Skip to content

Latest commit

 

History

History
127 lines (109 loc) · 5.88 KB

Configuration_RNAseq.md

File metadata and controls

127 lines (109 loc) · 5.88 KB

Installing packages and programs required for Applied Bioinformatics course: RNA-seq analysis

Jerry Zak

8 Oct 2018

Please follow these instructions and complete the steps by Thursday's class (11 October 2018).

Unless stated otherwise, the instructions below should work for Mac and Win users. Please contact me or the other TAs via Slack if you run into problems. NB: You need at least 10 GB of free hard drive space, and 8 GB RAM is recommended. For those with less than 8 GB RAM, follow instructions in part 3 to build single-chromosome indices.

1. Install R packages

Windows/Ubuntu users, first run in terminal (Cmder session {WSL::bash}):

sudo apt install libxml2-dev

All users, open an R console (from terminal or Jupyter) and type the commands below. Please note that the installer might first prompt you to install required packages which may need to be compiled from source. Ask for help if you get stuck.

install.packages(c("RColorBrewer", "gplots", "ggplot2", "pheatmap", "plyr", "dplyr", "fastqcr"))

source("https://bioconductor.org/biocLite.R")
biocLite(pkgs = c("genefilter", "biomaRt", "DESeq2"))

NB: If you see a message that one of the libraries is protected, say 'No' to using a personal library. Such message normally means that you already have the package installed.

2. Download SRA toolkit and the raw reads for SRR5454079

NB: Make sure you have at least 3 GB of free disk space available.

Mac users: Download the appropriate version of NCBI SRA Tools from https://www.ncbi.nlm.nih.gov/sra/docs/toolkitsoft/

Windows/Ubuntu users: Using terminal from outside of the Applied Bioinformatics folder (e.g. your home directory), run

wget https://ftp-trace.ncbi.nlm.nih.gov/sra/sdk/current/sratoolkit.current-ubuntu64.tar.gz

Mac and Win shared instructions: From the folder in which you downloaded the .tar.gz file, run

tar -zxvf [put your sra filename here.tar.gz]

Now you should have a folder named sratoolkit.2.9.2-ubuntu64 or the equivalent for Mac. If you can see this folder, run

./sratoolkit.2.9.2-ubuntu64/bin/fastq-dump --split-files SRR5454079

This will take a while. Depending on your setup and connection speed it can take over an hour. You might see a warning Rejected xxx READS because READLEN < 1 - ignore this, the reads will be extracted correctly if you run the command above.

3. Download HISAT and the human reference indices

First, we need to download a software called HISAT

  • If you are a Mac user, go to https://ccb.jhu.edu/software/hisat2/index.shtml and find the box labeled Releases. Download the Mac version by clicking the link.
  • If you are an ubuntu/WSL user, open a terminal, the execute this command wget ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/downloads/hisat2-2.1.0-Linux_x86_64.zip. (NB: if you get an error using wget, try replacing that with curl -O. NB: -O is the letter O not the digit 0. Same applies for all subsequent wget commands.)

For either operating system, unzip the file (this can be done using the unzip command from terminal) and keep the folder in a location where you can find it.

Next, Download the HISAT2 reference indices for the human genome

wget ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/grch38.tar.gz

This will take a while, please allow the download to complete, the index file is about 4 GB. (NB: again, if you get an error using wget, try replacing that with curl -O.) Once the download is complete, create a folder called HISAT_indices somewhere you can find it, and move the file there, then extract:

mv grch38.tar.gz HISAT_indices/
cd HISAT_indices
tar -zxvf HISAT_indices/grch38.tar.gz

If you have less than 8 GB RAM follow these instructions:

  • In terminal
wget ftp://ftp.ensembl.org/pub/release-94/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.chromosome.20.fa.gz
gunzip Homo_sapiens.GRCh38.dna.chromosome.20.fa.gz
  • Next, create a directory in your preferred location called HISAT_indices, then take note of the absolute path to your HISAT2 folder above and run the following (with the path adjusted to your system):
/path/to/hisat2-2.1.0/hisat2-build -f Homo_sapiens.GRCh38.dna.chromosome.20.fa HISAT_indices/grch38_chr20
  • The building procedure should conclude with a message about the Total time for call to driver() for forward index:. Now you have built indices for the human chromosome 20.

4. Install samtools

Use a terminal for the following steps. On a Mac:

brew install samtools

NB: Previous instructions included tapping homebrew/science but this has been deprecated so use the command above.

On Win/Ubuntu:

sudo apt install samtools

5. Install HTSeq

Mac users, execute:

pip install HTSeq

Alternatively, follow these instructions: https://htseq.readthedocs.io/en/release_0.10.0/install.html#installation-on-macos-x

Win/Ubuntu users, run from terminal:

sudo apt-get install build-essential python2.7-dev python-numpy python-matplotlib python-pysam python-htseq

Check your HTSeq version using

pip freeze | grep HTSeq

You should be running version 0.11 (although 0.6 and above should work). If the command above gives no output, try the following:

sudo pip uninstall htseq
sudo pip install htseq
pip freeze | grep HTSeq

If you see error messages about ownership of the package directory, try

sudo apt-get remove python-htseq
sudo pip install htseq

If pip freeze does not show the package version, try

pip show htseq

6. Check the location of the newly installed software

Using the bash commands introduced in Unit 1, reassure yourself that you can

  • find the location of the folder containing the SRA Toolkit (starts with sratoolkit)
  • find the location of the downloaded FASTQ file (SRR5454079_1.fastq)
  • be able to move SRR5454079_1.fastq to your homework folder (or the data subfolder of the homework folder)
  • find the location of the HISAT2 program files and indices