Differential gene expression analysis using RNA-seq
|
|
- Sabrina Holland
- 6 years ago
- Views:
Transcription
1 Differential gene expression analysis using RNA-seq Applied Bioinformatics Core, August 2017 Friederike Dündar with Luce Skrabanek & Ceyda Durmaz
2 Day 1: Introduction into high-throughput sequencing [many general concepts!] 1. RNA isolation & library preparation 2. Illumina s sequencing by synthesis 3. raw sequencing reads download quality control 4. experimental design
3 RNA-seq is popular, but still developing RNA%seq)is)not$a$mature$technology.)It$is$ undergoing$rapid$evolution$of)biochemistry) of)sample)preparation;)of)sequencing) platforms;)of)computational)pipelines;)and)of$ subsequent$analysis$methods$that$include$ statistical$treatments)and)transcript)model) building.) ) ENCODE&consortium& Reuter et al. ( 2015). Mol Cell. Goodwin, McPherson & McCombie (2016). Nat Gen, 17(6),
4 Analysis paralysis basically no generally accepted standard reference myriad tools! highly complex & specialized pipelines The ( ) flexibility and seemingly infinite set of options ( ) have hindered its path to the clinic. ( ) The fixed nature of probe sets with microarrays or qrt-pcr offer an accelerated path ( ) without the lure of the latest and newest analysis methods. Byron et al., 2016 Byron et al. Nat Rev Genetics (2016)
5 What to expect from the class Sample type & quality Library preparation Poly-A enrichment vs. ribo minus Strand information Sequencing Read length PE vs. SR Sequencing errors Biological question Expression quantification Alternative splicing De novo assembly needed mrnas, small RNAs. Experimental design Controls No. of replicates Randomization Bioinformatics Aligner Normalization DE analysis strategy NOT COVERED: novel transcript discovery transcriptome assembly alternative splicing analysis (see the course notes for references to useful reviews)
6 cells RNA fragments cdna with adapters RNA-seq workflow overview Total RNA extraction Fragmentation mrna enrichment Library preparation Sequencing Bioinformatics Cluster generation Sequencing by synthesis Image acquisition
7 Quality control of RNA extraction 28S:18S ratio avoid degraded RNA junk
8 QC! RNA-seq library preparation RNA extraction rrna depletion/mrna enrichment poly(a) enrichment or ribo-depletion fragmentation random priming and reverse transcription 3 adapter ligation second strand synthesis 5 adapter ligation U U U U UU U end repair, A- addition, adapter ligation end repair, A- addition, adapter ligation U U reverse transcription PCR PCR PCR classical Illumina protocol (unstranded) dutp stranded library preparation sequential ligation of two different adapters Van Dijk et al. (2014). Experimental Cell Research, 322(1), doi: /j.yexcr
9 RNA-seq workflow overview cells Total RNA extraction RNA fragments cdna with adapters Sequencing flowcell with primers
10 Cluster generation bridge amplification denaturation cluster generation removal of complementary strands! identical fragment copies remain
11 Image from Illumina Sequencing by synthesis labelled dntp 1. extend 1 st base 2. read 3. deblock repeat for bp generate base calls
12 Typical biases of Illumina sequencing sequencing errors miscalled bases PCR artifacts (library preparation) duplicates (due to low amounts of starting material) length bias GC bias sample-specific problems! RNA-seq-specific Figure from Love et al. (2016). Nat Biotech, 34(12). More details & refs in course notes (esp. Table 6).
13 General sources of biases (not inherently sample-specific) issues with the reference CNV mappability inappropriate data processing inclusion of multi-mapped reads exclusion of multi-mapped reads
14 RAW SEQUENCING READS Let the data wrangling begin!
15 Bioinformatics workflow of RNA-seq analysis Images.tif FASTQC Raw reads.fastq Aligned reads.sam/.bam Base calling & demultiplexing Bustard/RTA/OLB, CASAVA Mapping STAR Counting featurecounts Read count table.txt Normalized read count table.robj List of fold changes & statistical values.robj,.txt Downstream analyses on DE genes Normalizing DESeq2, edger DE test & multiple testing correction DESeq2, edger, limma Filtering Customized scripts
16 Where are all the reads? GenBank Sequence Read Archive DDBJ ENA The SRA is the main repository for publicly available DNA and RNA sequencing data of which three instances are maintained world-wide.
