Ambient temperature regulated flowering time

Similar documents
Analysis of Massively Parallel Sequencing Data Application of Illumina Sequencing to the Genetics of Human Cancers

genomics for systems biology / ISB2020 RNA sequencing (RNA-seq)

Transcriptome Analysis

RASA: Robust Alternative Splicing Analysis for Human Transcriptome Arrays

MODULE 4: SPLICING. Removal of introns from messenger RNA by splicing

Supplemental Figure S1. Expression of Cirbp mrna in mouse tissues and NIH3T3 cells.

Analyse de données de séquençage haut débit

Inference of Isoforms from Short Sequence Reads

Lecture 8 Understanding Transcription RNA-seq analysis. Foundations of Computational Systems Biology David K. Gifford

Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

P. Tang ( 鄧致剛 ); PJ Huang ( 黄栢榕 ) g( ); g ( ) Bioinformatics Center, Chang Gung University.

Transcript reconstruction

Supplemental Data. Integrating omics and alternative splicing i reveals insights i into grape response to high temperature

RNA-seq Introduction

RNA SEQUENCING AND DATA ANALYSIS

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR)

Accessing and Using ENCODE Data Dr. Peggy J. Farnham

Nature Biotechnology: doi: /nbt.1904

Small RNA-Seq and profiling

Phylogenomics. Antonis Rokas Department of Biological Sciences Vanderbilt University.

Hao D. H., Ma W. G., Sheng Y. L., Zhang J. B., Jin Y. F., Yang H. Q., Li Z. G., Wang S. S., GONG Ming*

Selective depletion of abundant RNAs to enable transcriptome analysis of lowinput and highly-degraded RNA from FFPE breast cancer samples

MODULE 3: TRANSCRIPTION PART II

Simple, rapid, and reliable RNA sequencing

Analysis and design of RNA sequencing experiments for identifying isoform regulation

Deep-Sequencing of HIV-1

Mechanisms of alternative splicing regulation

Daehwan Kim September 2018

AbSeq on the BD Rhapsody system: Exploration of single-cell gene regulation by simultaneous digital mrna and protein quantification

Widespread alternative and aberrant splicing revealed by lariat sequencing

Genomic structural variation

Transcriptome and isoform reconstruc1on with short reads. Tangled up in reads

BIMM 143. RNA sequencing overview. Genome Informatics II. Barry Grant. Lecture In vivo. In vitro.

Computational Identification and Prediction of Tissue-Specific Alternative Splicing in H. Sapiens. Eric Van Nostrand CS229 Final Project

BWA alignment to reference transcriptome and genome. Convert transcriptome mappings back to genome space

RNA- seq Introduc1on. Promises and pi7alls

Nature Genetics: doi: /ng.3731

York criteria, 6 RA patients and 10 age- and gender-matched healthy controls (HCs).

Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

Computational Analysis of UHT Sequences Histone modifications, CAGE, RNA-Seq

Gene finding. kuobin/

Iso-Seq Method Updates and Target Enrichment Without Amplification for SMRT Sequencing

Supplementary Material for IPred - Integrating Ab Initio and Evidence Based Gene Predictions to Improve Prediction Accuracy

RNA-Seq guided gene therapy for vision loss. Michael H. Farkas

Introduction to Systems Biology of Cancer Lecture 2

REGULATED SPLICING AND THE UNSOLVED MYSTERY OF SPLICEOSOME MUTATIONS IN CANCER

fl/+ KRas;Atg5 fl/+ KRas;Atg5 fl/fl KRas;Atg5 fl/fl KRas;Atg5 Supplementary Figure 1. Gene set enrichment analyses. (a) (b)

GENOME-WIDE DETECTION OF ALTERNATIVE SPLICING IN EXPRESSED SEQUENCES USING PARTIAL ORDER MULTIPLE SEQUENCE ALIGNMENT GRAPHS

Advance Your Genomic Research Using Targeted Resequencing with SeqCap EZ Library

RNA SEQUENCING AND DATA ANALYSIS

Supplementary Figure 1 Transcription assay of nine ABA-responsive PP2C. Transcription assay of nine ABA-responsive PP2C genes. Total RNA was isolated

An Analysis of MDM4 Alternative Splicing and Effects Across Cancer Cell Lines

A Practical Guide to Integrative Genomics by RNA-seq and ChIP-seq Analysis

AVENIO family of NGS oncology assays ctdna and Tumor Tissue Analysis Kits

High-Resolution Expression Map of the Arabidopsis Root Reveals Alternative Splicing and lincrna Regulation

Bio 111 Study Guide Chapter 17 From Gene to Protein

Tutorial: RNA-Seq Analysis Part II: Non-Specific Matches and Expression Measures

DNA-seq Bioinformatics Analysis: Copy Number Variation

Variant Classification. Author: Mike Thiesen, Golden Helix, Inc.

