GBiB: From download to BLAT at assembly hubs

Introduction

Genome Browser in a Box (GBiB) has some obvious advantages when compared to other options we have while working with genomic data:

• It is a genome browser with a lot of features and tools that do not exist on other available genome browsers.
• It is much easier to install, configure and maintain when compared with a full mirror of UCSC Genome Browser web site.
• It is a safe way to keep your private data inaccessible to unauthorized users while still collaborating with authorized personal.

Nonetheless, even after choosing GBiB as your genome browser, there is a lot of different choices to do. This wiki page explains how to install, configure and maintain an assembly hub (with a track hub and BLAT) using GBiB on a laptop running Kubuntu 15.04 (Vivid). Most commands are the same for other GNU/Linux distributions, with the differences probably being only relative to package names and crontab settings. Other architectures should involve the installation of GBiB on a server using only text interface and with specific ports enabled on the firewall to restrict the use of the data for just your network.

Preparing the raw data

• Create the directories that will store the assembly hub configuration files:

$> mkdir -p ~/var/gbib/work ~/var/gbib/hubs/geneNetwork/eboVir3

$> cd ~/var/gbib/work
$> rsync -avzP rsync://hgdownload.cse.ucsc.edu/goldenPath/currentGenomes/Ebola_virus/bigZips/eboVir3.2bit .
$> rsync -avzP rsync://hgdownload.cse.ucsc.edu/goldenPath/currentGenomes/Ebola_virus/bigZips/eboVir3.chrom.sizes .
$> rsync -avzP rsync://hgdownload.cse.ucsc.edu/goldenPath/currentGenomes/Ebola_virus/bigZips/KM034562v1.fa.gz .

GBiB installation

• Create a folder at your machine to place the installation files:

$> sudo mkdir /usr/local/src/gbib

• Download GBiB from UCSC Genome Browser virtual store:
  • Go to the Genome Store.
  • Click in "Login / Register".
  • Check if you agree with the terms and conditions.
  • Check if your hardware and software meet the basic requirements.
  • Click in "Add to cart" at the box relative to GBiB.
  • Click in "Cart (1)" on menu.
  • Click in "Proceed to checkout".
  • Click in "My products" on menu.
  • Copy the address of download (let's call it <download_link>).
  • Download GBiB to /usr/local/src/gbib, uncompress and delete it.

$> cd /usr/local/src/gbib
$> sudo wget <download_link>
$> sudo unzip gbib.zip
$> sudo rm gbib.zip

• Give user sufficient access to the three uncompressed files and to the folder and start VirtualBox in background:

$> sudo chmod o+rw /usr/local/src/gbib/*
$> sudo chmod o+w /usr/local/srb/gbib
$> virtualbox &

• Add GBiB to VirtualBox and boot it for the first time:
  • Machine ---> Add ---> /usr/local/src/gbib/browserbox.vbox ---> Start
  • Wait while the first update is done (it can takes more than 1 hour to finish the update process, depending of your internet connection speed).
  • Close GBiB terminal window.
  • Select "Send the shutdown signal".
  • Confirm by clicking "OK".

GBiB configuration

• Click at "Settings".
  • General ---> Description: Ebola virus genome assembly and track hubs.
  • System ---> Motherboard ---> Base Memory: 4.096 MB.
  • System ---> Processor ---> Processor(s): 2.
  • Display ---> Video ---> Video Memory: 32 MB.
  • Shared Folders ---> + ---> Folder Path: ~/var/gbib/work ---> Auto-mount ---> OK.
  • Shared Folders ---> + ---> Folder Path: ~/var/gbib/hubs ---> Read-only ---> Auto-mount ---> OK.
• Boot GBiB virtual machine:
  • Select "browserbox" on menu at left.
  • Click at "Start".
• Test if everything is working at the following URLs:
  • http://127.0.0.1:1234
  • http://127.0.0.1:1234/folders
• Login using ssh, for a faster access.
  • Open a terminal, like "konsole".
  • Password: browser

