Introduction to RNA-Seq in GeneSpring NGS Software · Introduction to RNA-Seq • In a few years,...

Learn more at www.genespring.com

Introduction to RNA-Seq in GeneSpring NGS Software

Dipa Roy Choudhury, Ph.D. Strand Scientific Intelligence and Agilent Technologies

Introduction to RNA-Seq

• In a few years, massively parallel cDNA sequencing, or RNA-seq,

has allowed many advances in the characterization and

quantification of transcriptomes.

• Rapidly decreasing sequencing cost and massively-parallel

sequencing technologies has resulted in a dramatic increase in

the quantity of data that needs to be analyzed.

• Therefore, the need of the day is to build a tool that will enable

the analysis and integration of data produced on multiple

platforms and using multiple methods.

• Agilent has designed NGS analysis in GeneSpring keeping in

mind the biologist, who is interested in answering REAL biological

questions and does not want to become a bioinformatics expert

just to do their work.

GeneSpring

Agilent SureSelect Target Enrichment

5 Map Reads to the

Ref. Genome 6 Quality Control on

Mapping 7 Detect

SNPs/InDels or

Diff. Spliced

8 Find Biological

Relevance for

your Results

GeneSpring NGS Provides Downstream

Analysis of Next-Gen Sequencing data

Data File (Reads + Quality)

Control Software

Data File (Reads + Quality)

ELAND/BIOSCOPE/BWA…

Reads aligned to genome

GeneSpring NGS

Primary Analysis Tertiary Analysis Secondary Analysis

FASTQ, … BAM, …

Questions we can seek to answer using

GeneSpring RNA-Seq Workflow are

What are the differentially expressed genes?

What are the differentially spliced genes?

Are there any SNPs in the transcriptome?

Can we identify gene fusion events?

Can we identify novel genes, novel exons, and novel splice junctions?

Import Data and Annotations

Download Human build HG18 from the

Agilent Server

Open Human

“tree” Select hg18 and

Homologene

Groups

Click [UPDATE]

to start the

download

Annotations files

can be quite

Baits and Targets for SureSelect Catalog

Kits are Pre-loaded

List of Agilent

SureSelect Catalog

Creating a new Experiment

Click “New

Experiment” icon to

start new experiment

Or select “New

Experiment” from

Project menu

Create New GeneSpring NGS Experiment

Provide some useful and descriptive name

Be sure to select “NGS” as the Analysis type

Importing Data: Choosing Metadata New organisms supported on demand

Indicate organism,

build and transcript

Prepackaged

annotations

available for a

variety of

organisms

Support for Illumina,

Life Technology, 454

Support for single end,

paired end, and mate

pair protocols

Provide Information on Sequencing platform and

library layout for SureSelect specific kits

Be sure to select the

previously loaded

Target region

GeneSpring NGS

Organization Of The Windows

Project and Experiment Navigator

Workflow Browser

Region View

Quality Control

Perform QC on reads and filter anomalous reads View reads by tile/lane and remove reads in anomalous tiles

qualities in

the same

tiles show

Perfect match

reads and too

mismatch

reads in this

Quality Inspection

Open Quality Control Manager

Press Compute to

calculate the Library

QC metrics

Press Compute to

calculate the Library

QC metrics

July 11 Page 18

QC Manager feature in SureSelect experiments

Off-target reads

can be removed

to focus analysis

Targeted Regions

(SureSelect Baits)

Off-target reads

can be removed

to focus analysis

Determine the expression values for

each gene

Run Quantification to determine raw gene counts Which reads contribute to a gene’s count?

These reads

do NOT

contribute

Only Reads

overlapping exonic

regions contribute to

the read count

Multiply mapping

reads contribute

fractionally to the

Quantification

Quantify Genes, Transcripts and Exons

Reverted back to the All

Aligned Reads list to

establish a base line. Feel

free to try a different

(filtered) read list

Unchecked will only count

reads falling completely

inside an exon.

