This is a customizable LAMP primer sets designing system: GLAPD (whole genome based LAMP primer design for a set of target genomes).
Introduction: LAMP(Loop-mediated isothermal amplification) is a simple and effective new method to amplify DNA sequence. One LAMP primer set contains four single LAMP primers from six primer regions in genome. The six regions are F3, F2, F1c, B1c, B2 and B3. Sequences from F1c and F2 are synthesized into primer FIP and sequences from B1c and B2 are synthesized into BIP. In order to accelerate the amplification, two additional loop primers(LF, LB) can be added.
GLAPD can design LAMP primer sets based on whole genome. It can design common primers for a set of target genomes and the primers are specific for background genomes. Firstly, GLAPD identifies all candiate single primer regions. Then those single primers are aligned to target genomes and background genomes. Thirdly, GLAPD combines candidate single primers into LAMP primer sets. At last the commonality and specificity check are calculated for LAMP primer sets with the information from alignment.
SYSTEM REQUIREMENTS: GLAPD now runs under Linux operation system, it needs perl and gcc. If run GPU version, the computer capability of GPU >=2.0.
OTHER SOFTWARES MAY BE NEEDED Bowtie, which could be downloaded from http://bowtie-bio.sourceforge.net/index.shtml CUDA driver, which could be downloaded from http://www.nvidia.com
Files and Directories: This system is packaged in one file, "GLAPD.tar.gz", which can be downloaded from http://cgm.sjtu.edu.cn/GLAPD/ and https://github.com/jiqingxiaoxi/GLAPD2.git. Important files inside this tar.gz file are listed here.
|-Par/ tm_nn_parameter.txt Parameters for calculating Tm. stab_parameter.txt Parameters for calculating the stability of single primers. .db, .ds Sixteen files for calculating the secondary structure of primers. |-par.pl Get the information of positions and mismatches for each single primer. |-single.c The CPU version of GLAPD for identify single primer regions. |-LAMP.c The CPU version of GLAPD for design LAMP primer sets. |-Makefile Makefile for CPU version. |-GPU/ single.cu The GPU version of GLAPD for identify single primer regions. LAMP.cu The GPU version of GLAPD for design LAMP primer sets. Makefile Makefile for GPU version. |-bowtie/ bowtie The bowtie program bowtie-build The bowtie-build program |-example/ example.fa Sequences as example, from "NC_002951.2 Staphylococcus aureus subsp. aureus COL chromosome". target-list.txt The list of names from target genomes. It contains 3 strains of S. aureus. background-list.txt The list of names from background genomes. It contains 3 strains of other bacteria. index.* The index files for Bowtie software.
INSTALLATION: tar -zxvf GLAPD.tar.gz If you want to use CPU version: cd GLAPD/ make If you want to use GPU version: cd GLAPD/GPU/ make
QUICK START:
If you want design LAMP primers for a sequence without taking care of commonality and specificity: cd GLAPD/ ./Single -in example/example.fa -out Test (Two files "Inner/Test" and "Outer/Test" are created.) ./LAMP -in Test -ref example/example.fa -out success.txt (Ten LAMP primer sets are designed successfully stored in "success.txt" file.)
If you want design common LAMP primers without taking care of specificity: cd GLAPD/ ./Single -in example/example.fa -out Test (Two files "Inner/Test" and "Outer/Test" are created.) perl par.pl --in Test --ref example/example.fa --bowtie Bowtie_path/bowtie --index example/index --common example/target-list.txt (Three files "Inner/Test-common_list.txt", "Inner/Test-common.txt" and "Outer/Test-common.txt" are created.) ./LAMP -in Test -ref example/example.fa -out success.txt -common (Ten LAMP primer sets are designed successfully stored in "success.txt" file.)
If you want design specific LAMP primers without taking care of commonality:
cd GLAPD/
./Single -in example/example.fa -out Test
(Two files "Inner/Test" and "Outer/Test" are created.)
perl par.pl --in Test --ref example/example.fa --bowtie Bowtie_path/bowtie --index example/index --specific example/background-list.txt
(Two files "Inner/Test-specific.txt" and "Outer/Test-specific.txt" are created.)
./LAMP -in Test -ref example/example.fa -out success.txt -specific
(Ten LAMP primer sets are designed successfully stored in "success.txt" file.)
If you want design common and specific LAMP primers: cd GLAPD/ ./Single -in example/example.fa -out Test (Two files "Inner/Test" and "Outer/Test" are created.) perl par.pl --in Test --ref example/example.fa --bowtie Bowtie_path/bowtie --index example/index --common example/target-list.txt --left (Five files "Inner/Test-common_list.txt", "Inner/Test-common.txt", "Inner/Test-specific.txt", "Outer/Test-common.txt" and "Outer/Test-specific.txt" are created.) ./LAMP -in Test -ref example/example.fa -out success.txt -common -specific (Ten LAMP primer sets are designed successfully stored in "success.txt" file.)
RUN THE SYSTEM:
1.Identify candidate single primer regions:
Command:
Single -in
Arguments:
-in
2.Align sequences from single primer regions(optional):
Command:
perl par.pl --in
Arguments:
--in
3.Design LAMP primer sets:
Command:
LAMP -in
Arguments:
-in
INPUT FILE FORMAT: 1) The reference genome must be fasta format. 2) The common list file and specific list file used in step 2 must be one genome name per line. They can be generated by taking the sequence names from target or background genomes directly. 3) The bowtie index can be generated by "bowtie-build" command, more details in http://bowtie-bio.sourceforge.net/tutorial.shtml. OUTPUT FILE FORMAT: 1) In the files generated by "Single" program, each line means a candidate single primer. For example: "pos:3 length:25 +:2 -:0 61.89" Each line has five fields seperated by tabs. From left to right, the fields are:
TIPS: 1) Select reference genome: The reference genome can be select one randomly from the group of target genomes, or the most expected genome amplified by the LAMP primer set. 2) Specific file: When run the step 2, if you have the "common file", you can use "--left" option to replace the "--specific". In this way, all genomes in database expect for those in "common file" are defined as the background genomes. 3) Fast mode: When run the step 3, use the "-fast" option can accelerate the designing. But this mode may lost some right LAMP primer sets.
If you have any questions, please contact with us: Ben Jia: chenmodexiaoxi@126.com Chaochun Wei: ccwei@sjtu.edu.cn