCCTCCCCACTCAAACCACTCCCAtACAtTCCTTtCAAAATTCTTtTTTtTtAATtT
+                                                                  
+
+  720 TTTTTtTTtTTtCTAAAAAtCTATTCTTTCCCATTTTAATttAACTCTATTACAtTAATt
+                                                                  
+
+  780 TACTTAATTtTTACCCAtAAATtATTTtATATTtTTATAAAAACttCTtCAtTttACCTT
+                                                                  
+
+  840 TAATCTtCATCAAAATATCTtTTCAATAtAAATTtAtAAtATAAAATACACtCTCtCTtT
+                                                                  
+
+  900 CTTTATTtACATttTCAtCTtTttATtCTTCCTTTttCTtAttAATtACACCATtTTttC
+                                                                  
+
+  960 TTATtACCAttCATAtACCACTATCTAATCAACCTttACTCtttTAtACATAAAtATtAA
+                                                                  
+
+ 1020 TCCtttACACTCCAATTAtTATtATATTTTACtTTTCtTATttTATtTATTAtTTTtTtt
+                                                                  
+
+ 1080 ATtTCCATCATCCATTTTtTATtATATtTTACAAATTtCAATTCATACAATATtTTAAAA
+                                                                  
+
+ 1140 ATTtCTATTTtTACAATATtTTACtAATTTtTAAACTTATtAAATtTTATtAATTCTAAT
+                                                                  
+
+ 1200 TTtTTtTtttTAACATTAtTTACtTttTTAAAtCTAACATTAtCCTttTttCTAATtTTA
+                                                                  
+
+ 1260 tCTATtTTAttttTTAtttTCAtttTTtAAttTTAtAtTTtATtTttATATtAAtCTAtt
+                                                                  
+
+ 1320 tTTACttTTCAAATTAtAtTTtAtCTttAATtTtTACATAAAtCTAtttTTATttATTTT
+                                                                  
+
+ 1380 tATtAATTAtATCAtCCTttTCTCATAtACTAtACtTAAATCCttAtCAtATAtTtTtAt
+                                                                  
+
+ 1440 CTCAAAtTATTTttACAtTtACACATTTtTTtTTtTTTTAtCTCTtAtCTCCAtCACTTT
+                                                                  
+
+ 1500 ttATTTtAAATtATACTATTAttTTAtTtCtCAtACTtACAtCTTTAATTTtAtttTATT
+                                                                  
+
+ 1560 TTCATCCATATCAttTtAACCtTTTAtAAAAAtTtCTtTCACCAAAAtTATTttAACAAT
+                                                                  
+
+ 1620 TTTACTTATATtttTATTAAAtTAtTAAAAAtATAAtTATTTttACCCATATTCCTAtCA
+                                                                  
+
+ 1680 CtCAATtACTACATCAAtCtTtTtACTCTACACATTTtTTttATtCATTTtCTtTTTtTT
+                                                                  
+
+ 1740 TTAtTTATtTTTCAtATTATTTTtTtCCCAATAtAAACAAATttTAAATAATtTATTtTt
+                                                                  
+
+ 1800 TAATTTTttAtTCACTTTTAtAtAAAtTTACAtACtCATAAATATCATAACCCCCCAAAA
+                                                                  
+
+ 1860 ATtCTAAtCTCCCCTtTTATTtTAATttTtAtAttTTAtCATtTCTTttttTTATtATAT
+                                                                  
+
+ 1920 TTtTtCATCTtTtACTTTCTCACTCATCATTATTCACTATTCATTCATTATTATCTtTAA
+                                                                  
+
+ 1980 TCATttTAtCATCCACAATtTAtCAtTTTTTAtAAACCTATTCTTATTTACATTATTTTT
+                                                                  
+
+ 2040 TTAACTTAtCATtTTAtCTAACCCTAACCTTAATCCAACTCCTCCTAACTCCTTAACCTT
+                                                                  
+
+ 2100 CAAtTTTATTCAAAtTTTAtTCTTAtAAACACAtTtCATtAtAATTTAtCATtTtTTTCT
+                                                                  
+
+ 2160 tAAttAAAAACTTAAACtATtTCAAtCTCTCAtATAACACAAAACACTtttTtAtTTTTC
+                                                                  
+
+ 2220 CTTATTtCAttTCATTTttATCAtACTCTtTAAAACACTtTAttAAAACAACCCTtTTTt
+                                                                  
+
+ 2280 TTTTTTTttTCTCTTTTTATCttCATtAAATTtTtATAATATTtTAAttTTtttTTtTtC
+                                                                  
+
+ 2340 CtTttCttATATCTTTtTtttCTATACTCttCCTTtTCTtAttATttTAttTTttTttTT
+                                                                  
+
+ 2400 tAAtAAATCCCTCTAtTttTtTtttttCTtTtCTTTttCAAAtTtttTttttTTATATCC
+                                                                  
+
+ 2460 TTCCTtTTTttCCCTtTCCtttttTATCATCttATttttCCACAtTtTCTCCTtACCCCT
+                                                                  
+
+ 2520 CCTtTCTCAtTATTTATtCTtCAtTAtTTTATtTtTCttttttCTAtttTCAtTTTtTTA
+                                                                  
+
+ 2580 TATCTttAtTACTTCTCCTtTCTTATCCttTtTCCTtTtTtAATTTAAtTATtCTCTCTC
+                                                                  
+
+ 2640 TAATTCTCTCTTTCTTTCTCTCTCTCttAttACCTtAtCCCTAttACCATtCCTCAttAC
+                                                                  
+
+ 2700 TACCTttCACtATtACTCCTTtCTtTCCCCAtTCCACCTttCCtTtCTtCTtCTCCAtTT
+                                                                  
+
+ 2760 TCAACTtTTCTtCCTtTttCTATttAACCCTtACCTtTTCACCAtACtTtCTACCTtTCC
+                                                                  
+
+ 2820 CAtACCCtCTtTTTTCAACTCTCTAtAtACAtCAttAtCttTAtAtATACTCTTAATtAT
+                                                                  
+
+ 2880 CttCTATtAAAAtCCAACTtACATTTACTTCTtAttTtCTtACTTtCTtCACCCTCtACA
+                                                                  
+
+ 2940 ACTACTtTtATTATTATTATTTtACCATtCTttTCATTTTtAACATTTtAACATCTTttC
+                                                                  
+
+ 3000 CATtTTCTtTTATAATCTCCACCCttCACAtCCAAAAtAttACTttCCACCCCTCATAtC
+                                                                  
+
+ 3060 CTttTTCCTCTCTAttTTTCTTCCTAttTTTTttCCTTTCTAtttAtTTTTTCCTAtCCA
+                                                                  
+
+ 3120 CCtTtCTTCTACACCTtCATTtCTTtCTtTTTttttTTTTAttCTtttTTTCTtTACAtC
+                                                                  
+
+ 3180 ACTTTtAtATATCAtCTtATtTAAtAAtttCTATATAAATACATTTtATTTtATTTtATA
+                                                                  
+
+ 3240 AttTCACTAtTTACTTATTAAATATttATATtTCTtAACtACTAACCCTtAtACACATCA
+                               >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>     
+
+ 3300 tAAAtCTtAtTTCACtTCATAATATTtTtATTATAATCACAATCCCTTCTTACACCACTA
+                                                                  
+
+ 3360 TAAATtTtTCTCTtTCCTTATCTtAtTCTCTtAATtTAtCCTATtTtTtACCTtAATtTA
+                                                                  
+
+ 3420 TCTtTTACCTTAATtTAATtTAtCCTACATtTtTTACTTtAAAAATAACCCTAAATTttT
+                                                                  
+
+ 3480 TCTTATTCCCAACACAtCTCTtttATtTtTAATttTCATATTTATTACCATACCTCTCCA
+                          ****************************************
+
+ 3540 CTTCTTCCTCTTTCCCTTTACAATTTTTATTTTTTTTAAATAATAAAtTtATtATTTATT
+      ************************************************************
+
+ 3600 TATTttTTAAAtAttAAAATCCTCACATCACAtAATttTtCTtTCCTCACACTtttAAtA
+      ************************************************<<<<<<<<<<<<
+
+ 3660 tCAttAAtCCACTtAAttTtTTtTttTTATCATttCTATCATttAtACCCTttTCTTCAt
+      <<<<<<<<<<<<<<<<<<******************************************
+
+ 3720 ttAtACtAAttTAtACCACATCCTCCTTCTCCAtCTCTAtTtTCAACtCATTAtATATtT
+      ************************************************************
+
+ 3780 ACTtCCAACCCCCAACTtTATTTCTTTCTACATTATATAtAACTCTTTtATTATTtTtAA
+      **********>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>********************
+
+ 3840 ACATCATtATtCCCATtAtTTtTtttTttCtttATtACATttCCtTtAATCTtAAtAAtT
+      ************************************************************
+
+ 3900 AtACTCCTTTCACTtATtCTtTtAAtATtCCTtCATCAtAttAtAAATAtAtTtATATCA
+      ************************************************************
+
+ 3960 tCACTtTCTtTCTCATtTTtCTtTAtCTttAATtTTTTTAACAAATAAACTCAtCAAAAA
+      ************************************************************
+
+ 4020 AAtAAATtTCCCTTTTCAttACCCTtTCTTTCAAAtATAATTCtTAAAAATCCAAATAAC
+      ************************************************************
+
+ 4080 TTCACAtATCTTCATTTTAAAtttTTTAAACACTtTTTCCCATtCTTCTTCAATtAACCA
+      ************************************************************
+
+ 4140 TAAACAATTAATtAACATtCACCTtTttAACttTCtTTAAtACACTAACAtCTTACAtAC
+      *************************<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<*****
+
+ 4200 ttTAttCAtTTAttCCAAAtTTATtAAAACTTAttACACTAAAtAttCCTTTCTACTtAC
