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    PROTOCOL TBro Amazon Lightsail Template 0.5
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    PROTOCOL TBro Amazon Lightsail Template 0.5

    Introduction

    This protocol will setup up TBro in Docker in Amazon AWS Lightsail. It will load in transcripts, peptides, and an InterProScan annotation, and create a BLAST database server. It does not yet include pathways and expression features of TBro but will in the future. Items in red must be replaced with your own information or files.

    Materials

    • Transcriptome transcripts and peptides
      • T1-transcriptome.tr
      • T1-transcriptome.aa
    • TBro peptide tables (see TBro documentation)
      • T1-tbro-table.tbl
    • InterProScan annotation (see InterProScan documentation)
      • T1-annotations.tsv
    • Zipped BLAST databases and md5sums (see TBro documentation)
      • T1-blastdb-TR.zip
      • T1-blastdb-AA.zip
      • list-zipped-blastdb-md5sums

    Procedure

    • REVIEW TBRO INSTALLATION AND USE
    1. TBro documentation
    • CREATE AMAZON AWS LIGHTSAIL INSTANCE
    1. Create an Amazon AWS account ( requires phone for security check )
    • https://aws.amazon.com/
    1. Create an Amazon AWS Lightsail instance ( under Services > OS only > Ubuntu )
    • https://lightsail.aws.amazon.com/ls/webapp/home/resources
    1. Create a static IP
    • https://lightsail.aws.amazon.com/ls/webapp/home/resources
    1. Assign the static IP to your Lightsail instance
    • Follow directions on Amazon AWS Lightsail
    1. SSH to Amazon AWS Lightsail instance
    • Click the button
    • INSTALL DOCKER
    1. Uninstall any old CE versions of Docker
    • sudo apt-get purge docker-ce
    • sudo rm -rf /var/lib/docker
    • sudo apt-get remove docker docker-engine
    1. Select Ubuntu docker installation instructions
    1. Install packages for apt
    • sudo apt-get install apt-transport-https ca-certificates curl software-properties-common
    1. Add Docker GPG key
    • curl -fsSL https://download.docker.com/linux/ubuntu/gpg | sudo apt-key add -
    1. Check that key fingeprint is 9DC8 5822 9FC7 DD38 854A E2D8 8D81 803C 0EBF CD88
    • sudo apt-key fingerprint 0EBFCD88
    1. Set up stable repository
    • sudo add-apt-repository "deb [arch=amd64] https://download.docker.com/linux/ubuntu $(lsb_release -cs) stable"
    1. Update apt
    • sudo apt-get update
    1. Install Docker CE Unbuntu AMD64
    • sudo apt-get install docker-ce
    1. Add your user to user group
    • sudo usermod -aG docker $USER
    1. Connect to your instance using ssh from Amazon Lightsail interface
    • close the Lightsail terminal and then re-connect by SSH from Lightsail
    1. Run hello world from your Docker user group user
    • docker run hello-world
    1. Configure system to start Docker when it boots up
    • sudo systemctl enable docker
    • INSTALL TBRO
    1. Pull Tbro Docker images
    • docker pull greatfireball/generic_postgresql_db
    • docker pull tbroteam/generic_chado_db_reload
    • docker pull tbroteam/tbro_worker_ftp
    • docker pull tbroteam/tbro_worker
    • docker pull tbroteam/tbro_apache
    1. Start CHADO database and install schema
    • docker run -d -e DB_NAME=chado -e DB_USER=tbro -e DB_PW=tbro --name "Chado_DB_4_TBro_official" greatfireball/generic_postgresql_db
    • sleep 60
    • docker run --rm -i -t --link Chado_DB_4_TBro_official:CHADO --name "Chado_DB_4_TBro_load_official" tbroteam/generic_chado_db_reload
    1. Start database container for BLAST worker
    • docker run -d -e DB_NAME=worker -e DB_USER=worker -e DB_PW=worker --name "Worker_DB_4_TBro_official" greatfireball/generic_postgresql_db
    1. Start FTP server to host BLAST database
    • docker run -d --name "Worker_FTP_4_TBro_official" -e FTP_USER="tbro" -e FTP_PW="ftp" tbroteam/tbro_worker_ftp
    1. Start worker to execute BLAST server
