Differential gene expression

[Pink Sherbet Photography]
RNAseq analysis: Differential gene expression (2/2)
Hopscotch and isoforms
August 25, 2011

Reads->alignment to reference genome->transcript assembly
Resulting file type: BAM, gff/bed
“What transcripts are in my samples?”
August 25, 2011
Transcript assembly
Projects
Fastq
Mapping
Quick recap: Mapping and transcript assembly
Garber M, Grabherr MG, Guttman M, Trapnell C. Computational methods for transcriptome annotation and quantification using RNA-seq. Nat Methods. 2011 PMID: 21623353.

RNAseq analysis question
Is there a difference in the transcriptome of two different conditions ?
Quantify expression
Quantify difference
August 25, 2011
Condition1 Condition2

RNAseqvsExpression Array
RNAseq can capture a larger dynamic range
RNAseq can handle degraded samples
Gain additional information
New transcripts
(New) isoforms
Variants
August 25, 2011
Flattening
out
Array
RNA-seq
Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009 PMID: 19015660

Challenges
Strand-specific methods still biased
Number of reads not necessarily correlate with transcript abundance
Longer transcripts have more reads (fragmentation).
Technical variability between runs causes different number of total reads.
Lowly abundant does not mean non-functional
How to quantify expression of isoforms
August 25, 2011
Ozsolak F, Milos PM. RNA sequencing: advances, challenges and opportunities. Nat Rev Genet. 2011 PMID: 21191423

Production Informatics and Bioinformatics
August 25, 2011
Produce raw sequence reads
Basic Production
Informatics
Map to genome and generate raw genomic features (e.g. SNPs)
Advanced
Production Inform.
Analyze the data; Uncover the biological meaning
Bioinformatics
Research
Per one-flowcell project

Quantifying expression in RNAseq
Long genes get more reads
Normalize: fragments per kilobase of transcript per million mapped reads (FPKM)
FPKM accounts for the dependency between paired-end reads
August 25, 2011
Oshlack A, Wakefield MJ. Transcript length bias in RNA-seq data confounds systems biology. Biol Direct. 2009 PMID: 19371405

Quantifying expression of overlapping isoforms
We do not know where reads of overlapping isoformsacutally belong
Alexa-Seq
counting only the reads that map uniquely to a single isoform
isoform-expression methods (cufflinks)
likelihood function modeling the sequencing process (not very accurate for lowly expressed transcripts)
'exon intersection method’ (analogous to expression microarrays)
counts reads mapped to its constitutive exons (reduce power for differential expression analysis)
'exon union method’
counts all reads mapped to any exon in any of the gene's isoforms (underestimates expression for alternatively spliced genes).
August 25, 2011

Differentially expression
What is a statistically significant difference between a set of measurements (expression of a gene) of two populations (conditions)
First, estimate variability
Observe biological variability (needs large numbers of replicates to sample the population).
model biological variability
model the count variance across replicates as a nonlinear function of the mean counts using various different parametric approaches (such as the normal and negative binomial distributions) (EdgeR, DESeq, Cuffdiff)
August 25, 2011

Three things to remember
RNAseq captures larger dynamic range (more sensitive)
Additional information compared to arrays (e.g. isoforms)
Need to make assumptions/compromises (quantification, few replicates)
August 25, 2011
[cabbit]

Next Weeks: NGS Discussion group Jake’s topic
August 25, 2011
Two Weeks:
Abstract: This session will focus on identifying SNPs from whole genome, exome capture or targeted resequencing data. The approaches of mapping, local realigment, recalibration, SNP calling, and SNP recalibration will be introduced and quality metrics discussed.

Differential gene expression

by Denis Bauer on Sep 07, 2011

Accessibility

Categories

Tags

Upload Details

Usage Rights

Statistics

Differential gene expression — Presentation Transcript