Trinity and Transcriptome Evaluation¶

Launch a BIG maching Maybe a c4.8xl that has 32 cores and 60GB RAM. Get 100Gb Hard Drive

Trinity: http://trinityrnaseq.github.io/

Transrate: http://hibberdlab.com/transrate/installation.html

RCorrector: https://github.com/mourisl/Rcorrector

Update Software

sudo apt-get update && sudo apt-get -y upgrade

Install other software Note that you can install a large amount of software from the Ubuntu “App Store” using a single command. Some of this software we will not use for this tutorial, but...

sudo apt-get -y install build-essential tmux git gcc make g++ python-dev unzip \
                        default-jre libcurl4-openssl-dev zlib1g-dev python-pip

Install Ruby Ruby is a computer language like Python or Perl.

cd
wget https://keybase.io/mpapis/key.asc
gpg --import key.asc
\curl -sSL https://get.rvm.io | bash -s stable --ruby
source /home/ubuntu/.rvm/scripts/rvm

Install LinuxBrew

sudo mkdir /home/linuxbrew
sudo chown $USER:$USER /home/linuxbrew
git clone https://github.com/Linuxbrew/brew.git /home/linuxbrew/.linuxbrew
echo 'export PATH="/home/linuxbrew/.linuxbrew/bin:$PATH"' >> ~/.profile
echo 'export MANPATH="/home/linuxbrew/.linuxbrew/share/man:$MANPATH"' >> ~/.profile
echo 'export INFOPATH="/home/linuxbrew/.linuxbrew/share/info:$INFOPATH"' >> ~/.profile
source ~/.profile
brew tap homebrew/science
brew update
brew doctor

INSTALL TRANSRATE

curl -LO https://bintray.com/artifact/download/blahah/generic/transrate-1.0.3-linux-x86_64.tar.gz
tar -zxf transrate-1.0.3-linux-x86_64.tar.gz
echo 'export PATH=$PATH:"$HOME/transrate-1.0.3-linux-x86_64"' >> ~/.profile
source ~/.profile

INSTALL BLAST

curl -LO ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.3.0/ncbi-blast-2.3.0+-x64-linux.tar.gz
tar -zxf ncbi-blast-2.3.0+-x64-linux.tar.gz
echo 'export PATH="$HOME/ncbi-blast-2.3.0+/bin:$PATH"' >> ~/.profile
source ~/.profile

INSTALL Augustus, BUSCO, Trinity, RCorrector, Skewer

brew install gcc augustus emboss Trinity --without-express Rcorrector Skewer busco --without-blast

Download data: For this lab, we’ll be using

#Open tumx window

tmux new -s trinity

mkdir $HOME/reads && cd /$HOME/reads/
curl -LO https://s3.amazonaws.com/NYGC_August2015/raw_data/382-Kidney_ACTTGA_BC6PR5ANXX_L008_001.R1.fastq.gz
curl -LO https://s3.amazonaws.com/NYGC_August2015/raw_data/382-Kidney_ACTTGA_BC6PR5ANXX_L008_001.R2.fastq.gz
zcat 382-Kidney_ACTTGA_BC6PR5ANXX_L008_001.R1.fastq.gz | head -8000000 > read1.fq
zcat 382-Kidney_ACTTGA_BC6PR5ANXX_L008_001.R2.fastq.gz | head -8000000 > read2.fq


# control-b d to get out of tmux window.


tmux attach -t trinity #to get back in tmux window.

Correct Reads

run_rcorrector.pl -k 31 -t 30 \
-1 $HOME/reads/read1.fq \
-2 $HOME/reads/read2.fq

Run Skewer

curl -LO https://s3.amazonaws.com/gen711/TruSeq3-PE.fa

skewer -l 25 -m pe -o skewerQ2 --mean-quality 2 --end-quality 2 -t 30 \
-x TruSeq3-PE.fa \
$HOME/reads/read1.cor.fq \
$HOME/reads/read2.cor.fq

Run Trinity

mkdir $HOME/assembly && cd $HOME/assembly


Trinity --seqType fq --max_memory 40G --left $HOME/reads/skewerQ2-trimmed-pair1.fastq \
--right $HOME/reads/skewerQ2-trimmed-pair2.fastq --CPU 30

Run BUSCO for assemblies: There are Eukaryote, Metazoa, Arthropod, Vertebrate, Plant references for use with other genomes.

mkdir $HOME/busco && cd $HOME/busco

export AUGUSTUS_CONFIG_PATH=/home/ubuntu/.linuxbrew/Cellar/augustus/3.2.2_1/libexec/config/

#Download busco database

curl -LO http://busco.ezlab.org/files/vertebrata_buscos.tar.gz
tar -zxf vertebrata_buscos.tar.gz

busco -m trans -in $HOME/assembly/trinity_out_dir/Trinity.fasta \
--cpu 30 -l vertebrata -o trin.assem

less run*/short*

Run Transrate

mkdir $HOME/transrate && cd $HOME/transrate
transrate -a $HOME/assembly/trinity_out_dir/Trinity.fasta -t 30 \
--left $HOME/reads/read1.cor.fq \
--right $HOME/reads/read2.cor.fq

Terminate your instance¶

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