| Author: | Tamer Mansour |
|---|---|
| Date: | Nov 3, 2015 |
This tutorial shows how to build a new transcriptome with Cufflinks and share it as a custom track on the UCSC Genome Browser, using github to store the files.
Background documentation:
https://genome.ucsc.edu/goldenPath/help/hgTrackHubHelp.html
Go ahead and open the EC2 service to lunch a suitable instance. Change your location on the top right corner of the page to be US East (N. Virginia). Push the Launch Instance button then follow these instructions:
- On "Step 1: Choose an Amazon Machine Image (AMI)": The protocol was prepared to run on "Ubuntu Server 14.04 LTS (HVM)"
- On "Step 2: Choose an Instance Type": You can run this protocol on m3.xlarge instance (4 vCPU and 15 GiB memory).
- On "Step 3: Configure Instance Details": accept the default settings
- On "Step 4: Add storage": accept the default settings
- On "Step 5: Tag Instance": Give your instance a name.
- On "Step 6: Configure Security Group": Create a new security group and add security rules to enable ports 22, 80, and 443 (SSH, HTTP, and HTTPS).
- On "Step 7: Review Instance Launch": Review the information of your instance. You will see an alarm about the security of your instance. This is ok, once you click launch, you will be able to make a security key-pair (or use yours if you already have one)
- After you launch your instance, the confirmation page will show your instance ID. Click the instance ID to watch your instance status. In the lower half of the page, you will description info of your instance. Copy the public IP address to use in the next step.
You need an SSH client to connect from you own computer to the Amazon instance you just started in the cloud:
- Download PuTTY and PuTTYgen from: http://www.chiark.greenend.org.uk/~sgtatham/putty/download.html
- Run PuTTYgen, Find and load your '.pem' file, and Now, "save private key"
- Run PuTTY, paste the public DNS in the host name. In the left category panel, expand the SSH entry, select Auth, find your private key and click open
- click yes if prompted then Log in as "ubuntu"
- create a folder to contain all the files of this experiment and define this path
Define your working directory:
mkdir evalTrans workingPath=$"/home/ubuntu/evalTrans" export workingPath
Install prerequisites
cd ~ sudo apt-get update sudo apt-get install gcc g++ pkg-config wget make
install cufflinks/2.1.1
cd /usr/src/ sudo wget http://cole-trapnell-lab.github.io/cufflinks/assets/downloads/cufflinks-2.1.1.Linux_x86_64.tar.gz sudo tar -zxvf cufflinks-2.1.1.Linux_x86_64.tar.gz cd /usr/bin sudo ln -s /usr/src/cufflinks-2.1.1.Linux_x86_64/cufflinks . sudo ln -s /usr/src/cufflinks-2.1.1.Linux_x86_64/cuffmerge . sudo ln -s /usr/src/cufflinks-2.1.1.Linux_x86_64/cuffcompare . sudo ln -s /usr/src/cufflinks-2.1.1.Linux_x86_64/cuffdiff . sudo ln -s /usr/src/cufflinks-2.1.1.Linux_x86_64/gffread . sudo ln -s /usr/src/cufflinks-2.1.1.Linux_x86_64/gtf_to_sam .
cd $workingPath mkdir data && cd data curl -L -O https://github.com/ngs-docs/2015-nov-adv-rna/raw/master/cufflinkData/control1.tar.gz curl -L -O https://github.com/ngs-docs/2015-nov-adv-rna/raw/master/cufflinkData/control2.tar.gz curl -L -O https://github.com/ngs-docs/2015-nov-adv-rna/raw/master/cufflinkData/control3.tar.gz curl -L -O https://github.com/ngs-docs/2015-nov-adv-rna/raw/master/cufflinkData/test1.tar.gz curl -L -O https://github.com/ngs-docs/2015-nov-adv-rna/raw/master/cufflinkData/test2.tar.gz curl -L -O https://github.com/ngs-docs/2015-nov-adv-rna/raw/master/cufflinkData/test3.tar.gz for f in *.tar.gz; do tar zxvf $f;done
for f in $(pwd)/*.bam; do
label=${f%.bam}
mkdir ${label}_cufflinks && cd ${label}_cufflinks
cufflinks --label ${label} --num-threads 4 $f
done