New genes and exons are

discovered using

conservation data

(Conservation track in

Annotations)

Filter genes by RPKM: Results of Filtering

Profile plot of

genes that pass

the filter criteria

Profile plot of

genes that pass

the filter criteria

Handling overlapping genes Which reads contribute to a gene’s count?

These reads contribute to both

genes except for ABI data

which is strand specific

Overlapping gene on

negative strand

Gene on positive

strand

Determine expression values normalized across samples

Scatter Plot between Two Replicates

Detection of differentially expressed genes

Output of Differential Expression Analysis

P-values,

Corrected p-

values and fold

changes

Volcano Plot

Determine differentially spliced genes

detected

Identifying Differentially Spliced Genes

Compute the proportion of a

gene’s count that can be ascribed to

a particular transcript.

If the proportion for a particular

transcript changes substantially across conditions, the gene

is said to be differentially spliced.

The Challenge: Deconvoluting Transcript Read Counts

Which of the 4

transcripts do

these reads

come from?

Differential Splicing Analysis

View Results in Gene View

Ensure Splicing

Analysis Results

Entiy list is selected

Click “Gene

View” icon to

show Gene

Differential Splicing Analysis

Gene View

This transcript

is expressed

less in Tumor

Gene’s

4 Transcript

Transcript

This transcript

is expressed

Introduction to RNA-Seq in GeneSpring NGS Software · Introduction to RNA-Seq • In a few years,...

Documents

Transcript of Introduction to RNA-Seq in GeneSpring NGS Software · Introduction to RNA-Seq • In a few years,...

MaRS - Matrix of RNA-Seq · 2016-11-30 · RNA-Seq collection • RNA-Seq libraries are collected from public databases (NCBI, EBI) • We selected libraries: -with comparable data

Introductiontodiﬀerentialgeneexpressionanalysisusing RNA-seq · Figure 1 RNA-seq work flow. (a) Schematic diagram of RNA-seq library construction. Total RNA is extracted from 300,000

Poster rna seq-molecular_medtriconf_2011_a_vladimirova

BGi RNA-Seq Analysis

Analysis of RNA-seq Data - University of Hong Kongcgs.hku.hk/portal/files/GRC/Events/Seminars/2017/20170208/rna-seq.pdf · Outline • What is RNA-seq? • What can RNA-seq do? •

RNA-Seq Analysis Overview

Machine Learning Methods for RNA-seq-based Transcriptome ...rivals/SHD-2010/SPEECH/Raetsch-rnaseq-SHD-240310.pdfRNA-Seq Pipeline Deep RNA Sequencing (RNA-Seq) fml RNA-Seq allows ...

Biases in RNA- Seq data October 30, 2013 NBIC Advanced RNA- Seq course

Update - AH diagnostics...Advanta RNA-Seq NGS Library Prep on Juno RNA sequencing (RNA-seq) is the gold standard for hypothesis-free profiling of the transcriptome. Advanta™ RNA-Seq

RNA- Seq Lab

ERANGE RNA-Seq pipeline

RNA-Seq - · PDF fileWhat is RNA-seq? • RNA-seq is the high-throughput sequencing of the cDNA! • It’s used to measure the RNA expression! • It’s the NGS equivalent of

RNA-Seq analysis workshop - · PDF fileOutline • Background of RNA-Seq • Application of RNA-Seq (what RNA-Seq can do?) • Available sequencing platforms and strategies and which

Bioinformatics for DNA - seq and RNA- seq experiments

1.RNA Seq Part1 WorkingToTheGoal

RNA-seq data

RNA- seq Analysis in Galaxy

Rna seq pipeline

RNA-seq data analysis - CSC · Analysis tool overview 250 NGS tools for • RNA-seq • single cell RNA-seq • miRNA-seq • exome/genome-seq • ChIP-seq • FAIRE/DNase-seq •

RNA-seq experiments for bioinformaticians