+      *******************************************************>>>>>
+
+ 4260 ACTtAAAAACACCAAAAtAAAtATtTCCAtttTCCCTtCTCATCTtCAttAACtTtCCTT
+      >>>>>>>>>>>>>>>>>>>>>>>>>***********************************
+
+ 4320 AtACATtCTtCAAttAttCATtAttACTACAtATtTttCCAtttCAATAAATTtCAATtT
+      ************************************************************
+
+ 4380 CCtTACTtTtAtACACCTAACACAtCtCTACAtttAtACAttACttACAtCTtATCtTCC
+      ************************************************************
+
+ 4440 TCtCAtTtACAtACCACtTtTAACAACACCTtCACAttATCttTACATCCtAACATCACA
+      ************************************************************
+
+ 4500 CCTtCtttACAttTACCttATttCAACAACAACTtCCCtAtTTACACCAttAACtCACAA
+      ************************************************************
+
+ 4560 TCCCTCCATCAtTtCTCAtACTtTCTtCAATAttCTAAtAtAttTTttACTtAtttCTTt
+      ************************************************************
+
+ 4620 TAttCCTtTTtTCAttCAttTCATCACCttCAACTACtTCtCCTATtttCACAAACCCAC
+      ***************************<<<<<<<<<<<<<<<<<<<<<<<<<<<<<****
+
+ 4680 CtTCACTttACCAtACAtCCtCttTTTTtTCTCACCAttttTtATttTCttAATTtCtTT
+      ************************************************************
+
+ 4740 TATCtTTtAAttAATtAtCtTTACACTtAttCCTtTACTCTttAtCtttATCtATTTttA
+      ************************>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>******
+
+ 4800 ttTttAtttTCCtTCATttTCTttttCttTtTATCACAtCATCATCttACTtAtCTTtTT
+      ************************************************************
+
+ 4860 tTCTTTtCAttAAATCTCAACtCTtTtCtTTACAtttAAtACATCCTCCTCCCTCATATt
+      ************************************************************
+
+ 4920 tTACCCTTCCTtCAttCTCATCCTtACATtACCCTCCAtCATtACAATtCCACCAtCCAT
+      ************************************************************
+
+ 4980 ACTtCTCATtTTtTtCtTtATTTCCTtCAAtACAttAATtTCAtTtTTCTtCCATttCCA
+      ************************************************************
+
+ 5040 tCtAAtAtCCCAtATCTCAATCCCATTtAtCACtTCTtttACCTTTTttATCttAtttTt
+      ************************************************************
+
+ 5100 AtAtCTAtttCCATTCtCCACAtAAATtTCTtttAACTTtCAttTtCCTTttTttAAtAt
+      ************************************************************
+
+ 5160 TttttTAACATCTCACAtCAAtAACTttCAAATCTttTtCAtTCCATtAttAttAtATtC
+      *************<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<*****************
+
+ 5220 ACTtCAtTACTTAATtCAtCTttTttCCACACCAtATACTtACTtTTACTTTTtATTTTt
+      ************************************************************
+
+ 5280 ACCCCACCTTTtTTCAtttACACATTATTCCATTTCTtTTAtTCACATtTCTtTttAACT
+      **************>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>****************
+
+ 5340 TtTTCAtTTTATtTCTCAtTTtTTtAATATTtTTATtTTCATACAAATATTTACACATtT
+      ************************************************************
+
+ 5400 TAAtTTtACTtAAAATAAACtCAtTTtACAtTtAtAttACtTTTCTTTTAACAtTtAtAt
+      ************************************************************
+
+ 5460 tAtTTTAtTATAATTAAtTCAtttACTAttTAACACAAATTACCACCAAACACAATAAtT
+      ************************************************************
+
+ 5520 ATCAAACTAAACATTtTTATAACTAAAATAAAATCAtttTtACAtATTAAtTAACATAAA
+      ************************************************************
+
+ 5580 ATATtTCATTAAAAAAAATtACCtTAtACATACAtTACTAtTCAAAAtTTTttACATACT
+      ************************************************************
+
+ 5640 TAtAttTtTtttTTTTTCTTTATTTTTACTATTTTCTACATTtTAtCATAATAtTtAAtA
+      ************************************************************
+
+ 5700 CATAAACTATtAAATAACAAATATttAATCATtTAtTAACCAAAATAtTATTTAAAAAAT
+      ************************************************************
+
+ 5760 CTAAATATATTTCAtATTTTTCAAAtTAtCCACCCTTTtCCTCtATtACAtCTTTtCACT
+      ****>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>**************************
+
+ 5820 CTCTTttCATTCTCTCTTCACCTttAATtCTTTTCCAAtAtTtTtCAAAACTtTCATCAA
+      ************************************************************
+
+ 5880 ttCAAAtttTtACTACTTTAAAtAATtTCAAATATAAAATATATTTTtATTTtTTTTATA
+      ************************************************************
+
+ 5940 CTTTTTtttTTACTACATtATTCCATATtTtTTATTTAATAtTTTTtAAtTCTTCACTAT
+      ************************************************************
+
+ 6000 TATTCTACAATtTAtAAAATAtTAAAATAAAtAATCCCTttAATAAtTAttTtTtTCCAA
+      ************************************************************
+
+ 6060 ACTTTTtACTttTACTtTACtTtTTTttTATCATATAtCTAtTAtTTACAAAAtTAAACT
+      ************************************************************
+
+ 6120 CATTTAAAAtCTTtAAACtTAtAATTTtTCCCTCAtAATTATtTCCACTCTtAAAATCTA
+      *******************************<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
+
+ 6180 tTtTCTTATCATtATTTTCAAAtTAAAATATAAAACAAtTTAtTtAAtTAAAATTATATA
+      <***********************************************************
+
+ 6240 CAtAtATTACTtAtAAACATtCAtCAAAATCATCCCAtAAtAATTACATTAAATTtCATT
+      *****************************>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>*
+
+ 6300 CATAAAAAATTATtATtAtATtACTtACTCAATATTTttCATTtTTttATtAATtTTATt
+      ************************************************************
+
+ 6360 CAACATTCAAATttATTTCCCTCTTtTTAtTTAtTACCTtTAATTtttTTtTAttCCTTt
+      ************************************************************
+
+ 6420 CCtATtTTttTtATtACTCTACTtAAtATCAtTtTAtTAtCAtTACTtAATttACCTACt
+      ************************************************************
+
+ 6480 TTTCCAtAtTCAtTCAAACCAtCAtAtAAttCCACAtTTttTCTtCCTtTtAAAAtAAtT
+      ************************************************************
+
+ 6540 TtATTATTTAAAAttACAATCTtAtATTtAAATAACAACAAttCAAATtCTTACTTATCC
+      ************************************************************
+
+ 6600 ACCTtTATCAATCCAATTTACAAATtCCTtATATTTCTtACATtACCTtCTTTCTCTTTC
+      ************************************************************
+
+ 6660 TCCAtCTCTTCCACCTTtCTCTTCTttAtCTtCAtTTCAtTCTTttTtACtCTCAtCTCC
+      *************<<<<<<<<<<<<<<<<<<<<<<<<<<<<<******************
+
+ 6720 TtTCTCTtTTCCACCATCATtTCTCTCAtCTCCTCCAtCTTAtACCAtATtTCAttttTC
+      ***********************************>>>>>>>>>>>>>>>>>>>>>>>>>
+
+ 6780 TTtTtttCCtTCTCTtTAtCTTCAtCCCATtCCCCAAACAtAtAAAACAtCAtCAtAtCT
+      >>>>>*******************************************************
+
+ 6840 ACAtCACCTCTCATTCTtAAACtACACATCTTCTtAAATtATCTAtTCTTTtAttCTAtA
+      ************************************************************
+
+ 6900 AATTTACAttATTAAAtTAttAtCTATATCTtTtTtAAATATATTtTtTCTCTtTttAAA
+      **********************************************************  
+
+ 6960 AAAtAtttTTCTTAAATATCAtttAtTATTATTTtATTAttATTACTttTAttATTCTTT
+                                                                  
+
+ 7020 ATTTtTTTTTACTttCAATtAttTtAAttTTTCTTtCtTCATTCACCAtTTtTTtTTACT
+                                                                  
+
+ 7080 tTATTTCtTCATACTTAATTTTtATAtTCCCCTAtATAtttAtTtttCtTACACATCACA
+                                                                  
+
+ 7140 tACAAACTAAAATttTCCACACAtACAtTtTttTAAAtAAttAAttCCAAAAAtATCATC
+         <<<<<<<<<<<<<<<<<<<<<<<<<<<<<                            
+
+ 7200 AAttACAACAACCACCCtAtCCACTtCCTtTTCACACTtCTATCATCCAtAAttTtAttT
+                                                                  
+
+ 7260 CAtTACAttTACATCAAtCTtttACCtAtAtATTtAtAAACAtCTTCTATCTCAAttCCA
+                                                                  
+
+ 7320 TCAtACTtCTAAACAtCAATCATTAACTtAtAtAtTCTtCTtCCCACATTtAtAACCAAT
+                                                                  
+
+ 7380 CACAttACACTTTAATAAATttATCACTAtTCACTTTAAACAATtCCACTTTAAATAATt
+                                                                  
+
+ 7440 tCACTTTAATAATtCTTAAATATCTTACATTACTCATTTCACATtTATATACTtTATTTT
+                                                                  
+
+ 7500 ATACCATCTACTtCACCTTtCCTATtCCtCTCtCATTAACATCTtCTAACCATtTtTATt
+                                                                  
+
+ 7560 TtACCAATAAtATTTtATTTtATATCTtTAAATtATCTACAtATtATCAAACTAAtAAtA
+                                                                  
+
+ 7620 AACTATtAACTtCAACTCTtTTtATATTCAACTtCTTtTTAtttTTATttCAAttTCtAA
+                                                                  
+
+ 7680 TTttCCtTCTtCCAAATttACTttACTTTTTTTTTTAttTttTTtAtCCttTCtAAATTt
+                                                                  
+
+ 7740 AAAAAAATTATATTATATACATATATACAAAATCCATATTtCtATAACAATAAATCtAAC
