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
BUSCO: http://busco.ezlab.org/
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