17 Let s download! We will work with a data set submitted by Gierlinski et al. they deposited the sequence files with SRA we will retrieve it via ENA ( accession number: ERP Course See Section 2 (Raw Data) for download instructions etc. ls mkdir wget cut grep awk
18 FASTQ file format = FASTA + quality scores 1 read " 4 lines! DHKW5DQ1:219:D0PT7ACXX:2:1101:1590:2149/1 GATCGGAAGAGCGGTTCAGCAGGAATGCCGAGATCGGAAGAGCGGTTCAGC >HHHEGHIIIGGIFFGIBFAAGAFHA 5?B@D ID and sequencing run information 2. sequence 3. + (additional description possible) 4. quality scores
19 Base quality DHKW5DQ1:219:D0PT7ACXX:2:1101:1590:2149/1 GATCGGAAGAGCGGTTCAGCAGGAATGCCGAGATCGGAAGAGCGGTTCAGC >HHHEGHIIIGGIFFGIBFAAGAFHA 5?B@D base error probability p, e.g. 10e-4! -10 x log10(p) turn score into ASCII symbol Phred score, e.g.: 40 FASTQ score, e.g.: (
20 Base quality scores each base has a certain error probability (p) Phred score = -10 x log10(p) Phred scores are ASCII-encoded, e.g.,! COULD represent Phred score 33 SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS......XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX......IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII......JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ... LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL...!"#$%&'()*+,-./ :;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{ }~ S - Sanger Phred+33, raw reads typically (0, 40) X - Solexa Solexa+64, raw reads typically (-5, 40) I - Illumina 1.3+ Phred+64, raw reads typically (0, 40) J - Illumina 1.5+ Phred+64, raw reads typically (3, 40) with 0=unused, 1=unused, 2=Read Segment Quality Control Indicator (bold) (Note: See discussion above). L - Illumina 1.8+ Phred+33, raw reads typically (0, 41) also see Table 2 in the course notes image from
21 Quality control of raw reads: FastQC FastQC aims to provide a simple way to do some quality control checks on raw sequence data coming from high throughput sequencing pipelines. It provides a modular set of analyses which you can use to give a quick impression of whether your data has any problems of which you should be aware before doing any further analysis. The main functions of FastQC are: Import of data from BAM, SAM or FastQ files (any variant) Providing a quick overview to tell you in which areas there may be problems Summary graphs and tables to quickly assess your data Export of results to an HTML based permanent report Offline operation to allow automated generation of reports without running the interactive application not specific for RNA-seq data! $ mat/software/fastqc/fastqc $ mat/software/anaconda2/bin/multiqc
22 EXPERIMENTAL DESIGN How to avoid spurious signals and drowning in noise
23 How deep is deep enough? for DGE (logfc~ 2) in mammals: mio SR, 75 bp Goals that require more, longer, and possibly pairedend reads: quantification of lowly expressed genes identification of genes with small changes between conditions investigation of alternative splicing/isoform quantification identification of novel transcripts, chimeric transcripts de novo transcriptome assembly
24 doi: /nmeth.2613 Why do we need replicates? Goal: Identify differences in expression for every gene. and differences should preferably be due to our experiment, not noise! Samples are our windows to the population, and their statistics are used to estimate those of the population. Martin Krzywinski & Naomi Altman
25 Gierliński et al. (2015). Bioinformatics, 31(22), & Schurch et al. (2016) RNA. Invest in replicates! recommended: 6 biological replicates per condition for DGE of strongly changing genes (logfc >= 2) [based on insights from the fairly simple yeast transcriptome] Gene X log2 Counts condition 1 condition 2
26 also see course notes and Blainey et al. (2014) Nature Methods, 1(9) Technical replicates Replicates library prep sequencing lane sequencing lane RNA extraction library prep sequencing lane sequencing lane Biological replicates RNA from an independent growth of cells/tissue sequencing lane RNA extraction sequencing lane library prep RNA extraction sequencing lane sequencing lane
27 Lin, Lin, and Snyder (2014). PNAS 111:48 Gilad & Mizrahi-Man (2015). F1000Research 4:121 Batch effects can happen everywhere Overall,)our)results)indicate)that)there)is) considerable$rna$expression$diversity$ between$humans$and$mice,)well)beyond) what)was)described)previously,)likely) reilecting)the)fundamental)physiological) differences)between)these)two)organisms.) ) Once$we$accounted$for$the$batch$effect$[i.e.,) mouse)and)human)samples)being)sequenced)on)two) different)machines])( ),)the)comparative)gene) expression)data)no)longer)clustered)by)species,)and) instead,)we)observed)a$clear$tendency$for$ clustering$by$tissue. ))
28 ENCODE s* study design was not optimal Tissue was confounded with (at least): sequencer sex age tissue handling human data: deceased organ donors mouse data: 10-week-old littermates A very good read (including the reviews and comments that discuss many scientific as well as ethical issues: * not just ENCODE: see e.g. Leek et al. (2010) Nat Rev Gen 11(10) or Jaffe & Irizarry (2014) Genome Biol 15(R31) 1 9
29 Completely randomized design Avoiding bias Restricted randomized design Blocked & randomized design WEIGHT Block what you can, randomize what you cannot. What factors are of interest? Which ones might introduce noise? Which nuisance factors do you absolutely need to account for? Krzywinski & Altman (2014) Nature Methods 11(7)
30 Auer & Doerge (2010). Genetics, 185(2), Typical RNA-seq set-up keep the technical nuisance factors (harvest date, RNA extraction kit, sequencing date ) to a minimum cover only as much of the biological variation as needed (just keep possible restrictions about your conclusions in mind for later) Make sure the sequencing core multiplexes all samples!
