1. An Introduction to Studying Expression Data Through RNA-seq
By Jason Van Houten

2. Outline Why do we study RNA? What is RNA-seq? Issues with RNA quality
Brief overview on how to make RNA-seq libraries
Choices to make (depth, paired-end, cost, strand specificity, ect…)
Examples

3. Before we start… Please ask questions!!
If I’m not clear or have a comment, don’t be afraid to stop me.
There is more than one right way to do this type of analysis
What I present is only a variation
I am not the expert! I am always learning new things as well so if you have a thought or opinion don’t be afraid to chime in as well

4. Gene Expression The Central Dogma Each level can tell us
something different
GIVE CREDIT

5. Why Study RNA over DNA? Functional studies
Genome may be constant but an experimental condition has a pronounced effect on gene expression
e.g. Drug treated vs. untreated cell line
e.g. Wild type versus knock out mice
Some molecular features can only be observed at the RNA level
Alternative isoforms, fusion transcripts, RNA editing
Predicting transcript sequence from genome sequence is difficult
Alternative splicing, RNA editing, etc.
EDIT!!! NOT MY WORDS!!

6. Why Study RNA over DNA?
Interpreting mutations that do not have an obvious effect on protein sequence
‘Regulatory’ mutations that affect what mRNA isoform is expressed and how much
e.g. splice sites, promoters, exonic/intronic splicing motifs, etc.
Prioritizing protein coding somatic mutations (often heterozygous)
If the gene is not expressed, a mutation in that gene would be less interesting
If the gene is expressed but only from the wild type allele, this might suggest loss-of-function (haploinsufficiency)
If the mutant allele itself is expressed, this might suggest a candidate drug target
EDIT!!! NOT MY WORDS!!

7. What is RNA-seq Whole Transcriptome Shotgun Sequencing
High-throughput sequencing of cDNA to gain information about that samples RNA content.
“Transcription Snap-shot”
Know the expression levels of every gene in the genome at that particular point in time.

8. What is Next Gen Seq? Video!
Briefly discusses library prep and how sequencing works

9. We start by asking a question
Condition 1
(normal colon)
Condition 2
(colon tumor)

10. We start by asking a question
Condition 1
(normal colon)
Condition 2
(colon tumor)
What genes are turned on or off during these conditions?
What about whole gene pathways?
Change of expression of one gene effect the expression of many

11. RNA sequencing Overview
Fragment,generate cDNA, add adapters, size select, PCR amplify
Isolate RNAs
Samples of interest
Condition 1
(normal colon)
Condition 2
(colon tumor)
Sequence ends
Map to genome, transcriptome, and predicted exon junctions
EDIT and Give Credit
100s of millions of paired reads
10s of billions bases of sequence
Downstream analysis

12. Challenges of Studying RNA
RNAs consist of small exons that may be separated by large introns
Mapping reads to genome is challenging
The relative abundance of RNAs vary wildly
105 – 107 orders of magnitude
Since RNA sequencing works by random sampling, a small fraction of highly expressed genes may consume the majority of reads
Ribosomal and mitochondrial genes
RNAs come in a wide range of sizes
Small RNAs must be captured separately
PolyA selection of large RNAs may result in 3’ end bias
RNA is fragile compared to DNA (easily degraded)

14. mRNA Selection

15. mRNA Selection

16. mRNA Selection

17. Quality Agilent Bioanalyzer Very good RNA, RIN of 10
Still good, RIN of 8.9
Starting to get worse, RIN of 6.3

18. Quality
RIN 3
RIN 2.2

19. Best Practice www.invitrogen.com
RNA is highly susceptible to degradation by RNAse enzymes. RNAse enzymes are present in cells and tissues and can be carried on hands, labware, or even dust. They are very stable and difficult to inactivate. For these reasons, it is important to follow best laboratory practices while preparing and handling RNA samples.
When harvesting total RNA, use a method that quickly disrupts tissue and isolates and stabilizes RNA
Wear gloves and use sterile technique at all times
Reserve a set of pipettes for RNA work. Use sterile RNAse-free filter pipette tips to prevent cross-contamination
Use disposable plasticware that has been certified to be RNAse-free.
All reagents should be prepared from RNAse-free components, including ultrapure water
Store RNA samples by freezing. Keep samples on ice at all times while working with them. Avoid extended pauses in the protocol until the RNA has been reverse transcribed into DNA
Use RNAse/DNAse decontamination solution to decontaminate work surfaces and equipment
EDIT to make shorter

21. Now we are ready to sequence!

22. Length of Reads/single vs. paired
Longer reads gives you better alignment confidence
Maximizes sequencing coverage on the flow cell
Average number of sequences representing a particular region of the transcriptome
Paired ends help deduce large insertions/deletions/rearrangements
Drawback- It costs more

23. Depth The number of reads per sample/library
More depth means more likely to see genes that are very low expressed
~200 million reads can be generated per lane on a flowcell
Nat Rev Genet , 57-63

24. How much depth do you need?
Depends on application
Differential gene expression, variant detection
10x – 30x coverage
If your interested in lower expressed genes, then you still might need more.
For applications like transcriptome assemblies
Much more depth needed
So we choose how much we want to add to a lane for sequencing depending on how much depth we need.

25. Multiplexing
Add a “barcode” to each sample/library then mix and sequence
A string of unique nucleotides within the adapter
Using barcode, sequenced reads can be traced back to their appropriate sample.
Barcodes
mixed
Sequencing B3 B1 B2 B4

26. Cost
In our lab, it only cost us about ~$30 a library to construct ourselves.
Additionally, you have sequencing costs
Depends on length(cycles)/paired end
Depends on facility and machine
$ per lane HiSeq2000
Again, multiplexing reduces cost per sample

27. Advantages of RNA-Seq compared with other transcriptomics methods

28. Typical Differential Gene Expression Workflow
Raw reads
Filter Reads
Assemble transcriptome
Align to reference genome/transcriptome
Count reads that map to genes
Run statistical tests
Evaluate genes that are differentially expressed

29. Strand Specificity
BMC Genomics 2012, 13:721
BMC Genomics 2012, 13:721

30. FPKM (RPKM): Expression Normalization
Fragments (Reads) Per Kilobase of exon model per Million mapped fragments
C= the number of reads mapped onto the gene's exons (raw counts)
N= total number of reads in the experiment
L= the sum of the exons in base pairs (size of gene).
Example 1: Large gene #1 with 100 reads and small gene #2 with 100 reads
Gene1<gene2
Example 2: library 1 has half the depth of library 2. Gene 1 has 50 reads in library 1 and 100 reads in library 2
Expression for gene1 is the same

31. Conclusions We know what RNA-seq is! RNA quality is very important
Library preparation
Next Generation Sequencing
RNA quality is very important
3’ bias
Tips to protect
Things to consider within the cost versus information balance
Introduced some analysis

32. Acknowledgements
I thank HHMI, the van der Knaap lab, Dr. Dean Fraga and everyone involved in this workshop for making this possible

33. Thank You!
Questions?