----------------------------------------------
RNA-Seq Analysis Pipeline: detecting mutations in RNA-Seq samples, exam genotype vs phenotype relationship
Author: Yang Hu
Department of Physiology and Biophysics
Weill Cornell Medicine
Email: yah2014@med.cornell.edu;Hu.Yang@nyu.edu
----------------------------------------------

I. Introduction

This RNA-Seq Analysis pipeline has four sections:

1. Alignment and Counts

Including STAR alignment, Samtools sort, HTSeq Count, and Cufflinks. Must be performed in the high-performance computing enviroment.

2. Call Somatic mutations and annotate vcf

Including Mpileup, Varscan, and SnpEff. Preferably be performed in the high-performance computing enviroment.

3. Summarize FPKM, QC and cluster

Including mutation data clean, FPKM data clean, QC, hclust, and mutation/expression comparison. Can be performed on a local machine.

4. DESeq for gene level comparision

DESeq and downstram analysis. Can be performed on a local machine.

II. Work flow

1). Alignment_and_Counts.sh

Input Materials:
-i Sample.fastq.gz

Output Materials:
-o samtools Sample_name_sorted.bam,Sample_sorted.bam,Sample_sorted.bam.bai
-o HTSeq Sample.bam.count
-o CuffLinks genes.fpkm_tracking,genes.fpkm_tracking, genes.fpkm_tracking, genes.fpkm_tracking

Linux bash shell script:Alignment and Counts This bash shell is written for analyzing multiple projects. One project will have multiple RNA-seq samples.

For example, there are total 155 Waldenström Macroglobulinemia (more than one terabyte) RNA-seq data from two projects("WTCHG" and "zhunter").
The PROJECT_NAME can be changed to fit different projects.

2). CallVar_and_Annotation.sh

Input Materials:
-i Sample_sorted.bam,Sample_sorted.bam.bai

Output Materials:
-o samtools mpileup ${PROJECT_NAME}.mpileup
-o VarScan ${PROJECT_NAME}_mutations.vcf
-o snpEff ${PROJECT_NAME}_Annotated.eff.vcf

Linux bash shell script:Call Somatic mutations and annotate vcf

2-1). If want to merge two Annotated.eff.vcf from two projects, run following script:

#merge multiple Annotated.eff.vcf files with bcftools:
file1=path to file1/${PROJECT_NAME}_Annotated.eff.vcf
file1=path to file2/${PROJECT_NAME}_Annotated.eff.vcf
bgzip $file1
bgzip $file1
file1=${file1}.gz
file2=${file2}.gz
tabix $file1
tabix $file2
bcftools merge -o {merged vcf name}.vcf $file1 $file2
grep -v "##" {merged vcf name}.vcf > {merged vcf name2}.vcf #remove header   

3) Summarize FPKM, QC and cluster

Input Materials:
-i {merged vcf name2}.vcf

Output Materials:
-o boxplot
-o hclust
-o Heatmap
R code:Summarize FPKM, QC and cluster R markdown: somatic variant analysis"

4). DESeq for gene level comparision

Input Materials:
-i Sample_table_${PROJECT_NAME}.csv (table with samples annotation)
-i Sample.bam.count (HTSeq-count files)

Output Materials:
-i gene expression analysis R code:DESeq for gene level comparision R markdown: Diferential expression analysis"

This is inspired by http://genomicsclass.github.io/book/pages/rnaseq_gene_level.html

III. Requirement

System requirements:

32 GB memory high-performance computing (HPC) environment, prefer Sun Grid Engine. Linux and Mac OS 64 bit system

Software requirement: R > 3.3
python 2.7 in server