31 Summary Day 1 RNA-seq analysis is not a completely solved issue but DE analysis on a gene level is decently mature and the field seems to gravitate towards some sort of standard no analysis tool can enforce (or replace!) common sense and knowledge about the biology behind the experiment crap in, crap out more replicates are often better investments than more reads FastQC and multiqc are great tools to detect possible technical nuisance factors
Differential gene expression analysis using RNA-seq
https://abc.med.cornell.edu/ Differential gene expression analysis using RNA-seq Applied Bioinformatics Core, March 2018 Friederike Dündar with Luce Skrabanek & Paul Zumbo Day 1: Introduction into high-throughput
More informationDifferential gene expression analysis using RNA-seq
https://abc.med.cornell.edu/ Differential gene expression analysis using RNA-seq Applied Bioinformatics Core, August 2017 Friederike Dündar with Luce Skrabanek & Ceyda Durmaz Day 4 overview (brief) theoretical
More informationDifferential gene expression analysis using RNA-seq
https://abc.med.cornell.edu/ Differential gene expression analysis using RNA-seq Applied Bioinformatics Core, August 2017 Friederike Dündar with Luce Skrabanek & Ceyda Durmaz Day 3 QC of aligned reads
More informationExperimental Design. Sequencing. Data Quality Control. Read mapping. Differential Expression analysis
-Seq Analysis Quality Control checks Reproducibility Reliability -seq vs Microarray Higher sensitivity and dynamic range Lower technical variation Available for all species Novel transcript identification
More informationExperimental Design. Dr. Matthew L. Settles. Genome Center University of California, Davis
Experimental Design Dr. Matthew L. Settles Genome Center University of California, Davis settles@ucdavis.edu What is Differential Expression Differential expression analysis means taking normalized sequencing
More informationRead Quality Assessment & Improvement. UCD Genome Center Bioinformatics Core Tuesday 14 June 2016
Read Quality Assessment & Improvement UCD Genome Center Bioinformatics Core Tuesday 14 June 2016 QA&I should be interactive Error modes Each technology has unique error modes, depending on the physico-chemical
More informationLecture 7. Next-generation sequencing technologies
Lecture 7 Next-generation sequencing technologies Next-generation sequencing technologies General principles of short-read NGS Construct a library of fragments Generate clonal template populations Massively
More informationRNA-Seq data analysis course September 7-9, 2015
RNA-Seq data analysis course September 7-9, 2015 Peter-Bram t Hoen (LUMC) Jan Oosting (LUMC) Celia van Gelder, Jacintha Valk (BioSB) Anita Remmelzwaal (LUMC) Expression profiling DNA mrna protein Comprehensive
More informationTranscriptomics analysis with RNA seq: an overview Frederik Coppens
Transcriptomics analysis with RNA seq: an overview Frederik Coppens Platforms Applications Analysis Quantification RNA content Platforms Platforms Short (few hundred bases) Long reads (multiple kilobases)
More informationNext Generation Sequencing
Next Generation Sequencing Complete Report Catalogue # and Service: IR16001 rrna depletion (human, mouse, or rat) IR11081 Total RNA Sequencing (80 million reads, 2x75 bp PE) Xxxxxxx - xxxxxxxxxxxxxxxxxxxxxx
More informationTranscriptome analysis
Statistical Bioinformatics: Transcriptome analysis Stefan Seemann seemann@rth.dk University of Copenhagen April 11th 2018 Outline: a) How to assess the quality of sequencing reads? b) How to normalize
More informationSO YOU WANT TO DO A: RNA-SEQ EXPERIMENT MATT SETTLES, PHD UNIVERSITY OF CALIFORNIA, DAVIS
SO YOU WANT TO DO A: RNA-SEQ EXPERIMENT MATT SETTLES, PHD UNIVERSITY OF CALIFORNIA, DAVIS SETTLES@UCDAVIS.EDU Bioinformatics Core Genome Center UC Davis BIOINFORMATICS.UCDAVIS.EDU DISCLAIMER This talk/workshop
More informationIntroduction to differential gene expression analysis using RNA-seq
Introduction to differential gene expression analysis using RNA-seq Written by Friederike Dündar, Luce Skrabanek, Paul Zumbo September 2015 updated March 20, 2018 1 Introduction to RNA-seq 4 1.1 RNA extraction............................................
