RNA-Seq analysis with AstrocyteDifferential expression and transcriptome assembly

Beibei Chen Ph.D

BICF

9/28/2016

Agenda

• Launch Workflows using Astrocyte

• BICF Workflows

• BICF RNA-seq Workflow

• Experimental Design Affecting Your Analysis

• Required Inputs

• Astrocyte demo

• Outputs

• Common Errors

• Visualization of Results using Vizapps

• Vizapp Demo

Agenda

• Launch Workflows using Astrocyte

• BICF Workflows

• BICF RNA-seq Workflow

• Experimental Design Affecting Your Analysis

• Required Inputs

• Astrocyte demo

• Outputs

• Common Errors

• Visualization of Results using Vizapps

• Vizapp Demo

Allows groups to give easy-access to their analysis pipelines via the web

Astrocyte – BioHPC Workflow Platform

Standardized Workflows

Simple Web Forms

Online documentation & results visualization*

Workflows run on HPC cluster without developer or user needing cluster knowledge

Slide contribution: David Trudgian@BioHPC

astrocyte.biohpc.swmed.edu

Agenda

• Launch Workflows using Astrocyte

• BICF Workflows

• BICF RNA-seq Workflow

• Experimental Design Affecting Your Analysis

• Required Inputs

• Astrocyte demo

• Outputs

• Visualization of Results using Vizapps

• Common Errors

• Vizapp Demo

Browse workflows

BICF Workflows

• RNASeq

• Differential Expression Analysis

• Germline Variation

• Fastq to annotated VCF

• DNA

• RNA

• Somatic Variation

• Fastq to annotated VCF

Workflow for Genome Analysis

Annotation Sources

• Gene Annotation (Genes, Regulation and TFBS)

• dbSNP, ExAC

• clinvar, gwas catalog

• cosmic

• dbNSFP

• SIFT, Polyphen2, LRT, MutationTaster, MutationAssessor, FATHMM, VEST3, CADD, MetaLR, MetaSVM, PROVEAN, DANN, fathmm-MKL, fitCons

• PhyloP x 2, phastCons x 2, GERP++ and SiPhy

• Allele frequencies in 1000 Genomes Project phase 3 data, UK10K cohorts data, ExAC consortium data and the NHLBI Exome Sequencing Project ESP6500 data

• genesets (MSigDB)

• CIVIC

• BROAD Target

Agenda

• Launch Workflows using Astrocyte

• BICF Workflows

• BICF RNA-seq Workflow

• Experimental Design Affecting Your Analysis

• Required Inputs

• Astrocyte demo

• Outputs

• Visualization of Results using Vizapps

• Common Errors

• Vizapp Demo

Everything's connected slide by Dündar et al. (2015)

General

RNA-seq

Workflow

Experimental Design Affecting Your Analysis

• Whole transcriptome vs mRNA

• Single end vs paired ends• Paired-end produces more accurate alignments

• Paired-end allows for alternative transcript analysis

• Single-end is cheaper

• Number of Reads• 10-50M is a good range

• Read Length• Longer reads produce better alignments, min 50 bp paired

or 100bp single

Experimental Design Affecting Your Analysis

• Number of Samples

• Your power to detect an effect depends on

–Effect size (difference between group means)

–Within group variance

– Sample size

• More Samples the better, min 3 per group

• Five samples sequenced to 20M reads each offer more power than 2 samples sequenced to 50M reads

• Stranded

• Can distinguish expression of overlapping genes

Strand-specific RNA-seq

image from GATC Biotech

How to decide strand

Reverse stranded

Stranded

RNASeq Analysis Pipeline

http://www.utsouthwestern.edu/labs/bioinformatics/services/data-analysis/rnaseq-pipeline.html

Create a new project

Add data to your project

Add data to your project

For NGS experiment, this is recommended.

Make your design file

Make your design file

• Use tab as delimiter

– Excel save as “Text (tab delimited)”

• If no SubjectID, use same number/character for all rows

• If no FqR2, leave them empty

• For all contents, no “-”

• For all contents, no spaces

• Columns names MUST be exactly the same as documented

Comparisons

• Comparisons are based on SampleGroup

– All pair-wise comparisons

– Could be identified by file name

• A_B.edgeR.txt

• Log fold change will be A/B

• If you want B/A, -1*logFC

Select your data files and submit

SELECT YOUR FILES

Project is running

Timeline of the whole run

Download/visualize your results

Vizapp need about 30s to start if there is no queue. You need to refresh the page.

You can also choose individual files to download to your local computer

Agenda

• Launch Workflows using Astrocyte

• BICF Workflows

• BICF RNA-seq Workflow

• Experimental Design Affecting Your Analysis

• Required Inputs

• Astrocyte demo

• Outputs

• Common Errors

• Visualization of Results using Vizapps

• Vizapp Demo

Common errors and solutions

• Make sure the delimiter is tab

• Make sure the column name are the same as mentioned in documentation

• Make sure the file names match

Common errors and solutions

• Not all files are uploaded

• It’s about the proxy setting

• Use auto-detect proxy

Agenda

• Launch Workflows using Astrocyte

• BICF Workflows

• BICF RNA-seq Workflow

• Experimental Design Affecting Your Analysis

• Required Inputs

• Astrocyte demo

• Outputs

• Common Errors

• Visualization of Results using Vizapps

• Vizapp Demo

Vizapp: QC general stat

Vizapp: QC MSD and PCA

Vizapp: Gene Compare

Vizapp: DEA

• Uses edgeR results• Filter gene list by different parameters• Sort by different columns• Data table downloading

Vizapp: DEA heatmap

• Filter gene list by different parameters• Choose different comparisons• Support user define gene list (gene official symbol)• Support pathway

Vizapp: alternative splicing

Vizapp: alternative splicing

Different transcripts’ expression in sample groups

Vizapp: alternative splicing

Vizapp: QuSAGE

• Introduction to BioHPC

– 10/5/2016

– 10:30AM-Noon @ NL6.215

• Please attend so you can get an account to try this out

Acknowledgement

• Brandi Cantarel

• David Trudgian

• BioHPC team

• BICF team

Agenda

• Launch Workflows using Astrocyte

• BICF Workflows

• BICF RNA-seq Workflow

• Experimental Design Affecting Your Analysis

• Required Inputs

• Astrocyte demo

• Outputs

• Visualization of Results using Vizapps

• Common Errors

• Vizapp Demo