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Readme.md


PanTools version 1.3-alpha,

is a java application based on Neo4j graph database community edition 3.3.1 for computational pan-genomics, developed by Siavash Sheikhizadeh in Wageningen university, the Netherlands. If you use PanTools please do not forget to cite it :

https://doi.org/10.1186/s12859-018-2362-4

https://github.com/Sheikhizadeh/pantools


Requirements

  • KMC: is a disk-based programm for counting k-mers from (possibly gzipped) FASTQ/FASTA files (http://sun.aei.polsl.pl/kmc). You need to download it and add the path to the appropriate version (linux, macos or windows) of kmc and kmc_tools executables to your OS path environment variable.

  • Java Virtual Machine version 1.8 or higher: Add the path to the java executable to your OS path environment variable.

  • MCL: The Markov Cluster Algorithm, is a fast and scalable unsupervised cluster algorithm for graphs (http://micans.org/mcl ) which is needed for group functionality of PanTools. You need to download, unzip and compile it (see README), and add the path to the mcl executable to your path environment variable.

Running the program

java -jar pantools.jar

pantools.jar is available in folder pantools/dist/ arguments is a list of key value pairs separated by whitespace.

JVM options

[-server] [-XX:+UseConcMarkSweepGC]
[-Xmx(a number followed by g or m)]

PanTools commands

<build_pangenome or bg> To build a pan-genome out of a set of genomes.

--database_path or -dp path to the pangenome database. --genomes-file or -gf a text file containing paths to FASTA files of genomes; each in a seperated line. --kmer-size or -ks the size of k-mers, if not given or is out of range (6 <= K_SIZE <= 255),an optimal value would be calculated automatically.

<build_panproteome or bp> To build a pan-proteome out of a set of proteins.

--database_path or -dp path to the pangenome database. --proteomes_file or -pf a text file containing paths to FASTA files of proteomes; each in a seperated line.

<add_genomes or ag> To add new genomes to an available pan-genome.

--database_path or -dp path to the pangenome database. --genomes-file or -gf a text file containing paths to FASTA files of the new genomes to be added to the pangeome; each in a seperated line.

<add_annotations or aa> To add new annotations to an available pan-genome.

--database_path or -dp path to the pangenome database. --annotations-file or -af a text file each line of which contains genome number and path to the corresponding GFF file seperated by one space. Genomes are numbered in the same order they have been added to the pangenome. The protein sequence of the annotated genes will be also stored in the folder "proteins" in the same path as the pangenome. --connect_annotations or -ca connect the annotated genomic features to the nodes of gDBG.

<retrieve_features of rf> To retrieve the sequence of annotated features from the pangenome. For each genome the resulting FASTA file will be stored in the current directory.

--database_path or -dp path to the pangenome database. --genome-numbers or -gn a text file containing genome_numbers for which the features will be retrieved. The resulting FASTA files have two parts separated by a dot. The first part determines the feature and the second determines the genome number; for example, genes.1.fasta. --feature-type or -ft (default = gene) the feature name; for example gene, mRNA, exon, tRNA, ...

<retrieve_regions or rr> To retrieve the sequence of some genomic regios from the pangenome. The results will be stored in the same folder as the pangenome.

--database_path or -dp path to the pangenome database. --regions-file or -rf a text file containing records with genome_number, sequence_number, begin and end positions seperated by one space for each region. The resulting FASTA file would have the same name with an additional .fasta extention.

<retrieve_genomes or rg> To retrieve the full sequence of some genomes. The results will be stored in the same folder as the pangenome itself.

--database_path or -dp path to the pangenome database. --genome-numbers or -gn a text file containing genome_numbers to be retrieved in each line. The resulting FASTA files are named like Genome_x.fasta. To add homology nodes which point to a groups of homologous proteins. --database_path or -dp path to the pangenome database. --intersection-rate or -ir (default = 0.09) the fraction of kmers needs to be shared by two intersecting proteins. Should be in range [0.001, 0.1]. --min-protein-identity or -mpi (default = 95) the minimum similarity score. Should be in range [1-99]. --mcl-inflation or -mi (default = 9.6) the MCL inflation. Should be in range ]1-19[. --contrast or -ct (default = 8) the contrast factor. Should be in range ]0-10[. --relaxation or rn (default 1) the relaxation in homology calls. Should be in range [1, 8], from strict to relaxed. --threads-number or -tn (default = 1) the number of parallel working threads To map single or paired-end reads to all or a sebset of constituent genomes. --database_path or -dp path to the pangenome database. -1 a text file containing path to the first short-read archive in FASTQ or FASTA format. -2 optionally, a text file containing path to the second short-read archive in FASTQ or FASTA format. --genome-numbers or -gn a text file containing genome_numbers to map reads against in each line. --output-path or -op (default: database path determined by -dp) path to the output files. --threads-number or -tn (default = 1) the number of parallel working threads --min-mapping-score or -mms (default = 20) the minimum of read mapping score --num-kmer-samples or -nks (default = 20) the number of kmers sampled from read --min-hit-length or -mhl (default = 17) the minimum acceptable length of alignment after soft-clipping --max-alignment-length or -mal (default = 1000) the maximum acceptable length of alignment --max-fragment-length or -mfl (default = 2000) the maximum acceptable length of fragment --max-num-locations or -mnl (default = 20) the maximum number of location of candidate hits to examine --alignment-bound or -ab (default = 7) the length of bound of banded alignment --clipping-stringency or -ci (default = 2) the stringency of soft-clipping 0 : no soft clipping 1 : low 2 : medium 3 : high --bam-format or -bf (default = FALSE) the alignment format (.sam or .bam) --alignment-mode or -am (default = 0) the alignment mode 0 : Competitive, only-best 1 : Competitive, all-bests 2 : Normal, only-best 3 : Normal, all-bests To show the versions of PanTools and Neo4j. To see this document.

Visualization in the Neo4j browser

Neo4j browser allows you to run Cypher queries and receive the results in a tabular or a graph representation mode. You need to download the appropriate version of Neo4j. To visualize the pangenome of two HIV strains provided as a sample data in pantools repositiory, take these actions on a linux machine. Windows users could also download the Neo4j desktop application for starting and stopping a server instead of usingn commandline.

  1. Add the path to the Neo4j /bin directory to the path environment variable.
  2. Hard-code the path to your pangenome in the configuration file ,NEO4J-DIRECTORY/conf/neo4j.conf, by: dbms.directories.data = PATH_TO_THE_PANGENOME_DATABASE
  3. Start the Neo4j database server by: neo4j start
  4. open an internet browser and Open the URL http://localhost:7474
  5. To visualize the whole pangenome of two HIV strains, type this simple Cypher command: MATCH (n) RETURN n
  6. To stop the Neo4j server type: neo4j stop
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