More informationAnalysis of RNA-seq Data. Feb 8, 2017 Peikai CHEN (PHD)
Analysis of RNA-seq Data Feb 8, 2017 Peikai CHEN (PHD) Outline What is RNA-seq? What can RNA-seq do? How is RNA-seq measured? How to process RNA-seq data: the basics How to visualize and diagnose your
More informationWheat CAP Gene Expression with RNA-Seq
Wheat CAP Gene Expression with RNA-Seq July 9 th -13 th, 2018 Overview of the workshop, Alina Akhunova http://www.ksre.k-state.edu/igenomics/workshops/ RNA-Seq Workshop Activities Lectures Laboratory Molecular
More informationDeep Sequencing technologies
Deep Sequencing technologies Gabriela Salinas 30 October 2017 Transcriptome and Genome Analysis Laboratory http://www.uni-bc.gwdg.de/index.php?id=709 Microarray and Deep-Sequencing Core Facility University
More informationIntroduction to RNA-Seq. David Wood Winter School in Mathematics and Computational Biology July 1, 2013
Introduction to RNA-Seq David Wood Winter School in Mathematics and Computational Biology July 1, 2013 Abundance RNA is... Diverse Dynamic Central DNA rrna Epigenetics trna RNA mrna Time Protein Abundance
More informationIntroduction to differential gene expression analysis using RNA-seq
Introduction to differential gene expression analysis using RNA-seq Written by Friederike Dündar, Luce Skrabanek, Paul Zumbo September 2015 updated August 28, 2017 Contents 1 Introduction to RNA-seq 4
More informationNext-generation sequencing and quality control: An introduction 2016
Next-generation sequencing and quality control: An introduction 2016 s.schmeier@massey.ac.nz http://sschmeier.com/bioinf-workshop/ Overview Typical workflow of a genomics experiment Genome versus transcriptome
More informationRNA-Seq Analysis. Simon Andrews, Laura v
RNA-Seq Analysis Simon Andrews, Laura Biggins simon.andrews@babraham.ac.uk @simon_andrews v2018-10 RNA-Seq Libraries rrna depleted mrna Fragment u u u u NNNN Random prime + RT 2 nd strand synthesis (+
More informationRNA-Seq Workshop AChemS Sunil K Sukumaran Monell Chemical Senses Center Philadelphia
RNA-Seq Workshop AChemS 2017 Sunil K Sukumaran Monell Chemical Senses Center Philadelphia Benefits & downsides of RNA-Seq Benefits: High resolution, sensitivity and large dynamic range Independent of prior
More informationTECH NOTE Pushing the Limit: A Complete Solution for Generating Stranded RNA Seq Libraries from Picogram Inputs of Total Mammalian RNA
TECH NOTE Pushing the Limit: A Complete Solution for Generating Stranded RNA Seq Libraries from Picogram Inputs of Total Mammalian RNA Stranded, Illumina ready library construction in
More informationSanger vs Next-Gen Sequencing
Tools and Algorithms in Bioinformatics GCBA815/MCGB815/BMI815, Fall 2017 Week-8: Next-Gen Sequencing RNA-seq Data Analysis Babu Guda, Ph.D. Professor, Genetics, Cell Biology & Anatomy Director, Bioinformatics
More informationHigh Throughput Sequencing the Multi-Tool of Life Sciences. Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center
High Throughput Sequencing the Multi-Tool of Life Sciences Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center Complementary Approaches Illumina Still-imaging of clusters (~1000
More informationIntegrated NGS Sample Preparation Solutions for Limiting Amounts of RNA and DNA. March 2, Steven R. Kain, Ph.D. ABRF 2013
Integrated NGS Sample Preparation Solutions for Limiting Amounts of RNA and DNA March 2, 2013 Steven R. Kain, Ph.D. ABRF 2013 NuGEN s Core Technologies Selective Sequence Priming Nucleic Acid Amplification
More informationIntroduction to RNA-Seq in GeneSpring NGS Software
Introduction to RNA-Seq in GeneSpring NGS Software Dipa Roy Choudhury, Ph.D. Strand Scientific Intelligence and Agilent Technologies Learn more at www.genespring.com Introduction to RNA-Seq In a few years,
More informationParts of a standard FastQC report
FastQC FastQC, written by Simon Andrews of Babraham Bioinformatics, is a very popular tool used to provide an overview of basic quality control metrics for raw next generation sequencing data. There are
More information1. Introduction Gene regulation Genomics and genome analyses
1. Introduction Gene regulation Genomics and genome analyses 2. Gene regulation tools and methods Regulatory sequences and motif discovery TF binding sites Databases 3. Technologies Microarrays Deep sequencing
More informationIllumina Sequencing Error Profiles and Quality Control
Illumina Sequencing Error Profiles and Quality Control RNA-seq Workflow Biological samples/library preparation Sequence reads FASTQC Adapter Trimming (Optional) Splice-aware mapping to genome Counting
More informationHigh Throughput Sequencing the Multi-Tool of Life Sciences. Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center
High Throughput Sequencing the Multi-Tool of Life Sciences Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center DNA Technologies & Expression Analysis Cores HT Sequencing (Illumina
More informationDATA FORMATS AND QUALITY CONTROL
HTS Summer School 12-16th September 2016 DATA FORMATS AND QUALITY CONTROL Romina Petersen, University of Cambridge (rp520@medschl.cam.ac.uk) Luigi Grassi, University of Cambridge (lg490@medschl.cam.ac.uk)
More informationData Basics. Josef K Vogt Slides by: Simon Rasmussen Next Generation Sequencing Analysis
Data Basics Josef K Vogt Slides by: Simon Rasmussen 2017 Generalized NGS analysis Sample prep & Sequencing Data size Main data reductive steps SNPs, genes, regions Application Assembly: Compare Raw Pre-
More informationNext Gen Sequencing. Expansion of sequencing technology. Contents
Next Gen Sequencing Contents 1 Expansion of sequencing technology 2 The Next Generation of Sequencing: High-Throughput Technologies 3 High Throughput Sequencing Applied to Genome Sequencing (TEDed CC BY-NC-ND
More informationWet-lab Considerations for Illumina data analysis
Wet-lab Considerations for Illumina data analysis Based on a presentation by Henriette O Geen Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center Complementary Approaches Illumina
More informationSequence Analysis 2RNA-Seq
Sequence Analysis 2RNA-Seq Lecture 10 2/21/2018 Instructor : Kritika Karri kkarri@bu.edu Transcriptome Entire set of RNA transcripts in a given cell for a specific developmental stage or physiological
More informationDNA concentration and purity were initially measured by NanoDrop 2000 and verified on Qubit 2.0 Fluorometer.