$> ssh browser@localhost -p 1235

• Install tools that allows file manipulations:

$> gbibAddTools

• Turn off every kind of automatic update:

$> gbibAutoUpdateOff

• Do not allow users to mirror tracks:

$> gbibMirrorTracksOff

• Turn on the offline mode:

$> gbibOffline

• Reboot the virtual machine

$> sudo shutdown -r now

Assembly hub configuration

• Log in again using ssh:

$> ssh browser@localhost -p 1235

• Forcing configuration files to be loaded again every time that the page is reloaded (instead of after at least 300 seconds):
  • Insert "udcTimeout=1&" right after http://genome.ucsc.edu/cgi-bin/hgTracks? at URL.
  • To disable this feature, click at "clear" on the message that appears at the top of the page.
• Fill the contents of hub.txt file:

$> cat > /usr/local/src/gbib/hubs/geneNetwork/hub.txt << EOI
   hub geneNetwork
   shortlabel Gene Network
   longlabel Gene Network Hub for Schistosoma mansoni
   genomesFile genomes.txt
   email admin-gene@iq.usp.br
   descriptionUrl geneNetwork.html

   EOI

• The following rules must be obeyed:
  • hub: name without spaces.
  • shortLabel: limited to 17 characters.
  • longLabel: limited to 80 characters.
• Fill the contents of genomes.txt:

$> cat > /usr/local/src/gbib/hubs/geneNetwork/genomes.txt << EOI
   genome schMan2
   trackDb schMan2/trackDb.txt
   twoBitPath schMan2/schMan2.2bit
   groups schMan2/groups.txt
   description Dec. 2011 (Sanger 5.2)
   organism Schistosoma mansoni
   defaultPos Sm.Chr_1.unplaced.SC_0010:312,104-379,754
   orderKey 2
   htmlPath schMan2/description.html
   scientificName Schistosoma mansoni
   blat 127.0.0.1 42422
   transBlat 127.0.0.1 42423

   EOI

• Verify if everything is OK with the hub:

$> hubPublickCheck hubPublic -addHub="/folders/sf_hub/geneNetwork/hub.txt"

• If the above command works, you will get the MySQL command that could be executed to insert the hub at the public hub table. For example:

    mysql> insert into hubPublic (hubUrl,descriptionUrl,shortLabel,longLabel,registrationTime,dbCount,dbList) values ("/folders/sf_hubs/geneNetwork/hub.txt","/folders/sf_hubs/geneNetwork/geneNetwork.html", "Gene Network", "Gene Network Hub for Schistosoma mansoni", now(),2, "schMan2,");

Track hub configuration

• Create the contents of trackDb.txt (track without spaces or dots and with the firts character as a letter, shortLabel <= 17 chars, longLabel <= 80 chars):

$> sudo cat > /usr/local/share/gbib/hubs/geneNetwork/schMan2/trackDb.txt << EOI
   track SMPs
   bigDataUrl schMan2.bb
   shortLabel SMPs v5.2
   longLabel Schistosoma mansoni predictions (SMPs), version 5.2
   type bigBed 12
   searchIndex name
   visibility full
   html schMan2-description
   boxedCfg on
   color 96,64,0
   altColor 128,64,32
   dataVersion Dec. 2011 Sanger 5.2
   # directUrl http://verjo-server-01.iq.usp.br/genome/pires/geneNetwork/schMan1/geneView/%s
   iframeUrl https://www.google.com.br/search?q=$$
   iframeOptions height='400' width='640' scrolling='yes'
   priority 100
   url http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?form=4&db=n&term=$$
   urlLabel NCBI Details:
   urls pmid="http://www.ncbi.nlm.nih.gov/pubmed/$$" spId="http://www.uniprot.org/uniprot/$$"

   track roche454-blat
   bigDataUrl roche454-blat.bb
   shortLabel Roche 454 Trinity
   longLabel Schistosoma mansoni RNA-Seq Roche 454 Trinity contigs mapped by Blat
   type bigBed 12
   searchIndex name
   visibility full
   color 64,0,96
   altColor 64,32,128

