PMPrimer 1.0.1

automated design of multiplex PCR primer pairs for diverse templates

PMPrimer - Automatically design multiplex PCR primer pairs for diverse templates

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0x0 Module Information

This software need third package : primer3-py, biopython, seaborn etc.

If want use BLAST, need local support ncbi-blast-2.13.0+.

This software running on Linux, not mean it cannot work on Windows.

• Input file
• Basic process for data
• Primer design and extract of multiple alignment
• Amplicon design and evaluate
• Debug mode

0x1 Installation

1. Install by code

   Download source code from this repo, and install primer3-py, biopython, seaborn in your devices, then you can run this software by use python3 pmprimer.py

2. Install by pip

   Install this software by use command pip(or pip3 for python3) install PMPrimer.

   If u use pip to install this software, pip will automatically install dependency packages and add a command pmprimer in your system.

0x2 Usage

1. Input file

   --file -f

   Use like : pmprimer -f seqs.fasta or --file seqs.fasta

2. Basic process for data

   --progress -p

   All parameter as follows :

   • notlen Do not processing sequences according to distribution of length
   • notsameseq Do not remove redanduncy sequences according to three levels
   • matrix Draw heatmap according to mismatch analysis.

   If length distribution is good, should use 'notlen' prevent removal of minority sequences.

   Add parameters behind -p, use like : -p notlen notsameseq matrix, order doesn't matter.

3. Primer design and extract of multiple alignment

   --alldesign -a

   All parameter as follows :

   • threshold Threshold for identify conservative region, default is 0.95, use like : threshold:0.95
   • minlen Minimum continues length when identify conservative regions, default is 15bp, use like : minlen:15
   • gaps Maximum rate of gaps, default is 0.1, use like : gaps:1.0
   • tm Default TM, default is 50, use like : tm:50.0
   • muscle Use MUSCLE to align sequences and save as another file
   • merge Use merge module to merge conservative regions
   • primer2 Primer design and extract
   • pdetail2 Information of primer2

   add parameters behind -a, like : -a threshold:0.96 minlen:16 merge primer2, order doesn't matter.

4. Amplicon design and evaluate

   --evaluate -e

   All parameter as follows :

   • hpcnt Maximum count of haploType, default is 10, use like : hpcnt:10
   • minlen Minimum length of amplicon, default is 150bp, use like : minlen:150
   • maxlen Maximum length of amplicon, default is 1500bp, use like : maxlen:1500
   • blast Use fasta to create blast db and search, use ',' split different files, use like : blast:../taxo1.fasta,homo.fasta
   • rmlow Remove TM lower than configure in Design module
   • save Save final results

   add parameters behind -e, like : -e hpcnt:50 maxlen:1200 save, order doesn't matter.

5. Debug mode

   --debuglevel -d

   parameter is number :

   • 1 Basic debug info
   • 2 Mocule debug info
   • 4 Complex debug info

   If need debug info, use -d 1 in normal conditions, this number use binary calculate.

0x3 Tips

1. If input file is alignments, use like :

   pmprimer -f seqs.fasta -a merge primer2 -e hpcnt:70 save

2. If input file need data process, use like :

   pmprimer -f seqs_need_filt.fasta -p default

   this command will process data according to major length and different third level when same sequences.

3. If input file need align, use like :

   pmprimer -f seqs_need_align.fasta -a muscle merge primer2 -e hpcnt:70 save

   this command will save alignments as 'seqs_need_align.mc.fasta'

0x4 Datasets In Paper

Dataset in paper can obtained by https://github.com/AGIScuipeng/PMPrimer_datasets

1. 16S ribosomal RNA (rRNA) genes of Archaea

   Archaea_16SrRNA.rep.mc.fasta is more than 200MB, so only upload original file to github, need use use MUSCLE5 to align sequences and save output file as Archaea_16SrRNA.rep.mc.fasta or use pmprimer -f Archaea_16SrRNA.rep.fasta -p notlen notsameseq muscle to align sequences.

   Command in paper is : pmprimer -f Archaea_16SrRNA.rep.mc.fasta -a threshold:0.85 gaps:1.0 merge primer2 haplo tm:45.0 -e hpcnt:600 save

2. hsp65 (groEL2) genes of Mycobacteriaceae

   Command in paper is : pmprimer -f Mycobacteriaceae_groEL2.filt.mc.fasta -a primer2 -e hpcnt:70 save

3. tuf genes of Staphylococci

   Command in paper is : pmprimer -f Staphylococcus_tuf.filt.mc.fasta -a threshold:0.995 minlen:5 merge primer2 -e save