DNA Preparation and QC Extraction DNA was extracted from whole blood or flash frozen post-mortem tissue using a DNA mini kit (QIAmp #51104 and QIAmp#51404, respectively) following the manufacturer s recommendations.
More informationChIP-seq and RNA-seq. Farhat Habib
ChIP-seq and RNA-seq Farhat Habib fhabib@iiserpune.ac.in Biological Goals Learn how genomes encode the diverse patterns of gene expression that define each cell type and state. Protein-DNA interactions
More informationIntroduction to NGS analyses
Introduction to NGS analyses Giorgio L Papadopoulos Institute of Molecular Biology and Biotechnology Bioinformatics Support Group 04/12/2015 Papadopoulos GL (IMBB, FORTH) IMBB NGS Seminar 04/12/2015 1
More informationHow to deal with your RNA-seq data?
How to deal with your RNA-seq data? Rachel Legendre, Thibault Dayris, Adrien Pain, Claire Toffano-Nioche, Hugo Varet École de bioinformatique AVIESAN-IFB 2017 1 Rachel Legendre Bioinformatics 27/11/2018
More informationQuantifying gene expression
Quantifying gene expression Genome GTF (annotation)? Sequence reads FASTQ FASTQ (+reference transcriptome index) Quality control FASTQ Alignment to Genome: HISAT2, STAR (+reference genome index) (known
More informationOvercome limitations with RNA-Seq
Buyer s Guide Simple, customized RNA-Seq workflows Evaluating options for next-generation RNA sequencing Overcome limitations with RNA-Seq Next-generation sequencing (NGS) has revolutionized the study
More informationsolid S Y S T E M s e q u e n c i n g See the Difference Discover the Quality Genome
solid S Y S T E M s e q u e n c i n g See the Difference Discover the Quality Genome See the Difference With a commitment to your peace of mind, Life Technologies provides a portfolio of robust and scalable
More informationChIP-seq analysis 2/28/2018
ChIP-seq analysis 2/28/2018 Acknowledgements Much of the content of this lecture is from: Furey (2012) ChIP-seq and beyond Park (2009) ChIP-seq advantages + challenges Landt et al. (2012) ChIP-seq guidelines
More informationChIP-seq and RNA-seq
ChIP-seq and RNA-seq Biological Goals Learn how genomes encode the diverse patterns of gene expression that define each cell type and state. Protein-DNA interactions (ChIPchromatin immunoprecipitation)
More informationIntroductory Next Gen Workshop
Introductory Next Gen Workshop http://www.illumina.ucr.edu/ http://www.genomics.ucr.edu/ Workshop Objectives Workshop aimed at those who are new to Illumina sequencing and will provide: - a basic overview
More informationNovel methods for RNA and DNA- Seq analysis using SMART Technology. Andrew Farmer, D. Phil. Vice President, R&D Clontech Laboratories, Inc.
Novel methods for RNA and DNA- Seq analysis using SMART Technology Andrew Farmer, D. Phil. Vice President, R&D Clontech Laboratories, Inc. Agenda Enabling Single Cell RNA-Seq using SMART Technology SMART
More informationApplications of short-read
Applications of short-read sequencing: RNA-Seq and ChIP-Seq BaRC Hot Topics March 2013 George Bell, Ph.D. http://jura.wi.mit.edu/bio/education/hot_topics/ Sequencing applications RNA-Seq includes experiments
More informationStatistical Genomics and Bioinformatics Workshop. Genetic Association and RNA-Seq Studies
Statistical Genomics and Bioinformatics Workshop: Genetic Association and RNA-Seq Studies RNA Seq and Differential Expression Analysis Brooke L. Fridley, PhD University of Kansas Medical Center 1 Next-generation
More informationSingle Cell Transcriptomics scrnaseq
Single Cell Transcriptomics scrnaseq Matthew L. Settles Genome Center Bioinformatics Core University of California, Davis settles@ucdavis.edu; bioinformatics.core@ucdavis.edu Purpose The sequencing of
More informationWhy QC? Next-Generation Sequencing: Quality Control. Illumina data format. Fastq format:
Why QC? Next-Generation Sequencing: Quality Control BaRC Hot Topics January 2017 Bioinformatics and Research Computing Whitehead Institute Do you want to include the reads with low quality base calls?