Alternative splicing. Biosciences 741: Genomics Fall, 2013 Week 6

RNA- seq Introduc1on. Promises and pi7alls

Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal cancer Gosia Komor

NGS in tissue and liquid biopsy

Supplementary Materials and Methods

TITLE: The Role Of Alternative Splicing In Breast Cancer Progression

CRISPR/Cas9 Enrichment and Long-read WGS for Structural Variant Discovery

Comprehensive Genome and Transcriptome Structural Analysis of a Breast Cancer Cell Line using PacBio Long Read Sequencing

SCIENCE CHINA Life Sciences

Introduction. Introduction

Supplemental Methods RNA sequencing experiment

Supplementary Figure 1 IL-27 IL

High Throughput TruSeq Stranded mrna Library Construction on the Biomek FX P

Results. Abstract. Introduc4on. Conclusions. Methods. Funding

Studying Alternative Splicing

SUPPLEMENTARY INFORMATION

RECENT ADVANCES IN THE MOLECULAR DIAGNOSIS OF BREAST CANCER

Histone Modifications Are Associated with Transcript Isoform Diversity in Normal and Cancer Cells

Supplementary Figures

Nature Structural & Molecular Biology: doi: /nsmb.2419

Circular RNAs (circrnas) act a stable mirna sponges

Multifactorial Interplay Controls the Splicing Profile of Alu-Derived Exons

Not IN Our Genes - A Different Kind of Inheritance.! Christopher Phiel, Ph.D. University of Colorado Denver Mini-STEM School February 4, 2014

Evaluation of MIA FORA NGS HLA test and software. Lisa Creary, PhD Department of Pathology Stanford Blood Center Research & Development Group

2/10/2016. Evaluation of MIA FORA NGS HLA test and software. Disclosure. NGS-HLA typing requirements for the Stanford Blood Center

Genomic DNA, extracted from PBMCs, was isolated from whole blood obtained at baseline, and at the

CONTRACTING ORGANIZATION: Joan & Sanford I. Weill Medical College of Cornell University New York, NY 10065

High-throughput transcriptome sequencing

Shatakshi Pandit, Yu Zhou, Lily Shiue, Gabriela Coutinho-Mansfield, Hairi Li, Jinsong Qiu, Jie Huang, Gene W. Yeo, Manuel Ares, Jr., and Xiang-Dong Fu

Identification and characterization of multiple splice variants of Cdc2-like kinase 4 (Clk4)

Supplemental Materials and Methods Plasmids and viruses Quantitative Reverse Transcription PCR Generation of molecular standard for quantitative PCR

Eukaryotic small RNA Small RNAseq data analysis for mirna identification

SUPPLEMENTARY FIGURES: Supplementary Figure 1

Supplementary Figure S1. Gene expression analysis of epidermal marker genes and TP63.

Characterisation of structural variation in breast. cancer genomes using paired-end sequencing on. the Illumina Genome Analyser

VirusDetect pipeline - virus detection with small RNA sequencing

ncounter Assay Automated Process Immobilize and align reporter for image collecting and barcode counting ncounter Prep Station

RECAP (1)! In eukaryotes, large primary transcripts are processed to smaller, mature mrnas.! What was first evidence for this precursorproduct

Transcriptional control in Eukaryotes: (chapter 13 pp276) Chromatin structure affects gene expression. Chromatin Array of nuc

Rapid genomic screening of embryos using nanopore sequencing

Alternative splicing control 2. The most significant 4 slides from last lecture:

Transcription:

Ambient temperature regulated flowering time Applications of RNAseq RNA- seq course: The power of RNA-seq June 7 th, 2013; Richard Immink

Overview Introduction: Biological research question/hypothesis Experimental set-up and preparations Data analysis Data confirmation Data interpretation: Back to the biological research question

Flowering time: When to flower?

Environmental factors influencing flowering time Floral integrator genes

Effects of ambient temperature fluctuations 23 C 27 C Floral integrator genes

The ambient temperature pathway For the majority of Regulators no temperature-dependent differential expression!? Balasubramanian et al, PLoS Genetics, 2006

Hypothesis Alternative splicing (AS) is a key mechanism in ambient temperature regulation of flowering time What is the genome-wide effect of temperature fluctuations on expression and splicing?