   EOI

• In the case that the fasta file is written with all nucleotides in lowercase, convert all the uppercase letters such that the genome do not be considered as if it was all masked. We can use the change_case command, from seq_crumbs:

$> change_case --in_format fasta --outfile schMan2.fasta --processes 80 -a upper Schistosoma_mansoni_v5.2.fa

• If the names of the chromosomes are very long, we need to make them shorter:

$> sed s/Schisto_mansoni/Sm/ schMan2.fasta > schMan2-shortChromNames.fasta

• Get the .2bit file from this fasta:

$> faToTwoBit schMan2-shortChromNames.fasta schMan2.2bit

• Get and sort from the largest to the shortest a file with the size of all chromosomes of the genome of interest:

$> twoBitInfo schMan2.2bit stdout | sort -k2rn > schMan2-chromSizes-sorted.txt

• The same substitution have to be done at the bed file of the track:

$> sed s/Schisto_mansoni/Sm/ smps.bed > smps-shortChromNames.bed

• The bed file of the track have to be sorted first by the name of the chromosome and after by the starting coordinate:

$> sort -k1,1 -k2,2n smps-shortChromNames.bed > smps-shortChromNames-sorted.bed

• Convert from bed to bigBed:

$> bedToBigBed -type=bed12 -tab -extraIndex=name smps-shortChromNames-sorted.bed schMan2-chromSizes-sorted.txt smps.bb

• Contents of groups.txt:

$> cat > /usr/local/src/gbib/hubs/geneNetwork/schMan2/groups.txt << EOI
   name custom
   label Custom
   priority 1
   defaultIsClosed 1

   name mapping
   label Mapping
   priority 2
   defaultIsClosed 1

   name genes
   label Genes
   priority 3
   defaultIsClosed 1

   name mrna
   label mRNA
   priority 4
   defaultIsClosed 1

   name regulation
   label Regulation
   priority 5
   defaultIsClosed 1

   name comparative
   label Comparative
   priority 6
   defaultIsClosed 1

   name variation
   label Variation
   priority 7
   defaultIsClosed 1

   name experimental
   label Experimental
   priority 8
   defaultIsClosed 0

   EOI

Blat configuration

• From the folder that contains the .2bit file, start two gfServer's, specifying the assembly hub ports that will be used to access the DNA sequence and the aminoacids sequence:

$> gfServer start 127.0.0.1 42422 -stepSize=5 schMan2.2bit &
$> gfServer start 127.0.0.1 42423 -trans schMan2.2bit &

• If the fasta file that was used to create the .2bit file was masked (i.e., it had aminoacids with lowercase letters), we can use the gfServer flag "-mask":

$> gfServer start 127.0.0.1 42423 -trans -mask schMan2.2bit &

• Edit the file genomes.txt of the assembly hub in order to include the lines relatives to blat and transBlat:

blat 127.0.0.1 42422
transBlat 127.0.0.1 42423

• Add this commands to cron.

Custom track configuration

track type=bigBed name="Name" description="Description"
bigDataUrl=http://verjo-server-01.iq.usp.br/genome/pires/Schistosoma_mansoni_v5.2.gff.bed.bb
color=204,51,51 altColor=204,51,51 visibility=full

GBiB maintenance

• Make an update of all softwares and data:

$> gbibOnline
$> gbibAutoUpdateOn
$> updateBrowser
$> gbibAutoUpdateOff
$> gbibOffline

References

See also:

• Track Hubs User Guide
• Assembly Hub Wiki
• Track Database Definitions
• Starting a Blat enabled Assembly Hub on GBiB
• Quick Start Guides to Basic Hubs and Organizing Hubs and Assembly Hubs