More informationRNA Sequencing: Experimental Planning and Data Analysis. Nadia Atallah September 12, 2018
RNA Sequencing: Experimental Planning and Data Analysis Nadia Atallah September 12, 2018 A Next Generation Sequencing (NGS) Refresher Became commercially available in 2005 Construction of a sequencing
More informationFFPE in your NGS Study
FFPE in your NGS Study Richard Corbett Canada s Michael Smith Genome Sciences Centre Vancouver, British Columbia Dec 6, 2017 Our mandate is to advance knowledge about cancer and other diseases and to use
More informationNext-Generation Sequencing: Quality Control
Next-Generation Sequencing: Quality Control Bingbing Yuan BaRC Hot Topics January 2017 Bioinformatics and Research Computing Whitehead Institute http://barc.wi.mit.edu/hot_topics/ Why QC? Do you want to
More informationRNA-Seq de novo assembly training
RNA-Seq de novo assembly training Training session aims Give you some keys elements to look at during read quality check. Transcriptome assembly is not completely a strait forward process : Multiple strategies
More informationAnalysis of RNA-seq Data. Bernard Pereira
Analysis of RNA-seq Data Bernard Pereira The many faces of RNA-seq Applications Discovery Find new transcripts Find transcript boundaries Find splice junctions Comparison Given samples from different experimental
More informationMatthew Tinning Australian Genome Research Facility. July 2012
Next-Generation Sequencing: an overview of technologies and applications Matthew Tinning Australian Genome Research Facility July 2012 History of Sequencing Where have we been? 1869 Discovery of DNA 1909
More informationWelcome to the NGS webinar series
Welcome to the NGS webinar series Webinar 1 NGS: Introduction to technology, and applications NGS Technology Webinar 2 Targeted NGS for Cancer Research NGS in cancer Webinar 3 NGS: Data analysis for genetic
More informationAn introduction to RNA-seq. Nicole Cloonan - 4 th July 2018 #UQWinterSchool #Bioinformatics #GroupTherapy
An introduction to RNA-seq Nicole Cloonan - 4 th July 2018 #UQWinterSchool #Bioinformatics #GroupTherapy The central dogma Genome = all DNA in an organism (genotype) Transcriptome = all RNA (molecular
More informationNextGen Sequencing Technologies Sequencing overview
Outline Conventional NextGen High-throughput sequencing (Next-Gen sequencing) technologies. Illumina sequencing in detail. Quality control. Sequence coverage. Multiplexing. FASTQ files. Shendure and Ji
More informationIntroduction to transcriptome analysis using High Throughput Sequencing technologies. D. Puthier 2012
Introduction to transcriptome analysis using High Throughput Sequencing technologies D. Puthier 2012 A typical RNA-Seq experiment Library construction Protocol variations Fragmentation methods RNA: nebulization,
More informationLong and short/small RNA-seq data analysis
Long and short/small RNA-seq data analysis GEF5, 4.9.2015 Sami Heikkinen, PhD, Dos. Topics 1. RNA-seq in a nutshell 2. Long vs short/small RNA-seq 3. Bioinformatic analysis work flows GEF5 / Heikkinen
More informationRNAseq Differential Gene Expression Analysis Report
RNAseq Differential Gene Expression Analysis Report Customer Name: Institute/Company: Project: NGS Data: Bioinformatics Service: IlluminaHiSeq2500 2x126bp PE Differential gene expression analysis Sample
More informationRNA-Sequencing analysis
RNA-Sequencing analysis Markus Kreuz 25. 04. 2012 Institut für Medizinische Informatik, Statistik und Epidemiologie Content: Biological background Overview transcriptomics RNA-Seq RNA-Seq technology Challenges
More informationDeep sequencing of transcriptomes
1 / 40 Deep sequencing of transcriptomes An introduction to RNA-seq Michael Dondrup UNI BCCS 2. november 2010 2 / 40 Transcriptomics by Ultra-Fast Sequencing Microarrays have been the primary transcriptomics
More informationC3BI. VARIANTS CALLING November Pierre Lechat Stéphane Descorps-Declère
C3BI VARIANTS CALLING November 2016 Pierre Lechat Stéphane Descorps-Declère General Workflow (GATK) software websites software bwa picard samtools GATK IGV tablet vcftools website http://bio-bwa.sourceforge.net/
More informationRNA-Seq Software, Tools, and Workflows
RNA-Seq Software, Tools, and Workflows Monica Britton, Ph.D. Sr. Bioinformatics Analyst September 1, 2016 Some mrna-seq Applications Differential gene expression analysis Transcriptional profiling Assumption:
More informationRNA-seq Data Analysis
Lecture 3. Clustering; Function/Pathway Enrichment analysis RNA-seq Data Analysis Qi Sun Bioinformatics Facility Biotechnology Resource Center Cornell University Lecture 1. Map RNA-seq read to genome Lecture
More informationSequencing applications. Today's outline. Hands-on exercises. Applications of short-read sequencing: RNA-Seq and ChIP-Seq