+                                                                  
+
+ 7800 AATAAtTTTATTACATTAATtTtCATCACAtTTTTTTTATTATCATTTAATCTATTACTA
+                                                                  
+
+ 7860 TTAtTTTtTTTtTAtTTTtTTTtTTAtCAAACTTATTTAATATCAAACTTCACTTTTCTC
+                                                                  
+
+ 7920 TAtCATAttCTACTTATCtTAATtAACtACATAAtCAAAATtCTTtCAATAAtTtATCtt
+                                                                  
+
+ 7980 AATttTTttTtTAtATAATTTTTtTTTTATCtTCCTAtCTtTAtTTCAAAtTCAAACtCt
+                                                                  
+
+ 8040 CCATCAtTtAtCCAtCCTATATTCCTACTAAAtTTtTtAtttAtAtACAtCTAAATtAtC
+                                                                  
+
+ 8100 ttAtTtAATTATttATtATtAttACAAAtAACCTtATTCTtTtTtCAAAtACAtTTCAAC
+                                                                  
+
+ 8160 TtCAtCCAAAttAACAtAAttTAATTCTtTCtAttTTtAAAACAtAtAtACATACTtACA
+                                                                  
+
+ 8220 tAtATCTCTAtTTttATtTCAAATtTTTTTtCATttACATTtCCATTtAtttCTTCCACC
+                                                                  
+
+ 8280 ATTTTAAAtTAtTCAACTtttTttttATTTCTATttATTtAtAATtATTAtCCAATtATC
+                                                                  
+
+ 8340 AtAtAATTATCTTAATCTTCAATTTttATTtCTTATttCTtTACATttCTTTAAtCTTCA
+                                                                  
+
+ 8400 TCACCACCATtAAtTttCCACAATAATTtAATtACAttACTCCAtCACTtCtttTttCAt
+                                                                  
+
+ 8460 TAAATCACCAATTTtTTATTTAACACtTTCTCATTTACAtCAACtACCTttttAATAtTT
+                                                                  
+
+ 8520 ACAttttAtAttAtttttATtAATTAtCCAATTtTAAACTttttATtATTAttTtACCAT
+                                                                  
+
+ 8580 tATttTATtAtttCCAtATTtttAATTTAtCCAtttCTAAACACCCCTACTCTTATAATA
+                                                                  
+
+ 8640 ATAATAATAATAATAATAATAATAATAAATtCCATtTtACCACAtAtTCAttACACCCAT
+                                                                  
+
+ 8700 TTAACATCCCATCCAAAAtACAtCAACCTACACAtttCAATtTCCCCAATCACTtCCTTt
+                                                                  
+
+ 8760 tttAATTtttATATTTTTTAtAtCAtACtAAAtAtTtCCTCCTACTttCCCtTCCAACAC
+                                                                  
+
+ 8820 CACTCCCAtCAtCATCTttTCTCCCATCCAtttCCCAACCTTtCTTAtCTTCAtAAtCAA
+                                                                  
+
+ 8880 tCCAtCAtTtttATttAtttTTtTATtCTTCTttCAAtCTAATtTTTAtTCCTtAACTTA
+                                                                  
+
+ 8940 TTCAttCTTtCCATAACAAAttttTTtAATACTTTTTtACTCAAtACATTTCAtCTTTTC
+                                                                  
+
+ 9000 ATTtTAAATACATTTtTAAAAATAAAAAAAATCCAACTAAATTCCACTTTtACATTtTAt
+                                                                  
+
+ 9060 ttTAATATtTtTAtATCAtTtACACAACATCTACATTTAATCAACTATATATTCAttCTt
+                                                                  
+
+ 9120 TCACACAACAAAATtTttAAAAAtTCAAttttAtTtAATACTTTTTTTTATAtCTTTtTT
+                                                                  
+
+ 9180 ATAAACATTATACTACATTATAATAACAACTTtCCAAAAAACTTtAAATttAAACAAATA
+                                                                  
+
+ 9240 tAATTTTACAACAAAATCAATtTTTCTtttACAAAATAttATTAtTtACAtCTtTAATTA
+                                                                  
+
+ 9300 AAttAAtATCTTCTttCTACAtTAACAATtACAtCTACATAAAAAAAAAAAAAAAAAtCT
+                                                                  
+
+ 9360 AtTCAttTAACTACtTAtTTAATtTTtCAATTtTTTACAtCTTTCAtATTTtCTATCATt
+                                                                  
+
+ 9420 AATTTtTTAAttCCAAtttTAAtAAttAAAATttCATtAATTttAACCTAATtACTAACT
+                                                                  
+
+ 9480 CCTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTt
+                                                                  
+
+ 9540 TtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTt
+                                                                  
+
+ 9600 TtTtTtTtTtTtTtTtTtTtTtTtTtCATACATtTTCATCTATCACTTTtTtTAtTTAtC
+                                                                  
+
+ 9660 TAtCAATTttCTACTAATtATTTtCCTtTCTACCCAtACTCCTtTCTACCCAtACtCCAA
+                                                                  
+
+ 9720 ACtTTATtCCACtCCCACttACtTTAtTTTATtTAtTtAACtACttAtCAtCtAtTtTtA
+                                                                  
+
+ 9780 tTCtTCAttCTAATTAATAtTCCTACTtCAACTtTCTTtATTATtAtTAtATCTACAAAt
+                                                                  
+
+ 9840 TATtCCTTCCTttCAttATtATATAATTAATATTATTTtTATCAATTtttTCtttATTTt
+                                                                  
+
+ 9900 TTTAACAAACTCTtCCATCtTttTAtTCTtCAtAAACATTtCTAtCCAAACACAtTCCTC
+                                                                  
+
+ 9960 TCTCtCTAtttttTTTATTCAtACCCAAtTTATACCCACATAAATATAATTtTATTCTCT
+                                                                  
+
+10020 TTTTATtCACTTTTCTCTCTATtCtTATTCTtACCTTtAACTTACTCATttttACAAtCC
+                                                                  
+
+10080 AtTtTCAtCAAttTACCCTtttTtAttttTtATACtCCAtTtTATTCTtACCTTtACTTA
+                                                                  
+
+10140 ATTCCCTACTAATTCTACAACCTTTATCAttCTAATATTCTtAATATCCTtTTCTCTCtA
+                                                                  
+
+10200 TATAtTCCAtTtAtTTTtTCAACAAAATTCAAATtATAttTAAACCtTATATTCAAttAT
+                                                                  
+
+10260 ttATAAAtATAAATtTACTtAAAATCCTATTtAATAAAAACTCCACAAtAAAATCTtTTt
+                                                                  
+
+10320 tCCAACAAtTtttAtttTTTTCAtCttTTtAATTAAATTTATTCAtAtCACtCCCAtCAt
+                                                                  
+
+10380 CAAAtCCCCAtCAtCAAAtACCtTTACACTACTtCATAAttTAAtTCTtAtTttTCCAAA
+                                                                  
+
+10440 CACACCAAtAAAtTTtTt
+                        
+
+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       3265   30   54.89   23.33    0.00   0.00    0.00 ttATATtTCTtAACtACTAACCCTtAtACA
+ 2 LEFT_PRIMER       3790   30   55.04   23.33    0.00   0.00    0.00 CCCAACTtTATTTCTTTCTACATTATATAt
+ 3 LEFT_PRIMER       4255   30   55.30   20.00    0.00   0.00    0.00 CTtACACTtAAAAACACCAAAAtAAAtATt
+ 4 LEFT_PRIMER       4764   30   55.09   23.33    0.00   0.00    0.00 ACTtAttCCTtTACTCTttAtCtttATCtA
+ 5 LEFT_PRIMER       5294   30   55.72   23.33    0.00   0.00    0.00 CAtttACACATTATTCCATTTCTtTTAtTC
+ 6 LEFT_PRIMER       5764   30   54.46   20.00    0.00   0.00    0.00 ATATATTTCAtATTTTTCAAAtTAtCCACC
+ 7 LEFT_PRIMER       6269   30   55.20   20.00    0.00   0.00   31.63 TCATCCCAtAAtAATTACATTAAATTtCAT
+ 8 LEFT_PRIMER       6755   30   54.98   20.00    0.00   0.00    0.00 CAtCTTAtACCAtATtTCAttttTCTTtTt
+
+                     start  len      tm     gc%  any_th  3'_th hairpin seq
+ 1 RIGHT_PRIMER      3677   30   55.32   20.00    0.00   0.00   42.14 aaTTaAGTGGaTTaaTGaTaTTaaaAGTGT
+ 2 RIGHT_PRIMER      4194   30   54.81   20.00    0.00   0.00    0.00 TAAGaTGTTAGTGTaTTAAaGAaaGTTaaA
+ 3 RIGHT_PRIMER      4675   29   59.24   34.48    0.00   0.00    0.00 GTTTGTGaaaATAGGaGAaGTAGTTGaaG
+ 4 RIGHT_PRIMER      5202   30   57.40   23.33    0.00   0.00    0.00 AaTGaAaaAGATTTGaaAGTTaTTGaTGTG
+ 5 RIGHT_PRIMER      6180   30   55.05   23.33    0.00   0.00    0.00 aTAGATTTTaAGAGTGGAaATAATTaTGAG
+ 6 RIGHT_PRIMER      6701   29   57.64   31.03    0.00   0.00    0.00 GAaTGAAaTGaAGaTaaAGAAGAGaAAGG
+ 7 RIGHT_PRIMER      7171   29   56.70   20.69    2.75   0.00    0.00 aAaAaTGTaTGTGTGGAaaATTTTAGTTT
+
+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    3303     0     0     0     0   974     0     9   185      0     0     0    61     0     8
+Right   3607     0     0     0     0  1486     0     3   460      0     0     0    34     0     7
+Pair Stats:
+considered 0, ok 0
+libprimer3 release 2.6.1
+

Looks like we have two primers we can use for sequencing. Neither overlaps a potential CpG, and both are outside of the C1q gene sequence.