Experimental set-up: How to detect and quantify AS events? AS characteristics: - Events can be rear - Differences can be limited (few nt only) Plant species related questions: - Genome sequence available? - Arrays available?

Tiling array or RNA-seq?

Correlation of expression levels

Exon Boundary detection

RNA-seq!... But, which method? Seq length (nt) Read quality Throughput fixed Illumina Hiseq 50/100 +++ 275 Gb/flowcell fixed SOLiD 5500 75 ++++ 120 Gb/flowcell range 454/Roche av~750 ++ 1 Mb range Ion Torrent av~170 + 70 Mb/chip Pac Bio range +/- 300.000/run ~3,5 kb

Illumina Hiseq Further considerations: cdna - Sample multiplexing? How many? - 50 nt or 100 nt run? - Paired-end or Single read?

RNAseq to search for temperature affected alternative splicing (AS) 23 C 16 C 23 C 27 C Paired-end 100nt Illumina Hiseq runs in duplo, 200-300 bp length fragments, (multiplexing of 4 samples/lane) Comparison of transcriptome of 23-23 ºC control vs. 23-27º switch and vs 23-16º C switch (after 24 hrs!) Identify genes that are differentially alternatively spliced (and/or differentially expressed).

Sample preparation (Elio Schijlen) Delivery of 2-10 μg total RNA

Data analysis (Edouard Severing) Mapping to the genome Transcriptome assembly, quantification of events, identification of differentially regulated events

Mapping to the genome: Tophat 2.0 Splice junction Settings: Insert size: (~220; sd ~20; between two adapters) No discordant alignments Alignments have fulfill the expectations given the insert size and map orientation Alignments have to be paired (no mix mode) Reads aligned to unique regions of the genome are considered Min max intron size: 50-11000.

Identification of differential expression/splicing: Cufflinks Settings: Only use of uniquely mapped reads Existing annotation is provided to Cufflinks Min max intron size: 50-11000

Results: reproducibility (duplo)

Results Total amount of differentially alternatively spliced genes 23 C 27 C: 1107 genes 23 C 16 C: 897 genes Reciprocal: 135 genes (15%) Differentially alternatively spliced flowering genes 23 C 27 C: 17 genes 23 C 16 C: 15 genes Overlap: 7 genes Reciprocal: 4 genes (including FLM) Relative expression cold Control heat Reciprocal

RNA-seq results: FLM alternative splicing (mutually exclusive exon) FLMβ FLMδ 23 C 27 C 23 C 16 C

RNAseq results: FLM alternative splicing (mutually exclusive exon) FLMβ FLMδ 16 C 23 C 27 C

Alternative splicing in MAF3 (alternative 3 splice site)

Alternative splicing in MAF3 (alternative 3 splice site 23 27 C 23 16 C

Problem I: Strange events! At5G65050 = MAF2 At5G65060 = MAF3 MAF2 MAF3 MAF2 and MAF3 joined as single gene

Solution Divide genes in variable and constitutive blocks (excluding terminal regions). For each variable region we group the isoforms based on their local composition V-region 1 3-groups V-region 2 2-groups

Problem II: Mixed reads

Data confirmation: qrt-pcr - Primer design! - Blast primers against RNA-seq data! - Note: Analyse based on isoform ratio s and not absolute values! (semi-quantitative) (in case to complex: amplify start till end clone mix of isoforms sequence individual clones

Data interpretation: back to our hypothesis Alternative splicing (AS) is a key mechanism in ambient temperature regulation of flowering time

The FLM case FLMβ FLMδ 16 C 27 C - Both FLMβ and FLMδ are predicted not to be targetted by NMD - Both FLMβ and FLMδ transcripts are translated (hence a protein is produced)

Functional analysis FLM isoforms Flowering time FLMβ = repressor of flowering FLMδ = activator of flowering Col0 35S::FLMβ 35S::FLMδ David Posé and Markus Schmid, submitted

Conclusion: the ambient temperature dependent flowering time control can be explained by FLM AS!

Acknowledgements PRI-Bioscience-PDS Leonie Verhage Gerco Angenent MPI Tuebingen David Posé Markus Schmid WUR-Bioinformatics Edouard Severing Elio Schijlen Horizon Breakthrough

2024 © healthdocbox.com Privacy Policy | Terms of Service | Feedback