This is manual for command line programs

See Manual for GUI-based program CpGPNP Manual Download
- Install perl and R package on your computer Perl: https://www.perl.org/get.html R package: https://www.r-project.org/

A. CpG Island predition

1. CpGPredictor.pl

[Usage] > perl CpGPredictor.pl -f INPUTFILE[FASTA] (-l INT -w INT -m STRING -g INT -o INT) -s OUTPUTFILE [Parameter] -f[--file] : INPUT file (FASTA format) -l[--searchlength] : Length of CpG island, [default 500] -w[--slide] : moving window, [default 1] -m[--motif] : motif which user searches -g[--GC] : Threshold of GC contents percent, [default 55] -o[--OBEX] : Threshold of Observed /Expected CpG Ratio, [default 0.65] -fa : make FASTA file, [y or yes] -s[--save] : OUTPUT file (.txt) [Example] For Gardiner-Garden and Frommer algorithm (1987), > perl CpGPredictor.pl -f test.fa -l 200 -g 50 -o 0.6 -s test_result * If you want to get a fasta file, type 'y' or 'yes' with the [-fa] option. [Result] .rawdata : all detected CpG islands results (1st result) .groups : the result grouped from redundant CpG islands of .rawdata (2nd result) .contig : CpG island results merged between the overlapped groups from .groups (Final result) .summary : result and parameter summary file .graph : Files to make graphs using R .contig result example:
No. START LAST Length GC_Contents obs/exp_CpG_ratio SEQEUNCE
11032711348102270.150.95taacccctaaccctaaccctaaccctaccctaaccctaaccctaaccctaacc
216469 16668 200 51.5 0.60 ACCAAGTAGAACAAGATATTTGAAATGGAAACTATTCAAAAAATTGAGAA
328625 29807 1183 70.07 0.84 ATTCTTTTTAAGTGACAAAACTTGTACATGTGTATCGCTC
451434 51995 562 54.80 0.86 GAATCACACGTATTGGAAAACCAGCGGAAGAGTAAGTCT
5121169 121614 446 51.34 0.64 GTGCAAGTAAAGAGCCTTACTGCTGATGAGGTTTGAGG

2. graphMake.pl

[Usage] >perl graphMake.pl test.graph [Result] .pdf : graph visualization pdf file

B. Primer design

1. standardPCR.pl

[Usage] >perl standardPCR.pl [Parameter] - FASTA file : fasta sequence file - START position : start of target region - END position : end of target region - PRIMER LENGTH (minimum, maximum) : set minimum/maximum primer length - Tm threshold [Result] .stdPrimer .stdPrimer result example:
CategoryForward/ReverseFirstPositionLastPosition5'-primer-3'TmGC(%)PrimerLength5'Position3'Position hair-pin_structureproduct_size
primer 1Forward150500CCTCTCAGCCTTTGAAAGAAA50.2442.8521129150...<.............>... 434
Reverse150500AACAGGAGGAGCAGAGAGCGAAGCG62.216025609584..<........<.>........>..
primer 2Forward150500AGCCTTTGAAAGAAAGAAAGGG52.0140.9022135157..<................>..427
Reverse150500AACAGGAGGAGCAGAGAGCGAAGCG62.216025609584..<........<.>........>..
primer 3Forward150500AGCCTTTGAAAGAAAGAAAGGG52.0140.9022135157..<................>..427
Reverse150500AACAGGAGGAGCAGAGAGCGAAGCG62.216025609584..<........<.>........>..

2. standardPCRmulti.pl

h4> [Usage] >perl standardPCRmulti.pl -p [POSITIONFILE] (-minlen [int] -maxlen [int] -mintm[int] -maxtm [int] -opt [int]) -r [FASTAFILE] -o [OUTPUTFILE] [Parameter] -p[--position] : the POSITION file(.groups, .contigs) resulted from CpGPredictor-2.0.pl -minlen[--minlength] : minimum primer length, [default 20] -maxlen[--maxlength] : maximum primer length, [default 30] -mintm[--mintm] : the minimum Tm, [default 50] -maxtm[--maxtm] : the maximum Tm, [default 65] -opt[--optTm] : the optimum Tm, [default 55] -r[--reference] : the REFERECE FASTA file -o[--output] : the OUTPUT file * The POSITION file can use the result file(.groups or .contig) obtained from CpGPrediction.pl, or user can manually make the POSITION file as follows start end 54 523 39 540 46 1707

