TASUKE - Installation

Installation

Outline

Here we explain commands of TASUKE for new installation and making database. If you want to update the TASUKE+ you are already using, please see "How to update".

The time for creation of databases depends on genome size, number of samples and the power of server computer. Steps 5 and 7 are repeated for each accession.

We prepared a shell script as unified tool for installation. It automatically finds the files from specified directory and conducts these process. (it does not support GWAS and System phylogenetic tree.)
More detail: Unified installer

0. System requirements

TASUKE browser requires the LAMP server. And it requires the Linux server that has Apache, MySQL5.0 or later with mysqli module and PHP5.3 or later.

First, configure PHP settings. Allows PHP code to be executed on .html file.

For CentOS8(Almalinux8) or later, set as follows.
If "php-fpm" is not installed, please install and enable it. If the "yum list php-fpm" command returns "Installed", it is installed.

$ yum install php-fpm
$ systemctl start php-fpm
$ systemctl enable php-fpm

Modifying '/etc/httpd/conf.d/php.conf'

# Redirect to local php-fpm (no mod_php in default configuration)
<IfModule !mod_php.c>
  ......
  <FilesMatch \.(php|phar|html)$>
  ......
</IfModule>

If mod_php was installed by default, you can use its "IfModule" tag in php.conf. If the settings are separate for each PHP version, configure for your PHP version. Alternatively, you can modify all "FilesMatch" tags.

•using php7.x
<IfModule mod_php7.c>
  ......
  <FilesMatch \.(php|phar|html)$>
  ......
</IfModule>

Modifying '/etc/php-fpm.d/www.conf' (Remove the commentout ";" at beginning of line)

;security.limit_extensions = .php .php3 .php4 .php5 .php7

security.limit_extensions = .php .php3 .php4 .php5 .php7 .html

Then restart "httpd" and "php-fpm".

$ systemctl restart httpd
$ systemctl restart php-fpm

For CentOS7(RHEL7) and earlier, the settings were as follows.
Modifying '/etc/httpd/conf.d/php.conf' and restart httpd:

•using php5.x
AddHandler php5-script .php AddHandler php5-script .php .html

•using php7.x
AddHandler php7-script .php AddHandler php7-script .php .html

For Ubuntu, add the following to the end of '/etc/apache2/apache2.conf' and restart httpd:

<FilesMatch ".+\.html?$">
SetHandler application/x-httpd-php
</FilesMatch>

Next, install additional modules as needed. These are often installed by default, but if they are not, please install them.

If php-mysql is not installed, install it. If the result of the command "php -m" contains "mysqlnd" or "mysqli", it is installed.

•CentOS php5.4 or earlier
$ yum install php-mysql

•CentOS php5.5 or later
$ yum install php-mysqlnd

•Ubuntu
$ apt install php-mysql

php-mysql and php-mysqlnd are in conflict. Please remove the previously installed one and then install the new one.

If php-json is not installed(you're using php5.1(or earlier) or php7.x), please install it. If the result of the command "php -m" contains "json", it is installed.

$ yum install php-json

If php-curl is not installed(Ubuntu etc.), please install it. If the result of the command "php -m" contains "curl", it is installed.
e.g.) If you want to install curl module on Ubuntu PHP7.4.

$ apt-get install php7.4-curl

Perl modules "FindBin" and "DBD::mysql" are required. If you can view the documentation with "perldoc FindBin" or "perldoc DBD::mysql", they are already installed. If not, please install them.

$ yum install perl-FindBin
$ yum install perl-DBD-MySQL

Additionally, when dealing with large datasets (hundreds of accessions or more), additional settings may be required for MySQL and PHP. See here.

[Optional]. Upgrade database schema(MySQL) from legacy TASUKE to TASUKE+

If you are currently using legacy TASUKE and you are updating it to TASUKE+, you will need to upgrade your database schema. This operation is not necessary if you are installing TASUKE+ newly or are already using TASUKE+.
Update your database schema as follows:

Command

$ tasuke_update_for_gwas.pl -db <database name> -u <user> -p <password>

-db <database name> : Database name for TASUKE
-u <user> : User name
-p <password> : Password for the database

After that, by updating the web content, the update of TASUKE will be completed.

1. Create a database(MySQL)

Here you create a MySQL database. First, you log-in to mysql with root authority and create a database. "database name" used here is used following installation steps.

$ mysql -u <user> -p

> Enter password: <password>

$ mysql> create database <database name>;

$ mysql> exit;

2. Initialization of database

This tool creates several tables on your database for TASUKE.