+
+
+
+
+

6 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. +
+
+Li, Heng, Bob Handsaker, Alec Wysoker, Tim Fennell, Jue Ruan, Nils Homer, Gabor Marth, Goncalo Abecasis, and Richard Durbin. 2009. “The Sequence Alignment/Map Format and SAMtools.” Bioinformatics 25 (16): 2078–79. https://doi.org/10.1093/bioinformatics/btp352. +
+
+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. +
+
+ + +
+ +
+ +
+ + + + + \ No newline at end of file diff --git a/docs/search.json b/docs/search.json index 51e8b2237..4b6f87f64 100644 --- a/docs/search.json +++ b/docs/search.json @@ -12073,5 +12073,68 @@ "title": "Daily Bits - July 2024", "section": "20240701", "text": "20240701\n\nIn lab\n\nFinished the last of the project-gigas-carryover (GitHub Repo) RNA isolation and quantification (Notebook)." + }, + { + "objectID": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html", + "href": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html", + "title": "Primer Design - Lake Trout C1q Gene Bisulfite Sequencing Primers Using Primer3", + "section": "", + "text": "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)), after bisulfite treatment.\nThis process also utilizes pyfaidx (Shirley et al. 2015) and samtools (Li et al. 2009).\nThis 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)), after bisulfite treatment.\nThis process also utilizes pyfaidx (Shirley et al. 2015) and samtools (Li et al. 2009)." + }, + { + "objectID": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#download-the-actual-files", + "href": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#download-the-actual-files", + "title": "Primer Design - Lake Trout C1q Gene Bisulfite Sequencing Primers Using Primer3", + "section": "2.1 Download the actual files", + "text": "2.1 Download the actual files\n# Load bash variables into memory\nsource .bashvars\n\nfor file in ${ncbi_gff_gz} ${ncbi_fasta_gz} ${ncbi_md5sums}\ndo\n wget \\\n --no-check-certificate \\\n --continue \\\n --quiet \\\n --directory-prefix=${data_dir} \\\n ${ncbi_url}${file}\ndone\n\nls -lh \"${data_dir}\"\ntotal 660M\n-rw-r--r-- 1 sam sam 642M Jan 13 2021 GCF_016432855.1_SaNama_1.0_genomic.fna.gz\n-rw-r--r-- 1 sam sam 18M Sep 28 2022 GCF_016432855.1_SaNama_1.0_genomic.gff.gz\n-rw-r--r-- 1 sam sam 33K Jul 26 06:11 md5checksums.txt" + }, + { + "objectID": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#check-md5-checkums", + "href": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#check-md5-checkums", + "title": "Primer Design - Lake Trout C1q Gene Bisulfite Sequencing Primers Using Primer3", + "section": "2.2 Check MD5 Checkums", + "text": "2.2 Check MD5 Checkums\n# Load bash variables into memory\nsource .bashvars\n\ncd \"${data_dir}\"\n\nfor file in *.gz\ndo\n grep \"${file}\" ${ncbi_md5sums} | md5sum -c -\ndone\n./GCF_016432855.1_SaNama_1.0_genomic.fna.gz: OK\n./GCF_016432855.1_SaNama_1.0_genomic.gff.gz: OK" + }, + { + "objectID": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#decompress-ncbi-files", + "href": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#decompress-ncbi-files", + "title": "Primer Design - Lake Trout C1q Gene Bisulfite Sequencing Primers Using Primer3", + "section": "2.3 Decompress NCBI files", + "text": "2.3 Decompress NCBI files\n# Load bash variables into memory\nsource .bashvars\n\ncd \"${data_dir}\"\n\nfor file in *.gz\ndo\n gunzip \"${file}\"\ndone\n\nls -lh\ntotal 2.6G\n-rw-r--r-- 1 sam sam 2.3G Jan 13 2021 GCF_016432855.1_SaNama_1.0_genomic.fna\n-rw-r--r-- 1 sam sam 373M Sep 28 2022 GCF_016432855.1_SaNama_1.0_genomic.gff\n-rw-r--r-- 1 sam sam 33K Jul 26 06:11 md5checksums.txt" + }, + { + "objectID": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#peek-at-gff", + "href": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#peek-at-gff", + "title": "Primer Design - Lake Trout C1q Gene Bisulfite Sequencing Primers Using Primer3", + "section": "3.1 Peek at GFF", + "text": "3.1 Peek at GFF\n# Load bash variables into memory\nsource .bashvars\n\nawk '$3==\"gene\"' \"${data_dir}/${genome_gff}\" | grep \"${sequence_ID}\"\nNC_052339.1 Gnomon gene 19244556 19248013 . - . ID=gene-LOC120027825;Dbxref=GeneID:120027825;Name=LOC120027825;gbkey=Gene;gene=LOC120027825;gene_biotype=protein_coding" + }, + { + "objectID": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#create-fasta-index", + "href": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#create-fasta-index", + "title": "Primer Design - Lake Trout C1q Gene Bisulfite Sequencing Primers Using Primer3", + "section": "3.2 Create FastA index", + "text": "3.2 Create FastA index" + }, + { + "objectID": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#format-region-and-extract-c1q-sequence-as-fasta.", + "href": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#format-region-and-extract-c1q-sequence-as-fasta.", + "title": "Primer Design - Lake Trout C1q Gene Bisulfite Sequencing Primers Using Primer3", + "section": "3.3 Format region and extract C1q sequence as FastA.", + "text": "3.3 Format region and extract C1q sequence as FastA.\n# Load bash variables into memory\nsource .bashvars\n\n# Format region for use with pyfaidx\nregion=$(awk '$3==\"gene\"' \"${data_dir}/${genome_gff}\" | grep \"${sequence_ID}\" | awk '{print $1\":\"$4\"-\"$5}')\n\necho \"${region}\"\necho \"\"\n\n# Extract region\n${pyfaidx} \"${data_dir}/${genome_fasta}\" \"${region}\" | tee \"${output_top}/${c1q_fasta}\"\nNC_052339.1:19244556-19248013\n\n>NC_052339.1:19244556-19248013\nTGGGATGTGTAATGGTCATATTTATTACCATACCTCTCCACTTCTTCCTCTTTCCctttacaatttttattttttttaaa\ntaataaagtGATGATTTATTTATTGGTTAAAGAGGAAAATCCTCACATCACAGAATGGTGCTGTCCTCACACTGGGAAGA\nGCAGGAAGCCACTGAAGGTGTTGTGGTTATCATGGCTATCATGGAGACCCTGGTCTTCAGGGAGACGAAGGTAGACCACA\nTCCTCCTTCTCCAGCTCTAGTGTCAACGCATTAGATATGTACTGCCAACCCCCAACTGTATTTCTTTCTACATTATATAG\nAACTCTTTGATTATTGTGAAACATCATGATGCCCATGAGTTGTGGGTGGCGGGATGACATGGCCGTGAATCTGAAGAAGT\nAGACTCCTTTCACTGATGCTGTGAAGATGCCTGCATCAGAGGAGAAATAGAGTGATATCAGCACTGTCTGTCTCATGTTG\nCTGTAGCTGGAATGTTTTtaacaaataaactcagcaaaaaaagaaatgtcccttttcaggaccctgtctttcaaagataa\nttcgtaaaaatccaaataacttcacagatcttcattttaaagggtttaaacactgtttcccatgcttcttcaatgaacca\ntaaacaattaatgaacatgcacctgtggaacggtcgttaagacactaacagcttacagacggtaggcagttAGGCcaaag\nttatgaaaacttaggacactaaagaggcctttctactgacactgaaaaacaccaaaagaaagatgtccagggtccctgct\ncatctgcaggaacgtgccttagacatgctgcaaggaggcatgaggactacagatgtggccagggcaataaattgcaatgt\nccgtactgtgagacacctaacacagcgctacagggagacaggacggacagctgatcgtcctcgcagtgacagaccacgtg\ntaacaacacctgcacaggatcggtacatccgaacatcacacctgcgggacaggtaccggatggcaacaacaactgcccga\ngttacaccaggaacgcacaatccctccatcagtgctcagactgtctgcaataggctaagagaggttggactgagggcttg\ntaggcctgttgtcaGGCAGGTCATCACCGGCAActacgtcgcctatgggcacaaacccaccgtcactggaccagacagcc\ngcggttttgtctcaccaggggtgatggtcggaattgcgtttatcgttgaaggaatgagcgttacactgaggcctgtactc\ntggagcgggatcgatttggaggtggagggtccgtcatggtctggggcggtgtatcacagcatcatcggactgagcttgtt\ngtctttgcaggaaatctcaacgctgtgcgttacagggaagacatcctcctccctcatatggtacccttcctgcaggctca\ntcctgacatgaccctccagcatgacaatgccaccagccatactgctcatgttgtgcgtgatttcctgcaagacaggaatg\ntcagtgttctgccatggccagcgaagagcccagatctcaatcccattgagcacgtctgggaccttttggatcggagggtg\nagagctagggccattcgccacagaaatgtctgggaacttgcaggtgccttggtggaagagtggggtaacatctcacagca\nagaactggcaaatctggtgcagtccatgaggaggagatgcactgcagtacttaatgcagctggtggccacaccagatact\ngactgttacttttgattttgaccccacctttgttcagggacacattattccatttctgttagtcacatgtctgtggaact\ntgttcagtttatgtctcagttgttgaatattgttatgttcatacaaatatttacacatgttaagttgactgaaaataaac\ngcagttgacagtgagaggacgtttcttttaaCAGTGAGAGGAGTTTAGTATAATTAAGTCAGGGACTAGGTAACACAAAT\nTACCACCAAACACAATAAGTATCAAACTAAACATTGTTATAACTAAAATAAAATCAGGGTGACAGATTAAGTAACATAAA\nATATGTCATTAAAAAAAATGACCGTAgacatacagtactagtcaaaagtttggacatacttagaggtgtgggtttttctt\ntatttttactattttctacattgtagcataatagtgaagacataaactatgaaataacaaatatggaatcatgtagtaac\ncaaaatagtatttaaaaaatctaaatatatttcagatttttcaaagtagccaccctttgcctcgatgacagctttgcact\nctcttggcattctctcttcacctggaatgcttttccaagagtgtgcaaaactgtcatcaaggcaaagggtgactacttta\naagaatgtcaaatataaaatatattttgatttgttttatactttttgggttactacatgattccatatgtgttatttaat\nagttttgaagtcttcactattattctacaatgtagaaaatagtaaaataaagaatccctggaataagtaggtgtgtccaa\nacttttgactggtactgtacgtgttTGGTATCATATAGCTAGTAGTTACAAAAGTAAACTCATTTAAAAGCTTGAAACGT\nAGAATTTGTCCCTCAGAATTATGTCCACTCTGAAAATCTAGTGTCTTATCATGATTTTCAAAGTAAAATATAAAACAAGT\nTAGTGAAGTAAAATTATATACAGAGATTACTGAGAAACATGCAGCAAAATCATCCCAGAAGAATTACATTAAATTGCATT\nCATAAAAAATTATGATGAGATGACTGACTCAATATTTGGCATTGTTGGATGAATGTTATGCAACATTCAAATGGATTTCC\nCTCTTGTTAGTTAGTACCTGTAATTGGGTTGTAGGCCTTGCCGATGTTGGTGATGACTCTACTGAAGATCAGTGTAGTAG\nCAGTACTGAATGGACCTACGTTTCCAGAGTCAGTCAAACCAGCAGAGAAGGCCACAGTTGGTCTGCCTGTGAAAAGAAGT\nTGATTATTTAAAAGGACAATCTGAGATTGAAATAACAACAAGGCAAATGCTTACTTATCCACCTGTATCAATccaattta\ncaaatgcctgatATTTCTGACATGACCTGCTTTCTCTTTCTCCAGCTCTTCCACCTTGCTCTTCTGGAGCTGCAGTTCAG\nTCTTGGTGACGCTCAGCTCCTGTCTCTGTTCCACCATCATGTCTCTCAGCTCCTCCAGCTTAGACCAGATGTCAGGGGTC\nTTGTGGGCCGTCTCTGTAGCTTCAGCCCATGCCCCAAACAGAGAAAACAGCAGCAGAGCTACAGCACCTCTCATTCTGAA\nACGACACATCTTCTGAAATGATCTAGTCTTTGAGGCTAGAAATTTACAGGATTAAAGTAGGAGCTATATCTGTGTGAAAT\nATATTGTGTCTCTGTGGA" + }, + { + "objectID": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#bisulfite-conversions", + "href": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#bisulfite-conversions", + "title": "Primer Design - Lake Trout C1q Gene Bisulfite Sequencing Primers Using Primer3", + "section": "5.1 Bisulfite conversions", + "text": "5.1 Bisulfite conversions\nIn this step we convert all lowercase to uppercase. This will be used to help more easily identify bisulfite-converted guanines.\nWe create two versions of bisuflte conversions:\n\nG -> t\nG -> N\n\nThe first conversion is a “realistic” representation of G -> U (t). We use a lowercase t to make them stand out.\nThe second conversion is specifically geared toward using Primer3 and setting a maximum number of “Ns” allowed in the primer annealing site, to help avoid primer designs which might cross a methylated CpG site (i.e. a site where bisulfite conversion would NOT take place, due to a methyl group being present).\n# Load bash variables into memory\nsource .bashvars\n\ntr '[:lower:]' '[:upper:]' < \"${output_top}/${c1q_buffer_fasta}\" | sed 's/G/t/g' > \"${output_top}/${c1q_bisulfite_t_fasta}\"\n\n\ntr '[:lower:]' '[:upper:]' < \"${output_top}/${c1q_buffer_fasta}\" | sed 's/G/N/g' > \"${output_top}/${c1q_bisulfite_N_fasta}\"\n\nhead ${output_top}/*BS_conversion*.fasta\n==> ../output/LOC120027825-BS_conversion-N-3500bp.left-3500bp.right.fasta <==\n>NC_052339.1:19241056-19251513\nTNNNNCTCNCCANNACAANNNNANCCCTNAAAANATNTNAANTTACACCCCANCTACCCANTCACCNTCTTTCACTATCC\nNCAACCCTCCACTNNNACAATCNTCAACAATACATTCAANCCTTCATNTACTCCNNNNCCNCNAATAATTTCANNNACCA\nANACTTCNCCANCNAATTACAAATCCCCTNCNTNAAATNTCCCATCCCTCTCCATCTNCTCCNNCCANTTNNNNTATCAN\nATCAANCCCATTCTACTNCANNTTNNNNTNAACCACTCANACTCTTANTTTTCTTTTNATCACTNCTCCCNANAACCTNC\nTTATCCTANNNTACCCCTNNCTANTNCTACATAACCCACAATTTTTCTNNTCCACNNNACACATNCTANACTNNNNTTNT\nTCAACATATNACATTAAAATNACANATCNCTATAATANTTNTACACTCATTAAANNCCCANTNCANTCAACNTTATNTNA\nTCANTNTTATTTCCTNATANTTNCTNNTTNAAAATACAATCTACACTNNACCTTTTAATCANCNNNTTTNCATNNNTNNN\nANTTTTNNCTTTCCATNATNACATCACCATNCNNTAAATTNATTAATANACCAATAACANANTTCCAAACTTCTTTNCCA\nATAACANCAATTTTTCANTTTTTCCCTCCCCACTCAAACCACTCCCANACANTCCTTNCAAAATTCTTNTTTNTNAATNT\n\n==> ../output/LOC120027825-BS_conversion-t-3500bp.left-3500bp.right.fasta <==\n>NC_052339.1:19241056-19251513\nTttttCTCtCCAttACAAttttAtCCCTtAAAAtATtTtAAtTTACACCCCAtCTACCCAtTCACCtTCTTTCACTATCC\ntCAACCCTCCACTtttACAATCtTCAACAATACATTCAAtCCTTCATtTACTCCttttCCtCtAATAATTTCAtttACCA\nAtACTTCtCCAtCtAATTACAAATCCCCTtCtTtAAATtTCCCATCCCTCTCCATCTtCTCCttCCAtTTttttTATCAt\nATCAAtCCCATTCTACTtCAttTTttttTtAACCACTCAtACTCTTAtTTTTCTTTTtATCACTtCTCCCtAtAACCTtC\nTTATCCTAtttTACCCCTttCTAtTtCTACATAACCCACAATTTTTCTttTCCACtttACACATtCTAtACTttttTTtT\nTCAACATATtACATTAAAATtACAtATCtCTATAATAtTTtTACACTCATTAAAttCCCAtTtCAtTCAACtTTATtTtA\nTCAtTtTTATTTCCTtATAtTTtCTttTTtAAAATACAATCTACACTttACCTTTTAATCAtCtttTTTtCATtttTttt\nAtTTTTttCTTTCCATtATtACATCACCATtCttTAAATTtATTAATAtACCAATAACAtAtTTCCAAACTTCTTTtCCA\nATAACAtCAATTTTTCAtTTTTTCCCTCCCCACTCAAACCACTCCCAtACAtTCCTTtCAAAATTCTTtTTTtTtAATtT" + }, + { + "objectID": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#design-primers", + "href": "posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html#design-primers", + "title": "Primer Design - Lake Trout C1q Gene Bisulfite Sequencing Primers Using Primer3", + "section": "5.2 Design primers", + "text": "5.2 Design primers\nQuick explanation: Primer3 requires a specially formatted input file. The file must be formatted similarly to this:\nSEQUENCE_ID=${seq_id}\nSEQUENCE_TEMPLATE=${sequence}\nPRIMER_TASK=generic\nPRIMER_PICK_LEFT_PRIMER=3\nPRIMER_PICK_RIGHT_PRIMER=3\nPRIMER_OPT_SIZE=18\nPRIMER_MIN_SIZE=15\nPRIMER_MAX_SIZE=21\nPRIMER_MAX_NS_ACCEPTED=1\nPRIMER_PRODUCT_SIZE_RANGE=75-150\nP3_FILE_FLAG=1\nPRIMER_EXPLAIN_FLAG=1\n=\nValues 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.\nThe 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-t-params.txt):\ncat << EOF > ${output_top}/primer3-t-params.txt\nEOF\nPrimer3 is run with the --format_output to make a nice, human-readable output format.\n\n5.2.1 Bisulfite lowercase t\nWe’ll use the bisulfite-converted sequence with the lowercase t to examine potential primers.\nThis will allow us to quickly identify any potential CpG that primers might anneal to (e.g. Ct).\nAdditionally, due to the low complexity of the bisulfite-converted sequence (very few G, due to conversion to U/T), I’ve changed the parameters to look for longer primers and lower melting temps than the default settings.\nI’ve also set Primer3 to look for sequencing primers and have defined the SEQUENCE_TARGET.