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 description.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

• Create the HTML page description for the hub:

$> cat > ~/var/gbib/hubs/geneNetwork/description.html << EOI
<HEAD><TITLE>Gene Network Hub</TITLE>
<BODY>
<P>
Ebola virus genome assembly and track hub.
<UL>
<LI><A HREF="http://www.ncbi.nlm.nih.gov/genome/4887" TARGET="_blank">
NCBI genome/4887 (Ebola virus)</A></LI>
</UL>
</P>
<BODY></HTML>

EOI

• Include an image of the organism.
• Check if everything is OK with the hub:

$> sudo ~browser/bin/hubCheck http://127.0.0.1:1234/folders/sf_hubs/geneNetwork/hub.txt

You will see an error message stating that it was not possible to open the file http://127.0.0.1:1234/folders/sf_hubs/geneNetwork/eboVir3/trackDb.txt. We have to configure at least one track at our track hub in order to have a working assembly hub.

Track hub configuration

• Create the contents of trackDb.txt (track without spaces or dots and with the first 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
group map
searchIndex name
visibility full
html schMan2-description
boxedCfg on
colorByStrand 150,100,30 230,170,40
color 150,100,30
altColor 230,170,40
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

# For bigWig data, we can use the new trackDb setting, negateValues on, to allow display on the Crick strand.

EOI

The name of each track ("track" field) must be unique at the entire file.

• Check again if everything is OK with the hub:

$> sudo ~browser/bin/hubCheck http://127.0.0.1:1234/folders/sf_hubs/geneNetwork/hub.txt

Now you will see an error message stating that it was not possible to open the file http://127.0.0.1:1234/folders/sf_hubs/geneNetwork/eboVir3/smpsWithoutUtrs.bb. We have to copy (link?) this file to the correct place.

$> cp smpsWithoutUtrs.bb ~/var/gbib/work/virusNetwork/eboVir3
$> cd ~/var/gbib/hubs/virusNetwork/eboVir3
$> ln -s ../../../sf_work/virusNetwork/eboVir3/smpsWithoutUtrs.bb

At last the hubCheck command will run without any error being identified. But, if you point your browser to http://127.0.0.1:1234 and go to: Genomes ---> group = Gene Network, you will see the following error:

"Couldn't open http://127.0.0.1:1234/folders/sf_hubs/geneNetwork/schMan2/description.html"

Let's compose a basic page to our organism of interest:

$> cat > ~/var/gbib/hubs/geneNetwork/eboVir3/description.html << EOI
<P>
Ebola virus genome assembly and track hub.
<UL>
<LI><A HREF="http://www.ncbi.nlm.nih.gov/genome/4887" TARGET="_blank">
NCBI genome/4887 (Ebola virus)</A></LI>
</UL>
</P>
<P>
<B>UCSC Genome Browser assembly ID:</B> araTha1<BR>
Use as an example: http://genome.ucsc.edu/goldenPath/help/examples/hubExamples/hubAssembly/plantAraTha1/araTha1/description.html
</P>
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 user
label Custom
priority 1
defaultIsClosed 1

name map
label Mapping
priority 2
defaultIsClosed 0

name genes
label Genes
priority 3
defaultIsClosed 0

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 varRep
label Variation
priority 7
defaultIsClosed 0

name x
label Experimental
priority 8
defaultIsClosed 1

EOI

• Let's compose an HTML page to our track:

$> cat > ~/var/gbib/hubs/virusNetwork/eboVir3/track-description.html << EOI
<H2>Description</H2>
<P>
Replace this text with a summary describing the
concepts or analysis represented by your data.

<H2>Methods</H2>
<P>
Replace this text with a description of the methods
used to generate and analyze the data.

<H2>Verification</H2>
<P>
Replace this text with a description of the methods
used to verify the data.

<H2>Credits</H2>
<P>
Replace this text with a list of the individuals 
and/or organizations who contributed to the collection
and analysis of the data.

<H2>References</H2>
<P>
Replace this text with a list of relevant literature
references and/or websites that provide background
or supporting information about the data.

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 -log=/var/log/gfServer.eboVir3.log eboVir3.2bit &
$> gfServer start 127.0.0.1 42423 -trans -log=/var/log/gfServer.eboVir3-trans.log eboVir3.2bit &

• 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, writing them just before the "exit" command at last line:

$> sudo su -
$> vim /etc/rc.local
@vim $>
@vim $> # Blat and transBlat daemons running against Ebola virus genome at ports 42422 and 42423, respectively.
@vim $> cd /folders/sf_hubs/virusNetwork/eboVir3
@vim $> ~browser/bin/blat/gfServer start localhost 42422 -stepSize=5 -log=/var/log/gfserver.eboVir3.log eboVir3.2bit &
@vim $> ~browser/bin/blat/gfServer start localhost 42423 -trans -log=/var/log/gfserver.eboVir3-trans.log eboVir3.2bit &

Custom track configuration

browser position chr22:20,100,000-20,100,900
browser hide all
track name="Track label" description="Chromossomes coordinates list" type=bigBed visibility=full color=200,50,50 itemRgb=On colorByStrand=0,0,50 0,50,0 useScore=1 altColor=100,200,200 group=x priority=1 db=eboVir3 url="http://verjo-server-01.iq.usp.br/genome/pires/smps.html#$$ htmlUrl="http://verjo-server-01.iq.usp.br/genome/pires/track-description.html" bigDataUrl=http://verjo-server-01.iq.usp.br/genome/pires/Schistosoma_mansoni_v5.2.gff.bed.bb

• The following rules must be obeyed:
  • name: can consist of up to 15 characters, and must be enclosed in quotes if the text contains spaces.
  • description: can consist of up to 60 characters, and must be enclosed in quotes if the text contains spaces.
  • visibility: values include: 0 - hide, 1 - dense, 2 - full, 3 - pack, and 4 - squish.
  • group: values include: custom, mapping, genes, mrna, regulation, comparative, variation, and x.

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