From ver.20241122 onwards, the DB mode option(-m) has been added. You can now select "Multi-sample VCF (mvcf)" mode instead of the conventional "Single sample VCF (svcf)" mode. The DB mode determined here will be automatically detected in the following steps (the user does not need to be aware of the current DB mode).
The features of each mode are as follows.

Single-sample VCF mode (default)

Even if it is a Multi-sample VCF, it will be converted to the contents of a Single-sample VCF for each accession(sample).
Since the site-common information (such as SnpEFF) is duplicated for each accession, increasing DB size.
GT values are converted to "0/1", "1/1", or "1/2" (poly-ploid GT is truncated to the first two alleles. e.g. 0/1/1 > 0/1 ).
You can add or remove accessions later without any conflicts

This mode does not support poly-ploid data. If you have single-sample VCFs for a poly-ploid (>3n) samples, please try merging the VCF files into one multi-sample VCF and register them using the multi-sample VCF mode as below.

Multi-sample VCF mode

The GT and Ploidy values in the VCF file can be displayed as is.
Since the site-common information (such as SnpEFF) is managed in a separate table, reducing DB size.
Generally, you cannot add or remove accessions(samples) later as this may result in inconsistent data.

Command

$ tasuke_init.pl -db <database name> -u <user> -p <password>

Required:
-db <database name> : Database name for TASUKE
-u <user> : User name
-p <password> : Password for the database
-m <DB mode> : svcf(default)/mvcf

Optional:
-h <remote host> : To connect remote host name
-r : Delete the tables from database.

> Input csv file about chromosome's information below.
> Where is the csv file? > <information of reference genome(.csv)>
> Are you sure creating database [y|n] > y

When registering the reference genomes containing more than 900 sequencens (e.g. scaffolds and contigs), you need to modify "tasuke_www/conf/config.php" after installing TASUKE as follows:
$autoDetectSerialTable = 1;

When you missed in chromosome list, you must create MySQL database again.

$ mysql -u <user> -p

> Enter password: <your password>

$ mysql> drop database <database name>;

$ mysql> create database <database name>;

$ mysql> exit;

3. Add an accession

This tool registers the accessions to database.

Command

$ tasuke_accession.pl -db <database name> -u <user> -p <password>

Required:
-db <database name> : Database name for TASUKE
-u <user> : User name
-p <password> : Password for the database

Optional:
-h <remote host> : To connect remote host name
-r : Delete the accessions from database.

> Input csv file about list of accessions below.
> Where is the csv file? > <accession list(.csv)>
:
> Are you sure adding or updating database?[y|n] > y

If an id with the same name is already exist, the accession in database is updated by this tool.

When you want to delete an accession. You can delete the accession with -r option.

$ tasuke_accession.pl -r -db <database name> -u <user> -p <password>

-----------------------------------
Deleting accession from database
-----------------------------------

Input csv file about list of accessions below.

* WARNING : This process deletes not only accession information but also depth and variant data.

4. Input a Reference Genome file (FASTA)

This tool sets reference genome to database.

Command

$ tasuke_ref.pl -db <database name> -u <user> -p <password> -f <reference genome>

Required:
-db <database name> : Database name for TASUKE
-u <user> : User name
-p <password> : Password for the database
-f <reference genome> : FASTA formatted reference genome file

Optional:
-h <remote host> : To connect remote host name
-r : Delete the reference genome from database.

The FASTA file needs line break letter per several ten to several hundred nucleotides. (e.g. 100bp/line)

When you want to input a reference genome again, you can delete it with '-r' option.

$ tasuke_ref.pl -r -db <database name> -u <user> -p <password>

5. Input a Variant (VCF) file

This tool sets variants to database.

Command:

$ tasuke_variant_vcf.pl -db <database name> -u <user> -p <password> -n <ID> -f <VCF file>
-t 'samtools' or 'freebayes' or 'gatk' or 'gatkm'

Required:
-db <database name> : Database name for TASUKE
-u <user> : User name
-p <password> : Password for the database
-n <ID> : Destination ID (accession). For "-t gatkm", a comma-separated list of IDs
-f <variant file> : Variant infromation (.VCF)
-t 'samtools' or 'gatk' or 'gatkm' : Set the program name that generated VCF file to this section
'gatkm' means multi sample VCF file generated by GATK.

Optional:
-z : For "-t gatkm". Register GT:0/0 variant(not by default)
-h <remote host> : To connect remote host name
-r : Delete the variants from database.

Apart from the above, there is a parallel wrapper script that is faster when registering large multi-sample VCF(tens to thousands of samples). Details are described later.