\n# Load bash variables into memory\nsource .bashvars\n\n# Get sequence only, by skipping the first record\n# Remove newlines so sequence is on a single line\nc1q_sequence=$(awk 'NR > 1' \"${output_top}/${c1q_fasta}\" | tr -d '\\n')\n\n# Determine length of C1q\nc1q_length=${#c1q_sequence}\n\n# Get sequence only, by skipping the first record\n# Remove newlines so sequence is on a single line\nc1q_buffer_sequence=$(awk 'NR > 1' \"${output_top}/${c1q_bisulfite_t_fasta}\" | tr -d '\\n')\n\n# Determine length of C1q with the 5'/3' buffer sequences included\nc1q_buffer_length=${#c1q_buffer_sequence}\n\n# Calculate combined length of 5'buffer regaion and C1q length\nc1q_buffer_and_seq_length=$((left_buffer + c1q_length))\n\n\n# Use heredoc to create Primer3 parameters file\ncat << EOF > ${output_top}/primer3-t-params.txt\nSEQUENCE_ID=${sequence_ID}\nSEQUENCE_TEMPLATE=${c1q_buffer_sequence}\nPRIMER_TASK=pick_sequencing_primers\nSEQUENCE_TARGET=${left_buffer},${c1q_length}\nPRIMER_MIN_TM=50\nPRIMER_OPT_TM=55\nPRIMER_PICK_LEFT_PRIMER=1\nPRIMER_PICK_RIGHT_PRIMER=1\nPRIMER_OPT_SIZE=30\nPRIMER_MIN_SIZE=25\nPRIMER_MAX_SIZE=36\nPRIMER_MAX_NS_ACCEPTED=0\nP3_FILE_FLAG=1\nPRIMER_EXPLAIN_FLAG=1\nPRIMER_THERMODYNAMIC_PARAMETERS_PATH=${primer3_config}\nPRIMER_NUM_RETURN=10\nPRIMER_PAIR_EXPLAIN=considered 0, ok 0\n=\nEOF\n\n\n# Run Primer3\n${primer3} \\\n--format_output \\\n--output=\"${output_top}/primer3-t-primers.txt\" \\\n\"${output_top}/primer3-t-params.txt\"\n\n\n# Pull out any primers falling outside of target\ntail -n 30 \"${output_top}/primer3-t-primers.txt\" \\\n| head -n -12 \\\n| awk -v left_buffer=\"$left_buffer\" -v target=\"$c1q_buffer_and_seq_length\" '$3 <= left_buffer || $3 >= target {print $0}'\n\necho \"\"\necho \"\"\necho \"----------------------------------------------------------------------------------\"\necho \"\"\necho \"Primer results file:\"\necho \"\"\necho \"\"\n\n# Print the full output file\ncat \"${output_top}/primer3-t-primers.txt\"\n start len tm gc% any_th 3'_th hairpin seq\n 1 LEFT_PRIMER 3265 30 54.89 23.33 0.00 0.00 0.00 ttATATtTCTtAACtACTAACCCTtAtACA\n\n start len tm gc% any_th 3'_th hairpin seq\n 7 RIGHT_PRIMER 7171 29 56.70 20.69 2.75 0.00 0.00 aAaAaTGTaTGTGTGGAaaATTTTAGTTT\n\n\n----------------------------------------------------------------------------------\n\nPrimer results file:\n\n\nPRIMER PICKING RESULTS FOR LOC120027825\n\nNo mispriming library specified\nUsing 0-based sequence positions\nWARNING: No right primer found in range 5634 - 5674\n\nSEQUENCE SIZE: 10458\nINCLUDED REGION SIZE: 10458\n\nTARGETS (start, len)*: 3500,3458\n 0 TttttCTCtCCAttACAAttttAtCCCTtAAAAtATtTtAAtTTACACCCCAtCTACCCA\n \n\n 60 tTCACCtTCTTTCACTATCCtCAACCCTCCACTtttACAATCtTCAACAATACATTCAAt\n \n\n 120 CCTTCATtTACTCCttttCCtCtAATAATTTCAtttACCAAtACTTCtCCAtCtAATTAC\n \n\n 180 AAATCCCCTtCtTtAAATtTCCCATCCCTCTCCATCTtCTCCttCCAtTTttttTATCAt\n \n\n 240 ATCAAtCCCATTCTACTtCAttTTttttTtAACCACTCAtACTCTTAtTTTTCTTTTtAT\n \n\n 300 CACTtCTCCCtAtAACCTtCTTATCCTAtttTACCCCTttCTAtTtCTACATAACCCACA\n \n\n 360 ATTTTTCTttTCCACtttACACATtCTAtACTttttTTtTTCAACATATtACATTAAAAT\n \n\n 420 tACAtATCtCTATAATAtTTtTACACTCATTAAAttCCCAtTtCAtTCAACtTTATtTtA\n \n\n 480 TCAtTtTTATTTCCTtATAtTTtCTttTTtAAAATACAATCTACACTttACCTTTTAATC\n \n\n 540 AtCtttTTTtCATtttTtttAtTTTTttCTTTCCATtATtACATCACCATtCttTAAATT\n \n\n 600 tATTAATAtACCAATAACAtAtTTCCAAACTTCTTTtCCAATAACAtCAATTTTTCAtTT\n \n\n 660 TTTCCCTCCCCACTCAAACCACTCCCAtACAtTCCTTtCAAAATTCTTtTTTtTtAATtT\n \n\n 720 TTTTTtTTtTTtCTAAAAAtCTATTCTTTCCCATTTTAATttAACTCTATTACAtTAATt\n \n\n 780 TACTTAATTtTTACCCAtAAATtATTTtATATTtTTATAAAAACttCTtCAtTttACCTT\n \n\n 840 TAATCTtCATCAAAATATCTtTTCAATAtAAATTtAtAAtATAAAATACACtCTCtCTtT\n \n\n 900 CTTTATTtACATttTCAtCTtTttATtCTTCCTTTttCTtAttAATtACACCATtTTttC\n \n\n 960 TTATtACCAttCATAtACCACTATCTAATCAACCTttACTCtttTAtACATAAAtATtAA\n \n\n 1020 TCCtttACACTCCAATTAtTATtATATTTTACtTTTCtTATttTATtTATTAtTTTtTtt\n \n\n 1080 ATtTCCATCATCCATTTTtTATtATATtTTACAAATTtCAATTCATACAATATtTTAAAA\n \n\n 1140 ATTtCTATTTtTACAATATtTTACtAATTTtTAAACTTATtAAATtTTATtAATTCTAAT\n \n\n 1200 TTtTTtTtttTAACATTAtTTACtTttTTAAAtCTAACATTAtCCTttTttCTAATtTTA\n \n\n 1260 tCTATtTTAttttTTAtttTCAtttTTtAAttTTAtAtTTtATtTttATATtAAtCTAtt\n \n\n 1320 tTTACttTTCAAATTAtAtTTtAtCTttAATtTtTACATAAAtCTAtttTTATttATTTT\n \n\n 1380 tATtAATTAtATCAtCCTttTCTCATAtACTAtACtTAAATCCttAtCAtATAtTtTtAt\n \n\n 1440 CTCAAAtTATTTttACAtTtACACATTTtTTtTTtTTTTAtCTCTtAtCTCCAtCACTTT\n \n\n 1500 ttATTTtAAATtATACTATTAttTTAtTtCtCAtACTtACAtCTTTAATTTtAtttTATT\n \n\n 1560 TTCATCCATATCAttTtAACCtTTTAtAAAAAtTtCTtTCACCAAAAtTATTttAACAAT\n \n\n 1620 TTTACTTATATtttTATTAAAtTAtTAAAAAtATAAtTATTTttACCCATATTCCTAtCA\n \n\n 1680 CtCAATtACTACATCAAtCtTtTtACTCTACACATTTtTTttATtCATTTtCTtTTTtTT\n \n\n 1740 TTAtTTATtTTTCAtATTATTTTtTtCCCAATAtAAACAAATttTAAATAATtTATTtTt\n \n\n 1800 TAATTTTttAtTCACTTTTAtAtAAAtTTACAtACtCATAAATATCATAACCCCCCAAAA\n \n\n 1860 ATtCTAAtCTCCCCTtTTATTtTAATttTtAtAttTTAtCATtTCTTttttTTATtATAT\n \n\n 1920 TTtTtCATCTtTtACTTTCTCACTCATCATTATTCACTATTCATTCATTATTATCTtTAA\n \n\n 1980 TCATttTAtCATCCACAATtTAtCAtTTTTTAtAAACCTATTCTTATTTACATTATTTTT\n \n\n 2040 TTAACTTAtCATtTTAtCTAACCCTAACCTTAATCCAACTCCTCCTAACTCCTTAACCTT\n \n\n 2100 CAAtTTTATTCAAAtTTTAtTCTTAtAAACACAtTtCATtAtAATTTAtCATtTtTTTCT\n \n\n 2160 tAAttAAAAACTTAAACtATtTCAAtCTCTCAtATAACACAAAACACTtttTtAtTTTTC\n \n\n 2220 CTTATTtCAttTCATTTttATCAtACTCTtTAAAACACTtTAttAAAACAACCCTtTTTt\n \n\n 2280 TTTTTTTttTCTCTTTTTATCttCATtAAATTtTtATAATATTtTAAttTTtttTTtTtC\n \n\n 2340 CtTttCttATATCTTTtTtttCTATACTCttCCTTtTCTtAttATttTAttTTttTttTT\n \n\n 2400 tAAtAAATCCCTCTAtTttTtTtttttCTtTtCTTTttCAAAtTtttTttttTTATATCC\n \n\n 2460 TTCCTtTTTttCCCTtTCCtttttTATCATCttATttttCCACAtTtTCTCCTtACCCCT\n \n\n 2520 CCTtTCTCAtTATTTATtCTtCAtTAtTTTATtTtTCttttttCTAtttTCAtTTTtTTA\n \n\n 2580 TATCTttAtTACTTCTCCTtTCTTATCCttTtTCCTtTtTtAATTTAAtTATtCTCTCTC\n \n\n 2640 TAATTCTCTCTTTCTTTCTCTCTCTCttAttACCTtAtCCCTAttACCATtCCTCAttAC\n \n\n 2700 TACCTttCACtATtACTCCTTtCTtTCCCCAtTCCACCTttCCtTtCTtCTtCTCCAtTT\n \n\n 2760 TCAACTtTTCTtCCTtTttCTATttAACCCTtACCTtTTCACCAtACtTtCTACCTtTCC\n \n\n 2820 CAtACCCtCTtTTTTCAACTCTCTAtAtACAtCAttAtCttTAtAtATACTCTTAATtAT\n \n\n 2880 CttCTATtAAAAtCCAACTtACATTTACTTCTtAttTtCTtACTTtCTtCACCCTCtACA\n \n\n 2940 ACTACTtTtATTATTATTATTTtACCATtCTttTCATTTTtAACATTTtAACATCTTttC\n \n\n 3000 CATtTTCTtTTATAATCTCCACCCttCACAtCCAAAAtAttACTttCCACCCCTCATAtC\n \n\n 3060 CTttTTCCTCTCTAttTTTCTTCCTAttTTTTttCCTTTCTAtttAtTTTTTCCTAtCCA\n \n\n 3120 CCtTtCTTCTACACCTtCATTtCTTtCTtTTTttttTTTTAttCTtttTTTCTtTACAtC\n \n\n 3180 ACTTTtAtATATCAtCTtATtTAAtAAtttCTATATAAATACATTTtATTTtATTTtATA\n \n\n 3240 AttTCACTAtTTACTTATTAAATATttATATtTCTtAACtACTAACCCTtAtACACATCA\n >>>>>>>>>>>>>>>>>>>>>>>>>>>>>> \n\n 3300 tAAAtCTtAtTTCACtTCATAATATTtTtATTATAATCACAATCCCTTCTTACACCACTA\n \n\n 3360 TAAATtTtTCTCTtTCCTTATCTtAtTCTCTtAATtTAtCCTATtTtTtACCTtAATtTA\n \n\n 3420 TCTtTTACCTTAATtTAATtTAtCCTACATtTtTTACTTtAAAAATAACCCTAAATTttT\n \n\n 3480 TCTTATTCCCAACACAtCTCTtttATtTtTAATttTCATATTTATTACCATACCTCTCCA\n ****************************************\n\n 3540 CTTCTTCCTCTTTCCCTTTACAATTTTTATTTTTTTTAAATAATAAAtTtATtATTTATT\n ************************************************************\n\n 3600 TATTttTTAAAtAttAAAATCCTCACATCACAtAATttTtCTtTCCTCACACTtttAAtA\n ************************************************<<<<<<<<<<<<\n\n 3660 tCAttAAtCCACTtAAttTtTTtTttTTATCATttCTATCATttAtACCCTttTCTTCAt\n <<<<<<<<<<<<<<<<<<******************************************\n\n 3720 