3. bisulfitePCR

[Usage] >perl bisulfitePCR.pl [Parameter] - FASTA file : fasta sequence file - START position : start of target region - END position : end of target region - PRODUCT SIZE : target size. It determines the direction of the primer design - PRIMER LENGTH (minimum, maximum) : set minimum/maximum primer length - Tm threshold [Result] .bisPrimer .bisPrimer result example:
CategoryForward/ReverseOriginal_Sequence5'-Primer-3'TmGC(%)PrimerLength5'Position3'PositionEnthalpyEnergySalt_concent(M)hair-pin_structureproduct_size
primer 1ForwardCGAGAAAGAAGGTGAGAAAGACAGAGCYGAGAAAGAAGGTGAGAAAGATAGAGT55.9240.74266895205.637.150.05.....<......<.>......>..... 244
ReverseCCCCACGGGAACCGCCCGTGCACACRAACRATTCCCRTAAAA54.4942.8520360339178.532.70.05.........<.>.........
primer 2 ForwardCGAGAAAGAAGGTGAGAAAGACAGAGCYGAGAAAGAAGGTGAGAAAGATAGAGT55.9240.74266895205.637.150.05 .....<......<.>......>..... 244
ReverseCCCCACGGGAACCGCCCGTGCACACRAACRATTCCCRTAAAA54.4942.8520360339178.532.70.05.........<.>.........
primer 3ForwardCGAGAAAGAAGGTGAGAAAGACAGAGCYGAGAAAGAAGGTGAGAAAGATAGAGT55.9240.74266895205.637.150.05 .....<......<.>......>..... 130
ReverseCGCCGTAAGGTGCCGCAGTCCCGAATGCATTCRAAACTACRACACCTTACRACR59.2448.1426252225215.240.250.05..<...<<..<.....>..>>...>..

4. bisulfitePCRmulti

[Usage] >perl bisulfitePCRmulti -p [POSITIONFILE] (-minlen [int] -maxlen [int] -mintm[int] -maxtm [int] -t [int]) -r [FASTAFILE] -o [OUTPUTFILE] [Parameter] -p[--position] : the POSITION file(.groups, .contigs) resulted from CpGPredictor-2.0.pl -minlen[--minlength] : minimum primer length, [default 20] -maxlen[--maxlength] : maximum primer length, [default 30] -mintm[--mintm] : the minimum Tm, [default 50] -maxtm[--maxtm] : the maximum Tm, [default 65] -t[--threshold] : the threshold of product size, [default 300] -r[--reference] : the REFERECE FASTA file -o[--output] : the OUTPUT file * The POSITION file is the same as that used in standardPCR-2.0.pl

5. msp.pl

[Usage] >perl msp.pl -f [FASTAFILE] -s [int] -e [int] -a [int] -o OUTPUTFILE [Parameter] -f[--fasta] : Input your fasta file -s[--start] : Start Position where you want to design MSP in your FASTA file -e[--end] : End Position where you want to design MSP in your FASTA file -a[--amplicon] : amplicon size (default 100) -o[--output] : the output file * If you do not specify start and end position, map to the entire fasta file. [Result] The MSP program produces two results as follows. 1) .CpGMAP 2) .msp.result 1) .CpGMAP - CpGMAP is a file that maps CG dimer positions
Map Of CpG site: GTAGGCGAGTCGGACGTCGTTCGTAGTATCGGAGAGGGCGTATTGTAAAGGCGGGTAGTAGATCGTGGAGAGTTCGGGAG CGGAGTTGGATATCGTTTCGGAGGGAAGAAATGAGGTAGCGGCGGTTTTCGGATTCGGTTATGTTCGTTTTTTGTTTTCG GAGTTTAGCGTCGTTTCGGTTAGGTTAGTTCGGATATTGAGCGGGTCGAGCGCGAGTTTTCGGCGTTCGGCGGAGCGAAG ATGTAGTGAGTTTTCGCGGGATTGTTGCGCGGGGTTCGTCGCGGTTAGTCGGATTTAGTATTCGATCGTATTTTGGGCGA GTTGTTGATTTGAGATTAGTTTAAACGGGGGGTTTTTTATTTTTAGTATTTTTCGGAGGTGGGGAGTATCGGTTTTTAGG TATATTCGTTGTAGAGTTTTCGCGGGTTTTGGTTTTAGTTTTGAGGGTTGTGTGTGTTAGGGGATTTATTTTACGTTCGT
Map of CpG : .....CG...CG..CG.CG..CG......CG.......CG...........CG..........CG.........CG.... CG...........CG...CG...................CG.CG.....CG....CG........CG...........CG ........CG.CG...CG............CG.........CG...CG..CGCG......CG.CG..CG.CG...CG... ..............CGCG.........CGCG.....CG.CGCG......CG...........CG..CG.........CG. .........................CG..........................CG..............CG......... ......CG............CGCG.................................................CG..CG.
Position of CpG : .....6....11...15..18...22.......30........39............52...........64........ ..75.....81............94....99....................120..123......130.....136.... .....146............159.........169..172....177.............191..........202.... 207...211.213.......221..224...228..231....236..................255.257......... .268.270......277..280.282.......290............303...307..........318.......... .................346...........................374...............390............ ....407.............421.423.......................................
2) .msp.result - The msp.result file shows the M and U primer set.
CpG_positionsense/antisenseM/U Primer5'-primer_sequence-3'Tm5'-end position3'-end position
30forwardM_PrimerTCGGACGTCGTTCGTAGTATC59.21829
forwardU_PrimerGGTGAGTTGGATGTTGTTTGTAGTATT54.18229
reverseUniversal(M&U)RCCTAARCCAATACAARCAA53.02150130
39forwardM_PrimerGTTCGTAGTATCGGAGAGGGC57.341738
forwardU_PrimerGTTGTTTGTAGTATTGGAGAGGGT53.251438
reverseUniversal(M&U)AATACAARCAAAAAACAAAARCC53.09162 139

1. positionFinder.pl

[Usage] >perl positionFinder.pl [Parameter] - FASTA file : fasta sequence file - starting motif : Position of Starting motif - end motif : Position of End motif