When you set "gatkm" for the program name(-t), Specify a comma-separated list of accessionIDs for "-n" (no spaces). The order of the accessionIDs corresponds to the order of the samples in the VCF file (sample names in the VCF are not used for DB registration). If IDs is less than the number of samples in VCF, ID is mapped from the first sample and the excess sample is ignored. If you want to ignore registering samples at the beginning or in the middle of the columns, write only commas like "-n ,,,ID1,ID2,,ID4".
A multi-sample VCF("-t gatkm") file contains a GT:0/0 variant, but it is not registered in DB by default as it will increase data size and reduce performance. Add '-z' option to register GT:0/0 variant. GT:0/0 variant will be displayed on the track in GT color mode.

When you want to input a VCF file again, you can delete it with '-r' option.

$ tasuke_variant_vcf.pl -r -db <database name> -u <user> -p <password> -n <ID>

If your VCF file format is multi-sample(gatkm) and the number of samples is large, we recommend the following script(tasuke_variant_vcf_multi.pl). This script simplifies operations and speeds up DB registration by executing multiple "tasuke_variant_vcf.pl" in parallel.
"-k" option only checks the correspondence between VCF samples and DB AccessionIDs. DB registration is not performed. It is strongly recommended to perform this check before DB registration.

Command:
$ tasuke_variant_vcf_multi.pl -db <database name> -u <user> -p <password> -f <VCF file>

Either required (*1):
-n <IDs> : Comma-separated AccessionID list (Corresponds to the order of VCF sample names) (*2)
-m <Path> : Path to "VCFsampleName > AccID" correspondence table file. VCFsampleName[,]AccID[\n]...
(none) : Consider VCF sample name as AccessionID.

Other options:
-h <remote host> :(default: localhost)
-z : Register GT:0/0 variant(not by default)
-t <num>: Number of threads(default: 4)
-k : TEST mode. Check AccessionIDs, and output commands for each thread, but do not perform DB registration.

Sub-action:
-r : Delete variant information for the specified AccessionIDs. AccessionID itself is not deleted. A multi-sample VCF previously used for DB registration must be specified with "-f".

(*1) priority is n > m, Only the AccIDs specified here will be registered.
(*2) See description in tasuke_variant_vcf.pl on this Wiki.

An example of the SampleName conversion table file (-m) is as follows. Samples not described here will not be registered. The order of name lines does not affect.

VcfSample1,DbAccId1
VcfSample2,DbAccId2
VcfSample3,DbAccId3
......

6. Input a Depth information file (Optional)

This tool sets depth information to database. First you need to create TSV files from your BAMs (see Preparation section).

Command:

$ tasuke_tsv_db.pl -db <database name> -u <user> -p <password> -n <ID> -f <depth file>

Required:
-db <database name> : Database name for TASUKE
-u <user> : User name
-p <password> : Password for the database
-n <ID> : Destination ID (accession)
-f <Depth file> : TSV formatted depth information file

Optional:
-h <remote host> : To connect remote host name
-r : Delete the variants from database.

A faster parallel wrapper script is available for registering a large number of samples (tens to hundreds). Details are below.

You can delete a TSV file with '-r' option.

$ tasuke_tsv_db.pl -r -db <database name> -u <user> -p <password> -n <ID>

If the number of samples is large, we recommend the following script(tasuke_tsv_db_multi.pl). This script simplifies operations and speeds up DB registration by executing multiple "tasuke_tsv_db.pl" in parallel.
"-k" option only checks the correspondence between TSV samples and DB AccessionIDs. DB registration is not performed. It is strongly recommended to perform this check before DB registration.

Command:
tasuke_tsv_db_multi.pl -db <database> -u <user> -p <password>

-d <DirPath> : Directory path where the TSV files are located. By default, filename(without extensions) is considered as AccessionID.
-m <Path>: Path to "TsvFilePath > AccID" correspondence table. If "-d" is specified, TsvFilePath is its relative path. TsvFilePath[,]AccID[\n]...

Other options:
-h <remote host>:(default: localhost)
-t <num>: Number of threads(default: 4)
-k : TEST mode. Check AccessionIDs, but do not perform DB registration.

Sub-action:
-r : Delete depth information for the specified AccessionIDs. AccessionID itself is not deleted.

An example of the FileName conversion table file (-m) is as follows. Samples not described here will not be registered. The order of name lines does not affect.

TsvFilePath1,DbAccId1
TsvFilePath2,DbAccId2
TsvFilePath3,DbAccId3
......