    • docker run -d --link Worker_DB_4_TBro_official:WORKER --link Worker_FTP_4_TBro_official:WORKERFTP --name "TBro_Worker_official" tbroteam/tbro_worker
    • docker exec -i -t TBro_Worker_official /home/tbro/worker_build_installation.sh
    1. Build TBro Docker container
    • docker run -d --link Chado_DB_4_TBro_official:CHADO --link Worker_FTP_4_TBro_official:WORKERFTP --link Worker_DB_4_TBro_official:WORKER --name "TBro_official" -p 80:80 tbroteam/tbro_apache
    • docker exec -i -t TBro_official /home/tbro/build_installation.sh
    1. Start TBro Docker container
    • docker exec -it TBro_official /bin/bash
    • PASSWORD PROTECTION
    1. Install apache2 utilities
    • cd /etc/apache2
    • sudo apt-get update
    • sudo apt-get install apache2-utils
    1. Set up user and password
    • sudo htpasswd -c /etc/apache2/.htpasswd USER
    • PASSWORD
    • PASSWORD
    1. Modify Apache(?) configuration file
    • sudo apt-get install nano
    • sudo nano /etc/apache2/sites-enabled/000-default.conf
    • ### FILE BEGIN
    • <VirtualHost *:80>
    • # The ServerName directive sets the request scheme, hostname and port that
    • # the server uses to identify itself. This is used when creating
    • # redirection URLs. In the context of virtual hosts, the ServerName
    • # specifies what hostname must appear in the request's Host: header to
    • # match this virtual host. For the default virtual host (this file) this
    • # value is not decisive as it is used as a last resort host regardless.
    • # However, you must set it for any further virtual host explicitly.
    • #ServerName www.example.com
    • ServerAdmin webmaster@localhost
    • DocumentRoot /var/www/html
    • # Available loglevels: trace8, ..., trace1, debug, info, notice, warn,
    • # error, crit, alert, emerg.
    • It is also possible to configure the loglevel for particular
    • # modules, e.g.
    • #LogLevel info ssl:warn
    • ErrorLog ${APACHE_LOG_DIR}/error.log
    • CustomLog ${APACHE_LOG_DIR}/access.log combined
    • # For most configuration files from conf-available/, which are
    • # enabled or disabled at a global level, it is possible to
    • # include a line for only one particular virtual host. For example the
    • # following line enables the CGI configuration for this host only
    • # after it has been globally disabled with "a2disconf".
    • #Include conf-available/serve-cgi-bin.conf
    • #### BEGIN: ADD THIS TEXT TO FILE
    • <Directory "/var/www/html">
    • AuthType Basic
    • AuthName "Restricted Content"
    • AuthUserFile /etc/apache2/.htpasswd
    • Require valid-user
    • </Directory>
    • #### END: ADD THIS TEXT TO FILE
    • </VirtualHost>
    • # vim: syntax=apache ts=4 sw=4 sts=4 sr noet
    • ### FILE END
    1. Test syntax and restart TBro
    • sudo apache2ctl configtest
    • exit
    • docker restart TBro_official
    • IMPORT TRANSCRIPTOMES
    1. Enter TBro Docker container
    • docker exec -it TBro_official /bin/bash
    1. Add species information to CHADO in TBro database
    • tbro-db organism insert --genus Octopus --species chierchiae --common_name pygmy octopus --abbreviation O.chierchiae
    1. Check organism table for organism id
    • tbro-db organism list
    1. OPTIONAL To update/correct errors in organism table
    • tbro-db organism update --genus Octopus --species chierchiae --common_name tiny octopus --abbreviation O.chierchiae
    1. Build directory system in TBro Docker container
    • mkdir -p octopus
    • cd octopus
    • mkdir -p transcripts
    • mkdir -p peptides
    • mkdir -p annotations
    • mkdir -p blastdbs
    1. Copy transcript files into TBro Docker container
    • cd transcripts
    • scp id@address:/path/to/transcripts/T1-transcriptome.tr .
    1. Create list of all identifiers for each transcriptome
    • grep ">" T1-transcriptome.tr | perl -pe 's/>(\S+).*/$1/' > T1-identifiers.ids
