Characterization of A. thaliana adenylate - isopentenyltransferases
Introductory RNA-seq Transcriptome Profiling of the hy5 mutation in Arabidopsis thaliana.
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Transcript of Introductory RNA-seq Transcriptome Profiling of the hy5 mutation in Arabidopsis thaliana.
Introductory RNA-seq Transcriptome Profiling of the hy5 mutation in
Arabidopsis thaliana
Before we start: Align sequence reads to the reference genome
The most time-consuming part of the analysis is doing the alignments of the reads (in Sanger fastq format) for all replicates against the reference genome.
Overview: This training module is designed to provide a hands on experience in using RNA-Seq for transcriptome profiling.
Question: How well is the annotated transcriptome represented in RNA-seq data in Arabidopsis WT and hy5 genetic backgrounds?
How can we compare gene expression levels in the two samples?
RNA-seq in the Discovery Environment
Scientific Objective
LONG HYPOCOTYL 5 (HY5) is a basic leucine zipper transcription factor (TF).
Mutations in the HY5 gene cause aberrant phenotypes in Arabidopsis morphology, pigmentation and hormonal response.
We will use RNA-seq to compare the transcriptomes of seedlings from WT and hy5 genetic backgrounds to identify HY5-regulated genes.
Samples
• Experimental data downloaded from the NCBI Short Read Archive (GEO:GSM613465 and GEO:GSM613466)
• Two replicates each of RNA-seq runs for Wild-type and hy5 mutant seedlings.
Specific Objectives
By the end of this module, you should
1)Be more familiar with the DE user interface
2)Understand the starting data for RNA-seq analysis
3)Be able to align short sequence reads with a reference genome in the DE
4)Be able to analyze differential gene expression in the DE
5)Be able to use DE text manipulation tools to explore the gene expression data
Quick Summary
Find D
iffere
ntially
Expre
ssed genes
Align to
Genom
e: TopHat
View Alig
nments
: IGV
Differe
ntial E
xpressio
n: CuffD
iff
Download R
eads from
SRA
Export Reads to
FASTQ
Import SRA data from NCBI SRA
Extract FASTQ files from the
downloaded SRA archives
Pre-Configured: Getting the RNA-seq Data
RNA-Seq Conceptual Overview
Image source: http://www.bgisequence.com
RNA-Seq Workflow Overview
Align the four FASTQ files to Arabidopsis genome using TopHat
Step 1: Align Reads to the Genome
TopHat
• TopHat is one of many applications for aligning short sequence reads to a reference genome.
• It uses the BOWTIE aligner internally.
• Other alternatives are BWA, MAQ, TopHat, Stampy, Novoalign, etc.
RNA-seq Sample Read Statistics
• Genome alignments from TopHat were saved as BAM files, the binary version of SAM ().
• Reads retained by TopHat are shown below
Sequence run WT-1 WT-2 hy5-1 hy5-2
Reads 10,866,702 10,276,268 13,410,011 12,471,462
Seq. (Mbase) 445.5 421.3 549.8 511.3
Index BAM files using SAMtools
Prepare BAM files for viewing
Using IGV in Atmosphere
1. We already Launched an instance of NGS Viewers in Atmosphere
2. Use VNClient to connect to your remote desktop
Pre-configured VM for NGS Viewers
The Integrative Genomics Viewer (IGV) is a high-performance visualization tool for interactive exploration of large, integrated genomic datasets. It supports a wide variety of data types, including array-based and next-generation sequence data, and genomic annotations.
Use IGV to inspect outputs from TopHat
http://www.broadinstitute.org/igv/
Integrated Genomics Viewer (IGV)
ATG44120 (12S seed storage protein) significantly down-regulated in hy5 mutantBackground (> 9-fold p=0). Compare to gene on right lacking differential expression
Other Ways to View Alignment Data WIG->Ensembl
RNA-Seq Workflow Overview
CuffDiff
• CuffLinks is a program that assembles aligned RNA-Seq reads into transcripts, estimates their abundances, and tests for differential expression and regulation transcriptome-wide.
• CuffDiff is a program within CuffLinks that compares transcript abundance between samples
Examining Differential Gene Expression
Examining the Gene Expression Data
Filter CuffDiff results for up or down-regulated gene expression in hy5 seedlings
Differentially expressed genes
Differentially expressed genes
Example filtered CuffDiff results generated with the Filter_CuffDiff_Results to1)Select genes with minimum two-fold expression difference2)Select genes with significant differential expression (q <= 0.05)3)Add gene descriptions