7. Input a TSV file to the general purpose track (Optional)

This tool inputs any kind of TSV formatted NGS data. To input the general purpose track, you can do it by using tasuke_tsv_db.pl with '-c' option. First you need to create TSV files from your BED or BEDgraph files (see Preparation section).

Command:

$ tasuke_tsv_db.pl -c -db <database name> -u <user> -p <password> -n <ID> -f <tsv file>

You can delete the file with '-r' option.

$ tasuke_tsv_db.pl -r -c -db <database name> -u <user> -p <password> -n <ID>

If you want to set any multiple conditions to the general purpose track, try following command. And load a TSV file using tasuke_tsv_db.pl.

Command:

$ tasuke_add_condition.pl -db <database name> -u <user> -p <password> -n <ID> -f <depth file>

Required:
-db <database name> : Database name for TASUKE
-u <user> : User name
-p <password> : Password for the database
-c <condition_id> : Condition ID(name)

Optional:
-h <remote host> : To connect remote host name
-r : Delete the conditon and tables from database.

8. Annotation track (Optional)

The annotation track on the TASUKE browser can be added from GFF files. Tasuke_screenshot

Command:

$ tasuke_track_gff.pl -db <database name> -u <user> -p <password> -f <annotation file> -t <track name>

Required:
-db <database name> : Database name for TASUKE
-u <user> : User name
-p <password> : Password for the database
-f <annotation file> : GFF(3) formatted file
-t <track name> : It sets here is directoly used for track name on TASUKE

Optional:
-h <remote host> : To connect remote host name
-r : Delete the annotations from database.

You can delete the file with '-r' option.

$ tasuke_track_gff.pl -r -db <database name> -u <user> -p <password> -t <track name>

9. Phenotype data (GWAS) (Optional)

The phenotype data can be added for using GWAS function on TASUKE.

Command:

$ tasuke_phenotype.pl -db <database name> -u <user>

Required:
-db <database name> : Database name for TASUKE
-u <user> : User name

Optional:
-h <remote host> : To connect remote host name
-r : Delete the phenotype data from database.

Enter db password: <password>
> Where is phenotype data csv file?
(File format: Breed Name,Phenotype,Phenotype Value)
# <phenotype data file(.csv)>
> Where is qqman output file?
# <qqman output file(.txt or .tsv)>
> What is a phenotype of the file?
# <phenotype>
> Completed.
> Read the next file? [y/N]
#

y: > Where is qqman output file?

# N: Done.

To enable GWAS function, you need to modify conf/config.php after installing TASUKE as follows:
$phenotypeFlg = "true";

10. Use System phylogenetic tree (Optional)

Tasuke_screenshot

To use the System phylogenetic tree, You need to create a distance matrix and/or Newick and set its file path in the config file.
There are several ways to create them. See here for details.

1. Use the script included in TASUKE+ package.

1-1. Create from DB contents (tasuke_tree_dmatrix.pl)

Distance matrix format
Newick format

1-2. Create from multi-sample VCF file (tree_dmatrix_fromVcf.pl)

Distance matrix format
Newick format

2. Create in your own way

Distance matrix format
Newick format

3. Create Newick from an existing distance matrix

Use TASUKE's "Export Current SystemTree" function
Use getNewick.php
Prepare Newick in your own way

Unified installer (Optional)

This tool supports installation of TASUKE. It automatically detects any datasets and load the data to a database. It treats each file name as registered ID. Before running the tool, confirm relation of file names and accession ID.
Unified installer does not support GWAS and system phylogenetic tree registration. Also, this tool assumes that multiple single-sample VCF files are used for Variant registration. Please perform DB registration by multi-sample VCF manually separately.

Command:

$ install.sh <TASK> <Option>

TASK (Required):
all : All installation processes
init : Setting defalut tables to a database
acc : Accession informtaion
ref : Reference sequence
ann : Annotation
var : Variants
tsv : Read depth or General purpose track (defalut: read depth)

Option:
-h : Help
-r : Delete specified datasets from the database.
-g : TSV file load to general purpose track.

Set your server environment to a 'install.conf' to run the 'install.sh'. And place the install.conf in same directory as install.sh.

Modifying install.conf

##### Configuration #####
#Path of 'tasuke_bin'
SCRIPTS='/PATH/tasuke_bin/'

#Database
#mysql or oracle
BACKEND='mysql or oracle'

#Database connection
DB=<database name>
USER=<user>
PASS=<password>

#For oracle
TABLESP=<tablespace name>

#Directory for datasets
# 'install.sh' searches for datasets in following directories. And it set the datasets to the database.
# For example, this tool searches for VCF file in './tasuke_sample_data/variants/', when setting variants to the database.