Sequencing applications Applications of short-read sequencing: RNA-Seq and ChIP-Seq BaRC Hot Topics March 2013 George Bell, Ph.D. http://jura.wi.mit.edu/bio/education/hot_topics/ RNA-Seq includes experiments
More informationRNA
RNA sequencing Michael Inouye Baker Heart and Diabetes Institute Univ of Melbourne / Monash Univ Summer Institute in Statistical Genetics 2017 Integrative Genomics Module Seattle @minouye271 www.inouyelab.org
More informationRNA standards v May
Standards, Guidelines and Best Practices for RNA-Seq: 2010/2011 I. Introduction: Sequence based assays of transcriptomes (RNA-seq) are in wide use because of their favorable properties for quantification,
More informationIntroduction to RNA sequencing
Introduction to RNA sequencing Bioinformatics perspective Olga Dethlefsen NBIS, National Bioinformatics Infrastructure Sweden November 2017 Olga (NBIS) RNA-seq November 2017 1 / 49 Outline Why sequence
More informationBST 226 Statistical Methods for Bioinformatics David M. Rocke. March 10, 2014 BST 226 Statistical Methods for Bioinformatics 1
BST 226 Statistical Methods for Bioinformatics David M. Rocke March 10, 2014 BST 226 Statistical Methods for Bioinformatics 1 NGS Technologies Illumina Sequencing HiSeq 2500 & MiSeq PacBio Sequencing PacBio
More informationIntroducing QIAseq. Accelerate your NGS performance through Sample to Insight solutions. Sample to Insight
Introducing QIAseq Accelerate your NGS performance through Sample to Insight solutions Sample to Insight From Sample to Insight let QIAGEN enhance your NGS-based research High-throughput next-generation
More informationExperimental design of RNA-Seq Data
Experimental design of RNA-Seq Data RNA-seq course: The Power of RNA-seq Thursday June 6 th 2013, Marco Bink Biometris Overview Acknowledgements Introduction Experimental designs Randomization, Replication,
More informationIntroduction of RNA-Seq Analysis
Introduction of RNA-Seq Analysis Jiang Li, MS Bioinformatics System Engineer I Center for Quantitative Sciences(CQS) Vanderbilt University September 21, 2012 Goal of this talk 1. Act as a practical resource
More informationZika infected human samples
Lecture 16 RNA-seq Zika infected human samples Experimental design ZIKV-infected hnpcs 56 hours after ZIKA and mock infection in parallel cultures were used for global transcriptome analysis. RNA-seq libraries
More informationIntroduction to RNA-Seq
Introduction to RNA-Seq Monica Britton, Ph.D. Bioinformatics Analyst September 2014 Workshop Overview of Today s Activities Morning RNA-Seq Concepts, Terminology, and Work Flows Two-Condition Differential
More informationMeasuring and Understanding Gene Expression
Measuring and Understanding Gene Expression Dr. Lars Eijssen Dept. Of Bioinformatics BiGCaT Sciences programme 2014 Why are genes interesting? TRANSCRIPTION Genome Genomics Transcriptome Transcriptomics
More informationRNA-sequencing. Next Generation sequencing analysis Anne-Mette Bjerregaard. Center for biological sequence analysis (CBS)
RNA-sequencing Next Generation sequencing analysis 2016 Anne-Mette Bjerregaard Center for biological sequence analysis (CBS) Terms and definitions TRANSCRIPTOME The full set of RNA transcripts and their
More informationFrancisco García Quality Control for NGS Raw Data
Contents Data formats Sequence capture Fasta and fastq formats Sequence quality encoding Quality Control Evaluation of sequence quality Quality control tools Identification of artifacts & filtering Practical
More informationSCALABLE, REPRODUCIBLE RNA-Seq
SCALABLE, REPRODUCIBLE RNA-Seq SCALABLE, REPRODUCIBLE RNA-Seq Advances in the RNA sequencing workflow, from sample preparation through data analysis, are enabling deeper and more accurate exploration
More informationNext Generation Sequencing Lecture Saarbrücken, 19. March Sequencing Platforms
Next Generation Sequencing Lecture Saarbrücken, 19. March 2012 Sequencing Platforms Contents Introduction Sequencing Workflow Platforms Roche 454 ABI SOLiD Illumina Genome Anlayzer / HiSeq Problems Quality
More informationNext-generation sequencing technologies
Next-generation sequencing technologies NGS applications Illumina sequencing workflow Overview Sequencing by ligation Short-read NGS Sequencing by synthesis Illumina NGS Single-molecule approach Long-read
More informationAnalysis of data from high-throughput molecular biology experiments Lecture 6 (F6, RNA-seq ),
Analysis of data from high-throughput molecular biology experiments Lecture 6 (F6, RNA-seq ), 2012-01-26 What is a gene What is a transcriptome History of gene expression assessment RNA-seq RNA-seq analysis
More informationGene Expression Profiling and Validation Using Agilent SurePrint G3 Gene Expression Arrays