ttAtACtAAttTAtACCACATCCTCCTTCTCCAtCTCTAtTtTCAACtCATTAtATATtT\n ************************************************************\n\n 3780 ACTtCCAACCCCCAACTtTATTTCTTTCTACATTATATAtAACTCTTTtATTATTtTtAA\n **********>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>********************\n\n 3840 ACATCATtATtCCCATtAtTTtTtttTttCtttATtACATttCCtTtAATCTtAAtAAtT\n ************************************************************\n\n 3900 AtACTCCTTTCACTtATtCTtTtAAtATtCCTtCATCAtAttAtAAATAtAtTtATATCA\n ************************************************************\n\n 3960 tCACTtTCTtTCTCATtTTtCTtTAtCTttAATtTTTTTAACAAATAAACTCAtCAAAAA\n ************************************************************\n\n 4020 AAtAAATtTCCCTTTTCAttACCCTtTCTTTCAAAtATAATTCtTAAAAATCCAAATAAC\n ************************************************************\n\n 4080 TTCACAtATCTTCATTTTAAAtttTTTAAACACTtTTTCCCATtCTTCTTCAATtAACCA\n ************************************************************\n\n 4140 TAAACAATTAATtAACATtCACCTtTttAACttTCtTTAAtACACTAACAtCTTACAtAC\n *************************<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<*****\n\n 4200 ttTAttCAtTTAttCCAAAtTTATtAAAACTTAttACACTAAAtAttCCTTTCTACTtAC\n *******************************************************>>>>>\n\n 4260 ACTtAAAAACACCAAAAtAAAtATtTCCAtttTCCCTtCTCATCTtCAttAACtTtCCTT\n >>>>>>>>>>>>>>>>>>>>>>>>>***********************************\n\n 4320 AtACATtCTtCAAttAttCATtAttACTACAtATtTttCCAtttCAATAAATTtCAATtT\n ************************************************************\n\n 4380 CCtTACTtTtAtACACCTAACACAtCtCTACAtttAtACAttACttACAtCTtATCtTCC\n ************************************************************\n\n 4440 TCtCAtTtACAtACCACtTtTAACAACACCTtCACAttATCttTACATCCtAACATCACA\n ************************************************************\n\n 4500 CCTtCtttACAttTACCttATttCAACAACAACTtCCCtAtTTACACCAttAACtCACAA\n ************************************************************\n\n 4560 TCCCTCCATCAtTtCTCAtACTtTCTtCAATAttCTAAtAtAttTTttACTtAtttCTTt\n ************************************************************\n\n 4620 TAttCCTtTTtTCAttCAttTCATCACCttCAACTACtTCtCCTATtttCACAAACCCAC\n ***************************<<<<<<<<<<<<<<<<<<<<<<<<<<<<<****\n\n 4680 CtTCACTttACCAtACAtCCtCttTTTTtTCTCACCAttttTtATttTCttAATTtCtTT\n ************************************************************\n\n 4740 TATCtTTtAAttAATtAtCtTTACACTtAttCCTtTACTCTttAtCtttATCtATTTttA\n ************************>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>******\n\n 4800 ttTttAtttTCCtTCATttTCTttttCttTtTATCACAtCATCATCttACTtAtCTTtTT\n ************************************************************\n\n 4860 tTCTTTtCAttAAATCTCAACtCTtTtCtTTACAtttAAtACATCCTCCTCCCTCATATt\n ************************************************************\n\n 4920 tTACCCTTCCTtCAttCTCATCCTtACATtACCCTCCAtCATtACAATtCCACCAtCCAT\n ************************************************************\n\n 4980 ACTtCTCATtTTtTtCtTtATTTCCTtCAAtACAttAATtTCAtTtTTCTtCCATttCCA\n ************************************************************\n\n 5040 tCtAAtAtCCCAtATCTCAATCCCATTtAtCACtTCTtttACCTTTTttATCttAtttTt\n ************************************************************\n\n 5100 AtAtCTAtttCCATTCtCCACAtAAATtTCTtttAACTTtCAttTtCCTTttTttAAtAt\n ************************************************************\n\n 5160 TttttTAACATCTCACAtCAAtAACTttCAAATCTttTtCAtTCCATtAttAttAtATtC\n *************<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<*****************\n\n 5220 ACTtCAtTACTTAATtCAtCTttTttCCACACCAtATACTtACTtTTACTTTTtATTTTt\n ************************************************************\n\n 5280 ACCCCACCTTTtTTCAtttACACATTATTCCATTTCTtTTAtTCACATtTCTtTttAACT\n **************>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>****************\n\n 5340 TtTTCAtTTTATtTCTCAtTTtTTtAATATTtTTATtTTCATACAAATATTTACACATtT\n ************************************************************\n\n 5400 TAAtTTtACTtAAAATAAACtCAtTTtACAtTtAtAttACtTTTCTTTTAACAtTtAtAt\n ************************************************************\n\n 5460 tAtTTTAtTATAATTAAtTCAtttACTAttTAACACAAATTACCACCAAACACAATAAtT\n ************************************************************\n\n 5520 ATCAAACTAAACATTtTTATAACTAAAATAAAATCAtttTtACAtATTAAtTAACATAAA\n ************************************************************\n\n 5580 ATATtTCATTAAAAAAAATtACCtTAtACATACAtTACTAtTCAAAAtTTTttACATACT\n ************************************************************\n\n 5640 TAtAttTtTtttTTTTTCTTTATTTTTACTATTTTCTACATTtTAtCATAATAtTtAAtA\n ************************************************************\n\n 5700 CATAAACTATtAAATAACAAATATttAATCATtTAtTAACCAAAATAtTATTTAAAAAAT\n ************************************************************\n\n 5760 CTAAATATATTTCAtATTTTTCAAAtTAtCCACCCTTTtCCTCtATtACAtCTTTtCACT\n ****>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>**************************\n\n 5820 CTCTTttCATTCTCTCTTCACCTttAATtCTTTTCCAAtAtTtTtCAAAACTtTCATCAA\n ************************************************************\n\n 5880 ttCAAAtttTtACTACTTTAAAtAATtTCAAATATAAAATATATTTTtATTTtTTTTATA\n ************************************************************\n\n 5940 CTTTTTtttTTACTACATtATTCCATATtTtTTATTTAATAtTTTTtAAtTCTTCACTAT\n ************************************************************\n\n 6000 TATTCTACAATtTAtAAAATAtTAAAATAAAtAATCCCTttAATAAtTAttTtTtTCCAA\n ************************************************************\n\n 6060 ACTTTTtACTttTACTtTACtTtTTTttTATCATATAtCTAtTAtTTACAAAAtTAAACT\n ************************************************************\n\n 6120 CATTTAAAAtCTTtAAACtTAtAATTTtTCCCTCAtAATTATtTCCACTCTtAAAATCTA\n *******************************<<<<<<<<<<<<<<<<<<<<<<<<<<<<<\n\n 6180 tTtTCTTATCATtATTTTCAAAtTAAAATATAAAACAAtTTAtTtAAtTAAAATTATATA\n <***********************************************************\n\n 6240 CAtAtATTACTtAtAAACATtCAtCAAAATCATCCCAtAAtAATTACATTAAATTtCATT\n *****************************>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>*\n\n 6300 CATAAAAAATTATtATtAtATtACTtACTCAATATTTttCATTtTTttATtAATtTTATt\n ************************************************************\n\n 6360 CAACATTCAAATttATTTCCCTCTTtTTAtTTAtTACCTtTAATTtttTTtTAttCCTTt\n ************************************************************\n\n 6420 CCtATtTTttTtATtACTCTACTtAAtATCAtTtTAtTAtCAtTACTtAATttACCTACt\n ************************************************************\n\n 6480 TTTCCAtAtTCAtTCAAACCAtCAtAtAAttCCACAtTTttTCTtCCTtTtAAAAtAAtT\n ************************************************************\n\n 6540 TtATTATTTAAAAttACAATCTtAtATTtAAATAACAACAAttCAAATtCTTACTTATCC\n ************************************************************\n\n 6600 ACCTtTATCAATCCAATTTACAAATtCCTtATATTTCTtACATtACCTtCTTTCTCTTTC\n ************************************************************\n\n 6660 TCCAtCTCTTCCACCTTtCTCTTCTttAtCTtCAtTTCAtTCTTttTtACtCTCAtCTCC\n *************<<<<<<<<<<<<<<<<<<<<<<<<<<<<<******************\n\n 6720 TtTCTCTtTTCCACCATCATtTCTCTCAtCTCCTCCAtCTTAtACCAtATtTCAttttTC\n ***********************************>>>>>>>>>>>>>>>>>>>>>>>>>\n\n 6780 TTtTtttCCtTCTCTtTAtCTTCAtCCCATtCCCCAAACAtAtAAAACAtCAtCAtAtCT\n >>>>>*******************************************************\n\n 6840 ACAtCACCTCTCATTCTtAAACtACACATCTTCTtAAATtATCTAtTCTTTtAttCTAtA\n ************************************************************\n\n 6900 AATTTACAttATTAAAtTAttAtCTATATCTtTtTtAAATATATTtTtTCTCTtTttAAA\n ********************************************************** \n\n 6960 AAAtAtttTTCTTAAATATCAtttAtTATTATTTtATTAttATTACTttTAttATTCTTT\n \n\n 7020 ATTTtTTTTTACTttCAATtAttTtAAttTTTCTTtCtTCATTCACCAtTTtTTtTTACT\n \n\n 7080 tTATTTCtTCATACTTAATTTTtATAtTCCCCTAtATAtttAtTtttCtTACACATCACA\n \n\n 7140 tACAAACTAAAATttTCCACACAtACAtTtTttTAAAtAAttAAttCCAAAAAtATCATC\n <<<<<<<<<<<<<<<<<<<<<<<<<<<<< \n\n 7200 AAttACAACAACCACCCtAtCCACTtCCTtTTCACACTtCTATCATCCAtAAttTtAttT\n \n\n 7260 CAtTACAttTACATCAAtCTtttACCtAtAtATTtAtAAACAtCTTCTATCTCAAttCCA\n \n\n 7320 TCAtACTtCTAAACAtCAATCATTAACTtAtAtAtTCTtCTtCCCACATTtAtAACCAAT\n \n\n 7380 CACAttACACTTTAATAAATttATCACTAtTCACTTTAAACAATtCCACTTTAAATAATt\n \n\n 7440 tCACTTTAATAATtCTTAAATATCTTACATTACTCATTTCACATtTATATACTtTATTTT\n \n\n 7500 ATACCATCTACTtCACCTTtCCTATtCCtCTCtCATTAACATCTtCTAACCATtTtTATt\n \n\n 7560 TtACCAATAAtATTTtATTTtATATCTtTAAATtATCTACAtATtATCAAACTAAtAAtA\n \n\n 7620 AACTATtAACTtCAACTCTtTTtATATTCAACTtCTTtTTAtttTTATttCAAttTCtAA\n \n\n 7680 TTttCCtTCTtCCAAATttACTttACTTTTTTTTTTAttTttTTtAtCCttTCtAAATTt\n \n\n 7740 AAAAAAATTATATTATATACATATATACAAAATCCATATTtCtATAACAATAAATCtAAC\n \n\n 7800 AATAAtTTTATTACATTAATtTtCATCACAtTTTTTTTATTATCATTTAATCTATTACTA\n \n\n 7860 TTAtTTTtTTTtTAtTTTtTTTtTTAtCAAACTTATTTAATATCAAACTTCACTTTTCTC\n \n\n 7920 TAtCATAttCTACTTATCtTAATtAACtACATAAtCAAAATtCTTtCAATAAtTtATCtt\n \n\n 7980 AATttTTttTtTAtATAATTTTTtTTTTATCtTCCTAtCTtTAtTTCAAAtTCAAACtCt\n \n\n 8040 CCATCAtTtAtCCAtCCTATATTCCTACTAAAtTTtTtAtttAtAtACAtCTAAATtAtC\n \n\n 8100 ttAtTtAATTATttATtATtAttACAAAtAACCTtATTCTtTtTtCAAAtACAtTTCAAC\n \n\n 8160 TtCAtCCAAAttAACAtAAttTAATTCTtTCtAttTTtAAAACAtAtAtACATACTtACA\n \n\n 8220 tAtATCTCTAtTTttATtTCAAATtTTTTTtCATttACATTtCCATTtAtttCTTCCACC\n \n\n 8280 ATTTTAAAtTAtTCAACTtttTttttATTTCTATttATTtAtAATtATTAtCCAATtATC\n \n\n 8340 AtAtAATTATCTTAATCTTCAATTTttATTtCTTATttCTtTACATttCTTTAAtCTTCA\n \n\n 8400 TCACCACCATtAAtTttCCACAATAATTtAATtACAttACTCCAtCACTtCtttTttCAt\n \n\n 8460 TAAATCACCAATTTtTTATTTAACACtTTCTCATTTACAtCAACtACCTttttAATAtTT\n \n\n 8520 ACAttttAtAttAtttttATtAATTAtCCAATTtTAAACTttttATtATTAttTtACCAT\n \n\n 8580 tATttTATtAtttCCAtATTtttAATTTAtCCAtttCTAAACACCCCTACTCTTATAATA\n \n\n 8640 ATAATAATAATAATAATAATAATAATAAATtCCATtTtACCACAtAtTCAttACACCCAT\n \n\n 8700 TTAACATCCCATCCAAAAtACAtCAACCTACACAtttCAATtTCCCCAATCACTtCCTTt\n \n\n 8760 tttAATTtttATATTTTTTAtAtCAtACtAAAtAtTtCCTCCTACTttCCCtTCCAACAC\n \n\n 8820 CACTCCCAtCAtCATCTttTCTCCCATCCAtttCCCAACCTTtCTTAtCTTCAtAAtCAA\n \n\n 8880 tCCAtCAtTtttATttAtttTTtTATtCTTCTttCAAtCTAATtTTTAtTCCTtAACTTA\n \n\n 8940 TTCAttCTTtCCATAACAAAttttTTtAATACTTTTTtACTCAAtACATTTCAtCTTTTC\n \n\n 9000 ATTtTAAATACATTTtTAAAAATAAAAAAAATCCAACTAAATTCCACTTTtACATTtTAt\n \n\n 9060 ttTAATATtTtTAtATCAtTtACACAACATCTACATTTAATCAACTATATATTCAttCTt\n \n\n 9120 TCACACAACAAAATtTttAAAAAtTCAAttttAtTtAATACTTTTTTTTATAtCTTTtTT\n \n\n 9180 ATAAACATTATACTACATTATAATAACAACTTtCCAAAAAACTTtAAATttAAACAAATA\n \n\n 9240 tAATTTTACAACAAAATCAATtTTTCTtttACAAAATAttATTAtTtACAtCTtTAATTA\n \n\n 9300 AAttAAtATCTTCTttCTACAtTAACAATtACAtCTACATAAAAAAAAAAAAAAAAAtCT\n \n\n 9360 AtTCAttTAACTACtTAtTTAATtTTtCAATTtTTTACAtCTTTCAtATTTtCTATCATt\n \n\n 9420 AATTTtTTAAttCCAAtttTAAtAAttAAAATttCATtAATTttAACCTAATtACTAACT\n \n\n 9480 CCTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTt\n \n\n 9540 TtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTt\n \n\n 9600 TtTtTtTtTtTtTtTtTtTtTtTtTtCATACATtTTCATCTATCACTTTtTtTAtTTAtC\n \n\n 9660 TAtCAATTttCTACTAATtATTTtCCTtTCTACCCAtACTCCTtTCTACCCAtACtCCAA\n \n\n 9720 ACtTTATtCCACtCCCACttACtTTAtTTTATtTAtTtAACtACttAtCAtCtAtTtTtA\n \n\n 9780 tTCtTCAttCTAATTAATAtTCCTACTtCAACTtTCTTtATTATtAtTAtATCTACAAAt\n \n\n 9840 TATtCCTTCCTttCAttATtATATAATTAATATTATTTtTATCAATTtttTCtttATTTt\n \n\n 9900 TTTAACAAACTCTtCCATCtTttTAtTCTtCAtAAACATTtCTAtCCAAACACAtTCCTC\n \n\n 9960 TCTCtCTAtttttTTTATTCAtACCCAAtTTATACCCACATAAATATAATTtTATTCTCT\n \n\n10020 TTTTATtCACTTTTCTCTCTATtCtTATTCTtACCTTtAACTTACTCATttttACAAtCC\n \n\n10080 AtTtTCAtCAAttTACCCTtttTtAttttTtATACtCCAtTtTATTCTtACCTTtACTTA\n \n\n10140 ATTCCCTACTAATTCTACAACCTTTATCAttCTAATATTCTtAATATCCTtTTCTCTCtA\n \n\n10200 TATAtTCCAtTtAtTTTtTCAACAAAATTCAAATtATAttTAAACCtTATATTCAAttAT\n \n\n10260 ttATAAAtATAAATtTACTtAAAATCCTATTtAATAAAAACTCCACAAtAAAATCTtTTt\n \n\n10320 tCCAACAAtTtttAtttTTTTCAtCttTTtAATTAAATTTATTCAtAtCACtCCCAtCAt\n \n\n10380 CAAAtCCCCAtCAtCAAAtACCtTTACACTACTtCATAAttTAAtTCTtAtTttTCCAAA\n \n\n10440 CACACCAAtAAAtTTtTt\n \n\nKEYS (in order of precedence):\n****** target\n>>>>>> left primer\n<<<<<< right primer\n^^^^^^ left primer / right primer overlap\n\n start len tm gc% any_th 3'_th hairpin seq\n 1 LEFT_PRIMER 3265 30 54.89 23.33 0.00 0.00 0.00 ttATATtTCTtAACtACTAACCCTtAtACA\n 2 LEFT_PRIMER 3790 30 55.04 23.33 0.00 0.00 0.00 CCCAACTtTATTTCTTTCTACATTATATAt\n 3 LEFT_PRIMER 4255 30 55.30 20.00 0.00 0.00 0.00 CTtACACTtAAAAACACCAAAAtAAAtATt\n 4 LEFT_PRIMER 4764 30 55.09 23.33 0.00 0.00 0.00 ACTtAttCCTtTACTCTttAtCtttATCtA\n 5 LEFT_PRIMER 5294 30 55.72 23.33 0.00 0.00 0.00 CAtttACACATTATTCCATTTCTtTTAtTC\n 6 LEFT_PRIMER 5764 30 54.46 20.00 0.00 0.00 0.00 ATATATTTCAtATTTTTCAAAtTAtCCACC\n 7 LEFT_PRIMER 6269 30 55.20 20.00 0.00 0.00 31.63 TCATCCCAtAAtAATTACATTAAATTtCAT\n 8 LEFT_PRIMER 6755 30 54.98 20.00 0.00 0.00 0.00 CAtCTTAtACCAtATtTCAttttTCTTtTt\n\n start len tm gc% any_th 3'_th hairpin seq\n 1 RIGHT_PRIMER 3677 30 55.32 20.00 0.00 0.00 42.14 aaTTaAGTGGaTTaaTGaTaTTaaaAGTGT\n 2 RIGHT_PRIMER 4194 30 54.81 20.00 0.00 0.00 0.00 TAAGaTGTTAGTGTaTTAAaGAaaGTTaaA\n 3 RIGHT_PRIMER 4675 29 59.24 34.48 0.00 0.00 0.00 GTTTGTGaaaATAGGaGAaGTAGTTGaaG\n 4 RIGHT_PRIMER 5202 30 57.40 23.33 0.00 0.00 0.00 AaTGaAaaAGATTTGaaAGTTaTTGaTGTG\n 5 RIGHT_PRIMER 6180 30 55.05 23.33 0.00 0.00 0.00 aTAGATTTTaAGAGTGGAaATAATTaTGAG\n 6 RIGHT_PRIMER 6701 29 57.64 31.03 0.00 0.00 0.00 GAaTGAAaTGaAGaTaaAGAAGAGaAAGG\n 7 RIGHT_PRIMER 7171 29 56.70 20.69 2.75 0.00 0.00 aAaAaTGTaTGTGTGGAaaATTTTAGTTT\n\nStatistics\n con too in in not no tm tm high high high high \n sid many tar excl ok bad GC too too any_th 3'_th hair- poly end \n ered Ns get reg reg GC% clamp low high compl compl pin X stab ok\nLeft 3303 0 0 0 0 974 0 9 185 0 0 0 61 0 8\nRight 3607 0 0 0 0 1486 0 3 460 0 0 0 34 0 7\nPair Stats:\nconsidered 0, ok 0\nlibprimer3 release 2.6.1\nLooks like we have two primers we can use for sequencing. Neither overlaps a potential CpG, and both are outside of the C1q gene sequence." } ] \ No newline at end of file diff --git a/docs/sitemap.xml b/docs/sitemap.xml index 3fd566f7e..3747db95f 100644 --- a/docs/sitemap.xml +++ b/docs/sitemap.xml @@ -6148,4 +6148,8 @@ https://robertslab.github.io/sams-notebook/index.html 2024-03-28T18:37:40.305Z + + https://robertslab.github.io/sams-notebook/posts/2024/2024-07-29-Primer-Design---Lake-Trout-C1q-Gene-Bisulfite-Sequencing-Primers-Using-Primer3/index.html + 2024-07-29T23:18:31.775Z +