rnaSeqFPro

rnaSeqFPro is a script that will do full processing of paired RNA-Seq data starting from fastq.gz files placed in the same folder. Script will sort files and process paired .fastq.gz files. rnaSeqFPro will perform Fastqc quality control, it will map paired fastq files to the reference genome hg19 using STAR's second pass mapping.

#Dependencies

Place fastqc.gz in a working folder

<pre> mkdir work.folder cp path-to-files/*fastq.gz work.folder </pre>

FastQC

Instalation (Linux), place FastQC folder in working directory:

<pre> cd work.folder wget http://www.bioinformatics.babraham.ac.uk/projects/fastqc/fastqc_v0.11.5.zip unzip fastqc_v0.11.5.zip chmod 755 ./FastQC/fastqc </pre>

STAR

Instalation (Linux):

<pre> # Get the latest STAR source wget https://github.com/alexdobin/STAR/archive/2.5.2b.tar.gz tar -xzf 2.5.2b.tar.gz cd STAR-2.5.2b # Build STAR make STAR # If you have a TeX environment, build the documentation make manual chmod 755 STAR cp STAR /usr/local/bin </pre>

Reference genome

Download the reference genome, in this example it is human hg19:

<pre> mkdir ~/reference_genomes cd ~/reference_genomes mkdir hg19 cd hg19 wget --timestamping 'ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips/hg19.2bit ' -O hg19.2bit wget http://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/twoBitToFa chmod 755 twoBitToFa ./twoBitToFa hg19.2bit hg19.fa </pre>

For the mouse genome:

<pre> mkdir mm10 cd mm10 wget --timestamping ' http://hgdownload.cse.ucsc.edu/goldenPath/mm10/bigZips/mm10.2bit' -O mm10.2bit ./twoBitToFa mm10.2bit mm10.fa </pre>

Indexing the reference genome

Use STAR to index the reference genome, use number of core on your machine, e.g. 64.

<pre> cd ~/reference_genomes STAR --runMode genomeGenerate --runThreadN 64 --genomeDir ./ --genomeFastaFiles hg19.fa </pre>

Download GENCODE transcript annotation and GENCODE fasta files to the working folder

For example for human hg19 genome:

<pre> cd work.folder wget ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_25/GRCh37_mapping/gencode.v25lift37.annotation.gtf.gz wget ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_25/GRCh37_mapping/gencode.v25lift37.transcripts.fa.gz </pre>

For mouse mm10 genome:

<pre> cd work.folder wget ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_mouse/release_M11/gencode.vM11.annotation.gtf.gz wget ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_mouse/release_M11/gencode.vM11.transcripts.fa.gz </pre>

Install featureCounts

Install featureCounts. Downoad Subread binary from Sourceforge.

<pre> wget https://sourceforge.net/projects/subread/files/subread-1.5.1/subread-1.5.1-Linux-x86_64.tar.gz/download </pre>

Download fileMulti2TableMod1

<pre> wget https://raw.githubusercontent.com/milospjanic/fileMulti2TableMod1/master/fileMulti2TableMod1.awk </pre>

In R install RGSEPD from Bioconductor

<pre> source("https://bioconductor.org/biocLite.R") biocLite("rgsepd") </pre>

In R install DESeq2

<pre> source("https://bioconductor.org/biocLite.R") biocLite("DESeq2") </pre>

In R install goseq

<pre> source("https://bioconductor.org/biocLite.R") biocLite("goseq") </pre>

Download Kallisto binary and create kallisto index for the corresponding GENCODE transcripts

<pre> wget https://github.com/pachterlab/kallisto/releases/download/v0.43.0/kallisto_linux-v0.43.0.tar.gz kallisto index -i GENCODE_transcripts_human gencode.v25lift37.transcripts.fa.gz kallisto index -i GENCODE_transcripts_mouse gencode.vM11.transcripts.fa.gz </pre>

Meta data

Providing a meta information placed in a file named meta.data is necessary for RGSEPD to perform analysis using DESeq2/goseq. Table 1 shows an example of a meta data sheet for SE reads. Note that sample names must be shown without .fastq.gz extension.

<pre> Sample Condition SHORTNAME file.name.1 A CONDITION1 file.name.2 A CONDITION1 file.name.3 B CONDITION2 file.name.4 B CONDITION2 </pre>

For paired end reads meta data table should contain the names of paired files separated with period (.)

<pre> Sample Condition SHORTNAME file.name.1.R1.file.name.1.R2 A CONDITION1 file.name.2.R1.file.name.2.R2 A CONDITION1 file.name.3.R1.file.name.3.R2 B CONDITION2 file.name.4.R1.file.name.4.R2 B CONDITION2 </pre>

Note: the order of the samples in meta.data has to the same as in command: ls -1 for the script to work. RGSEPD will stop if the orders do not match between meta.data and mastertable, therefore create meta.data in the same order as the mastertable is created, using ls -1 hierarchy

**Meta data needs to be tab separated to avoid errors. Run tabsep on your file if necessary. https://github.com/milospjanic/tabsep **

Running

rnaSeqFPro is composed of four pipelines that will run a RGSEPD version on either human genome hg19 or mouse genome mm10, using either paired-end (PE) or single-read (SR) sequences.

<pre> rnaSeqFPro.PE.hg19.sh rnaSeqFPro.PE.mm10.sh rnaSeqFPro.SR.hg19.sh rnaSeqFPro.SR.mm10.sh </pre>

Four additional pipelines are available to run a Kallisto version: PE hg19, SR hg19, PE mm10, and SR mm10.

<pre> rnaSeqFPro.PE.hg19.Kallisto.sh rnaSeqFPro.PE.mm10.Kallisto.sh rnaSeqFPro.SR.hg19.Kallisto.sh rnaSeqFPro.SR.mm10.Kallisto.sh </pre>

After placing files in the working folder run the script that is suitable for your experiment, e.g:

<pre> chmod 755 rnaSeqFPro.PE.hg19.sh ./rnaSeqFPro.PE.hg19.sh </pre>

If you are trying to re-run the pipe, e.g. you have already previously ran the pipe but for some reason it stopped, delete the GSEPD_OUTPUT folder as the RGSEPD needs to create this folder itself, it will stop if it encounters this folder already created

Don't forget to place the GENCODE gtf file, FastQC folder and meta.data into the working folder! These are the only 3 requirements neccesary to be in the working directory, in addition to the fastqc.gz files

For Kallisto script versions, in addition, you need to place the GENCODE fasta (fa.gz) file in the working folder

Dont forget that reference genome needs to be in your ~/reference_genomes folder, in case you switch to another user account script may not work because it searches for ~/reference_genomes folder.