#Datasets
DATADIR='/PATH/tasuke_sample_data/'

#Enter the fasta file name you use as reference genome. not a directory. DIR_FASTA='./reference.fasta'

#This scripts searches for '.gff' from in 'DIR_GFF'.
DIR_GFF='./'

#This scripts searches for '.vcf' from in 'DIR_VCF'.
DIR_VCF='./variants/'

#This scripts searches for '.tsv' from in 'DIR_TSV'.
DIR_TSV='./depth/'

#File format of your VCF files ['samtools' or 'gatk']
VCF='gatk'
#########################

In above case, the tool searches for any file from /PATH/tasuke_sample_data/ and load the file to the database.
e.g.) The tool searches for any files from '/PATH/tasuke_sample_data/variants/'. If the tool finds 'human001.vcf', it load the vcf to the table for human001 in your database.

Optimize tables (Optional)

If browsing TASUKE is extremely slow, such as "It takes several tens of seconds to scroll the track", MySQL table statistics may not have been created.
(It seems that it may occur when the database is loaded for a long time by registering a large dataset)
MySQL table statistics are normally created automatically, but you can create them manually with the script below:

Command
$ tasuke_optimize_tables.pl -db <database name> -u <user> -p <password> -a/-v/-d/-c/-s
Either required:
-a : optimize all tables(Same as specifying "-v -d -c -s")
-v : Optimize variant information tables
-d : Optimize depth information tables
-c : Optimize general purpose track tables
-s : Optimize other system-related tables
Other options:
-h : (default: localhost)
-k : TEST mode. Check AccessionIDs, and output SQLs, but do not execute it.

Running this script usually takes from tens of minutes to several hours depending on dataset size.

Installation of web contents

If you want to update already installed TASUKE+, please see "How to update".

Installation of web contents is as simple as copying files.
After download TASUKE package, copy the contents of "tasuke_www" to Apache document root under any name.
Below is an example command.

Unpack and copy files

$ tar xf ./tasuke-plus.tar

$ mkdir /var/www/html/tasuke

$ cp -r ./tasuke-plus/tasuke_www/* /var/www/html/tasuke/

Additional setting (Optional)

Exposing on the internet

Security setting for exposing on the internet.

1. Limited-mysql-user for security protection

$ mysql -u <user> -p

> Enter password: <password>

$ mysql> create user '<new user>'@'<hostname>';

$ mysql> set password '<new user>'@'<hostname>'=password('<new password>');

$ mysql> grant select on <database name>.* to '<new user>'@'<hostname>';

$ mysql> flush privileges;

$ mysql> exit;

Modifying conf/config.php

$user = <new user>;
$pswd = <new password>;

2. Access limiting for the configuration files
Modifying /etc/httpd/conf/httpd.conf

<Directory "<Apache document root>/conf" >
Require all denied
</Directory>

For Apache 2.2 and earlier, Modifying /etc/httpd/conf/httpd.conf

<Directory "<Apache document root>/conf" >
Order deny,allow
Deny from all
</Directory>

Compressing the database

Using database compression, data size will be reduce and the performance is slightly improve. Particularly TSV (depth and general-purpose) data size will be reduce to 1/2 to 1/6.

The TASUKE (database) does not work until finishing this processes.

Compressed database can not be update (read-only).

If you want to update data after making compressed database, decompressing is needed.

Compressing

Stop the mysql-server

$ service mysqld stop

Move to the database directory

$ cd <mysql database directory> (default: /var/lib/mysql/<database name>)

Compressing the tables
<tsv table> indicates dx_accession or dx_accession_cstm
Myisampack and myisamchk are repeated for each accession

$ myisampack -v <tsv table>

$ myisamchk -rq --sort-index --analyze <tsv table>.MYI

Start the mysql-server

$ service mysqld start

Load the tables

$ mysql -u <user> -p

> Enter password: <password>

$ mysql> flush tables;

$ mysql> exit;

Decompressing

Stop the mysql-server

$ service mysqld stop

Deompressing the tables

$ myisamchk --unpack <tsv table>

Start the mysql-server

$ service mysqld start

Load the tables

$ mysql -u <user> -p

> Enter password: <password>

$ mysql> flush tables;

$ mysql> exit;

TASUKE+ multiple genome browser for variants and GWAS

Installation

Outline

Starting TASUKE

Additional setting (Optional)

How to update