CALENDAR
June 12–13, 2020
Click here to download the schedule in PDF. The current schedule may suffer small alterations as we get closer to the start of the course. Please be sure to check it again later.
DAY 1
June 12, 2020
ARRIVAL AND INSTRUCTIONS
10:00 am
Introduce instructors and their background
Have students introduce themselves
SETUP/TROUBLESHOOT USER SYSTEMS
10:15 am
Make sure participants have Oracle VM VirtualBox and the course imaging running on their personal computers
Show the location of directories containing course materials (references, list of exercises, and examples)
EXPERIMENTAL DESIGN
11:15 am
Choosing among high-throughput RNA sequencing technologies
Strategies for RNA extraction, quantification, and storage
Considerations for sequencing library choice and preparation
Calculating expected coverage to optimize costs
Multiplexing strategies to optimize sequencing from multiple samples
Cost estimation
Exercise list #1 (coverage calculation and cost estimation)
LUNCH
12:00 am
On your own
TOOLS FOR RNA-SEQ DATA ANALYSIS AND QUALITY CONTROL
1:00 pm
Summary of the most used open-source/ free software for transcriptome assembly RNA-Seq analysis
Why and how to perform quality control (e.g., adapter trimming and filtering by size)
Detection and removal of technical sequences
Accessing the quality of each independent biological replication
Exercise list #2 (adapter trimming)
TRANSCRIPTOME ASSEMBLY
1:45 am
The bases of reference and de novo assembly
RNA-Seq de novo sequence assembly using Trinity
Estimating assembly completion using BUSCO
Exercise list #3 (sequence assembly)
NORMALIZATION AND DIFFERENTIAL EXPRESSION ANALYSIS
2:30 pm
Trimmed Mean of M-values (TMM) normalization method (edgeR)
Using RSEM to estimate transcript abundance
Identifying differentially expressed (DE) transcripts using edgeR
RPKM vs. FPKM
Exercise list #3 (estimating the abundance of the individual transcripts)
COFFEE BREAK
3:15 pm
On your own
SPLICING ANALYSIS AND MULTIPLE TESTING
3:30 pm
The BLAST like alignment tool (BLAT)
Spliced aligners (Tophat, GSNAP, SOAPsplice, MapSplice, TrueSite, star)
Aligners that can "clip" (Bowtie2's local mode, bwa-mem)
Exercise list #5 (an example BLAT analysis)
BIOLOGICAL INTERPRETATION
4:15 pm
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WRAP UP
5:00 pm
Gather preliminary feedback from participants and resolve any issues with the course image or the first five lists of exercises.
ADJOURN
5:15 pm
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DAY 2
June 13, 2020
8:30 am
INSTRUCTIONS FOR DAY TWO
Introduce speakers of day two and their backgrounds
Check that all course participants have a working image on their VMs
Show the location of directories containing course materials for this second day of the short course (references, list of exercises, and examples)
9:15 am
INTRODUCTION TO GENE ANNOTATION
Difference between gene prediction and annotation
The importance of gene annotation of comparative analysis
How to read annotation data from genomic sequences published in GenBank (.gb) format
Exercise list #6 (reading gene annotations)
10:00 am
COFFEE BREAK
On your own
10:30 am
AB INITIO GENE ANNOTATION
Definition of ab initio (intrinsic) annotation
Pros and cons (when to use it, why, and what to expect)
Sites we might want to predict (translation start, splice donor site, splice acceptor site, translation stop)
Hidden Markov Models (HMMs)
Exercise list #7 (examples of gene prediction using GENSCAN and Augustus)
11:15 am
HOMOLOGY-BASED GENE ANNOTATION
Definition of homology-based (extrinsic) annotation
Pros and cons (when to use it, why, and what to expect)
How to calculate and interpret the E-value and the bit-score
Exercise list #8 (examples of gene prediction using ORFfinder, BLASTN, and TBLASTN; comparison with results from exercise list #2)
12:00 am
LUNCH
On your own
1:00 pm
VISUALIZING GENE ANNOTATION
Examples of open-source or free tools to visualize gene annotation data
Exercise list #9 (Generic Genome Browser - GBrowser and Integrative Genomics Viewer - IGV)
1:45 pm
SHARING GENE ANNOTATION
Different formats for sharing gene annotation, with focus on the Generic Feature Format version 3 (GFF3)
Exercise list #10 (filtering, concatenating, and extracting information from GFF3 files using simple pre-written Python scripts)
2:30 pm
COFFEE BREAK
On your own
2:45 pm
MANAGING DATA FROM PUBLIC DATABASES
List of main public resources
How to leverage on EchinoDB for your research
The importance of using the correct gene names
Exercise list #11 (comparing your gene annotation with information available in EchinoDB to identify the gene of interest and compare it with orthologous sequences)
3:30 pm
WRAP UP
Gather feedback from participants on how the course will be of use to their work and general course feedback
3:45 pm
ADJOURN
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