cd ../ for dir in $(pwd)/*_cufflinks; do echo $dir/transcripts.gtf; done > assemblies.txt cuffmerge -o isofrac0.05 --num-threads 4 --min-isoform-fraction 0.05 assemblies.txt 2&> cuffisofrac0.05.log cuffmerge -o isofrac0.2 --num-threads 4 --min-isoform-fraction 0.2 assemblies.txt 2&> cuffisofrac0.2.log cuffmerge -o isofrac0.5 --num-threads 4 --min-isoform-fraction 0.5 assemblies.txt 2&> cuffisofrac0.5.log cuffmerge -o isofrac0.7 --num-threads 4 --min-isoform-fraction 0.7 assemblies.txt 2&> cuffisofrac0.7.log cuffmerge -o isofrac0.9 --num-threads 4 --min-isoform-fraction 0.9 assemblies.txt 2&> cuffisofrac0.9.log
mkdir $workingPath/UCSC_kent_commands cd $workingPath/UCSC_kent_commands wget -r --no-directories ftp://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/gtfToGenePred wget -r --no-directories ftp://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/genePredToBed wget -r --no-directories ftp://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/bedToBigBed wget -r --no-directories ftp://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/fetchChromSizes chmod 755 * cd $workingPath curl -L -O https://github.com/ngs-docs/2015-nov-adv-rna/raw/master/create_trackHub.sh curl -L -O https://github.com/ngs-docs/2015-nov-adv-rna/raw/master/edit_trackDb.sh
UCSCgenome=$"equCab2" hub_name=$"testhub1" shortlabel=$"CompIsoformFrac" longlabel=$"UCSC track hub to compare selection isoform fraction" email=$"youremail@somthing.com" mkdir -p $workingPath/track_hub/$UCSCgenome/BigBed cd track_hub bash $workingPath/create_trackHub.sh "$UCSCgenome" "$hub_name" "$shortlabel" "$longlabel" "$email"
cd $workingPath/data
$workingPath/UCSC_kent_commands/fetchChromSizes $UCSCgenome > chromSizes.txt
> $workingPath/data/UCSC_assemblies.txt
for assembly in $(pwd)/isofrac*; do
echo $assembly
cd $assembly
$workingPath/UCSC_kent_commands/gtfToGenePred merged.gtf merged.gpred
$workingPath/UCSC_kent_commands/genePredToBed merged.gpred merged.bed
sort -k1,1 -k2,2n merged.bed > merged_sorted.bed
$workingPath/UCSC_kent_commands/bedToBigBed merged_sorted.bed $workingPath/data/chromSizes.txt merged.BigBed
identifier=$(basename $assembly)
cp merged.BigBed $workingPath/track_hub/$UCSCgenome/BigBed/${identifier}.BigBed
echo $identifier >> $workingPath/data/UCSC_assemblies.txt
done
trackDb=$workingPath/track_hub/$UCSCgenome/trackDb_$shortlabel.txt > $trackDb bash $workingPath/edit_trackDb.sh "$trackDb" "$workingPath/data/UCSC_assemblies.txt"
Check:
ls -l $workingPath/track_hub/equCab2/BigBed
should yield something like:
total 352 -rw-rw-r-- 1 ubuntu ubuntu 69927 Nov 3 18:10 isofrac0.05.BigBed -rw-rw-r-- 1 ubuntu ubuntu 67022 Nov 3 18:10 isofrac0.2.BigBed -rw-rw-r-- 1 ubuntu ubuntu 65627 Nov 3 18:10 isofrac0.5.BigBed -rw-rw-r-- 1 ubuntu ubuntu 70916 Nov 3 18:10 isofrac0.7.BigBed -rw-rw-r-- 1 ubuntu ubuntu 70447 Nov 3 18:10 isofrac0.9.BigBed
Reference: https://help.github.com/articles/adding-an-existing-project-to-github-using-the-command-line/ :
1. Make a github account at https://github.com/
2. Create a new repository (https://help.github.com/articles/creating-a-new-repository/)
3. Install Git to your Amazon instance
sudo apt-get install git
4. Initialize the local directory as a Git repository
cd $workingPath git init
5. Add the files in your new local repository
git add track_hub
6. Commit the files that you've staged in your local repository.
git commit -m "upload track hub"
7. At the top of your GitHub repository's Quick Setup page, copy the remote repository URL(the HTTPS one).
8. In Terminal, add the URL for the remote repository
git remote add origin <remote repository URL>
9. Push the hub directory in your local repository to GitHub.
git push origin master
Get the URL of the raw hub_CompIsoformFrac.txt and add to your tracks on UCSC.
For example, you can add CTB's version of this to UCSC by going to the Track Data Hubs URL (http://genome.ucsc.edu/cgi-bin/hgHubConnect) on http://genome.ucsc.edu/, selecting "My hubs", and putting this URL in:
https://raw.githubusercontent.com/ctb/2015-nov-trackhub-example/master/track_hub/hub_CompIsoformFrac.txt
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