Gene Expression Profiling and Validation Using Agilent SurePrint G3 Gene Expression Arrays Application Note Authors Bahram Arezi, Nilanjan Guha and Anne Bergstrom Lucas Agilent Technologies Inc. Santa
More informationChapter 7. DNA Microarrays
Bioinformatics III Structural Bioinformatics and Genome Analysis Chapter 7. DNA Microarrays 7.9 Next Generation Sequencing 454 Sequencing Solexa Illumina Solid TM System Sequencing Process of determining
More informationHigh Throughput Sequencing the Multi-Tool of Life Sciences. Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center
High Throughput Sequencing the Multi-Tool of Life Sciences Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center DNA Technologies & Expression Analysis Cores HT Sequencing (Illumina
More informationSMARTer Ultra Low RNA Kit for Illumina Sequencing Two powerful technologies combine to enable sequencing with ultra-low levels of RNA
SMARTer Ultra Low RNA Kit for Illumina Sequencing Two powerful technologies combine to enable sequencing with ultra-low levels of RNA The most sensitive cdna synthesis technology, combined with next-generation
More informationAnalytics Behind Genomic Testing
A Quick Guide to the Analytics Behind Genomic Testing Elaine Gee, PhD Director, Bioinformatics ARUP Laboratories 1 Learning Objectives Catalogue various types of bioinformatics analyses that support clinical
More informationGuidelines Analysis of RNA Quantity and Quality for Next-Generation Sequencing Projects
Title: Protocol number: Guidelines Analysis of RNA Quantity and Quality for Next-Generation Sequencing Projects GAF S002 Version: Version 4 Date: December 17 th 2015 Author: P. van der Vlies, C.C. van
More informationQuality control for Sequencing Experiments
Quality control for Sequencing Experiments v2018-04 Simon Andrews simon.andrews@babraham.ac.uk Support service for bioinformatics Academic Babraham Institute Commercial Consultancy Support BI Sequencing
More informationObtain superior NGS library performance with lower input amounts using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina
be INSPIRED drive DISCOVERY stay GENINE TECHNICAL NOTE Directional rrna depletion Obtain superior NGS library performance with lower input amounts using the NEBNext ltra II Directional RNA Library Prep
More informationTranscriptome Assembly, Functional Annotation (and a few other related thoughts)
Transcriptome Assembly, Functional Annotation (and a few other related thoughts) Monica Britton, Ph.D. Sr. Bioinformatics Analyst June 23, 2017 Differential Gene Expression Generalized Workflow File Types
More informationObtain superior NGS library performance with lower input amounts using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina
be INSPIRED drive DISCOVERY stay GENINE TECHNICAL NOTE Directional rrna depletion Obtain superior NGS library performance with lower input amounts using the NEBNext ltra II Directional RNA Library Prep
More informationComputational & Quantitative Biology Lecture 6 RNA Sequencing
Peter A. Sims Dept. of Systems Biology Dept. of Biochemistry & Molecular Biophysics Sulzberger Columbia Genome Center October 27, 2014 Computational & Quantitative Biology Lecture 6 RNA Sequencing We Have
More informationHigh performance sequencing and gene expression quantification
High performance sequencing and gene expression quantification Ana Conesa Genomics of Gene Expression Lab Centro de Investigaciones Príncipe Felipe Valencia aconesa@cipf.es Next Generation Sequencing NGS
More informationApplication Note Selective transcript depletion
Application Note Selective transcript depletion Sample Authors Laura de Jager RED Scientist Michael Berry Bioinformatics Scientist Luke Esau RED Senior Scientist Ross Wadsworth RED Team Lead Roche Sequencing
More informationBasics of RNA-Seq. (With a Focus on Application to Single Cell RNA-Seq) Michael Kelly, PhD Team Lead, NCI Single Cell Analysis Facility
2018 ABRF Meeting Satellite Workshop 4 Bridging the Gap: Isolation to Translation (Single Cell RNA-Seq) Sunday, April 22 Basics of RNA-Seq (With a Focus on Application to Single Cell RNA-Seq) Michael Kelly,
More informationBioinformatics Advice on Experimental Design
Bioinformatics Advice on Experimental Design Where do I start? Please refer to the following guide to better plan your experiments for good statistical analysis, best suited for your research needs. Statistics
More informationAdvanced RNA-Seq course. Introduction. Peter-Bram t Hoen
Advanced RNA-Seq course Introduction Peter-Bram t Hoen Expression profiling DNA mrna protein Comprehensive RNA profiling possible: determine the abundance of all mrna molecules in a cell / tissue Expression
More information