    1. Import transcript sequence ids into TBro database
    • tbro-import sequence_ids --organism_id 13 --release octopus-T1 --file_type only_isoforms T1-identifiers.ids
    1. Import transcript sequences into TBro database
    • tbro-import sequences_fasta --organism_id 13 --release octopus-T1 T1-transcriptome.tr
    1. Copy TBro peptide fasta files into Docker TBro Docker container
    • cd ../peptides
    • scp id@address:/path/to/peptides/T1-transcriptome.aa .
    1. Copy TBro peptide tbl files into Docker TBro Docker container
    • scp id@address:/path/to/peptides/T1-tbro-table.tbl .
    1. Import peptide table into Tbro database
    • tbro-import peptide_ids --organism_id 13 --release octopus-T1 T1-tbro-table.tbl
    1. Import peptide sequences into Tbro database
    • tbro-import sequences_fasta --organism_id 13 --release octopus-T1 T1-transcriptome.aa
    • IMPORT ANNOTATIONS
    1. Copy InterProScan tsv files into TBro Docker container
    • cd ../annotations
    • scp id@address:/path/to/annotations/T1-annotations.tsv .
    1. Import InterProScan annotations into Tbro database
    • tbro-import annotation_interpro --organism_id 13 --release octopus-T1 -i interproscan-5.22-61 T1-annotations.tsv
    • BUILD BLAST DATABASE SERVER
    1. Pull zipped blast databases and md5sums files into TBro Docker container
    • cd ../blastdbs
    • scp id@address:/path/to/transcript/blastdbs/T1-blastdb-TR.zip .
    • scp id@address:/path/to/peptides/blastdbs/T1-blastdb-AA.zip .
    • scp id@address:/path/to/blastdb/md5sums/list-zipped-blastdb-md5sums .
    1. Copy zipped blast databases into Docker FTP container
    • curl --data-binary --ftp-pasv --user $WORKERFTP_ENV_FTP_USER:$WORKERFTP_ENV_FTP_PW -T T1-blastdb-TR.zip
    • curl --data-binary --ftp-pasv --user $WORKERFTP_ENV_FTP_USER:$WORKERFTP_ENV_FTP_PW -T T1-blastdb-AA.zip ftp://"$WORKERFTP_PORT_21_TCP_ADDR"/
    1. Update the queue_config.sql
    • cd /home/tbro
    • cp queue_config.example.sql queue_config.sql
    • nano queue_config.sql
    • ### BEGIN within file
    • -- database files available. name is the name it will be referenced by, md5 is the zip file's sum, download_uri specifies where the file can be retreived
    • INSERT INTO database_files (name, md5, download_uri) VALUES
    • ### BEGIN text to add 1
    • ### database_files( name ) is the name of the zipped blastdb file without '.zip' - and is referred to as program_database_relationships( database_name ) below
    • ('T1-blastdb-TR', 'becd11699b54377106482fdc7f54906d', 'ftp://172.17.0.4/T1-blastdb-TR.zip'),
    • ('T1-blastdb-AA', '52acbc6e906029a04ea3ac850bb75674', 'ftp://172.17.0.4/T1-blastdb-AA.zip');
    • ### END text to add 1
    • -- contains information which program is available for which program.
    • -- additionally, 'availability_filter' can be used to e.g. restrict use for a organism-release combination INSERT INTO program_database_relationships (programname, database_name, availability_filter) VALUES
    • ### BEGIN text to add 2
    • ### availability_filter is ( organism_id )_( release ) from earlier steps
    • ('blastn', 'T1-blastdb-TR', '13_octopus-T1'),
    • ('blastp', 'T1-blastdb-AA', '13_octopus-T1'),
    • ('blastx', 'T1-blastdb-AA', '13_octopus-T1'),
    • ('tblastn', 'T1-blastdb-TR', '13_octopus-T1'),
    • ('tblastx', 'T1-blastdb-TR', '13_octopus-T1');
    • ### END text to add 2
    • ### END within file
    1. Import configuration file
    • PGPASSWORD=$WORKER_ENV_DB_PW psql -U $WORKER_ENV_DB_USER -h $WORKER_PORT_5432_TCP_ADDR -p $WORKER_PORT_5432_TCP_PORT <queue_config.sql
    1. Log in to your TBro database
    • Enter your Amazon AWS Lightsail static IP address (see Step 4) into a web browser
    • Enter your user and password information (see Step 26)
    1. Explore
    • :)

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