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CAG EMEAI | Agilent Restricted | Page 1 Life Sciences & Diagnostics Group | Agilent Technologies | Page 1
Back-to-Basics:
RNA Sequencing 101
Presented By:
Jean Jasinski, Ph.D. Field Application Scientist
Agilent Technologies
Life Sciences & Diagnostics Group
CAG EMEAI | Agilent Restricted | Page 2 Life Sciences & Diagnostics Group | Agilent Technologies | Page 2
Back-to-Basics: Agilent’s Five Part 101
eSeminar Series
CAG EMEAI | Agilent Restricted | Page 3 Life Sciences & Diagnostics Group | Agilent Technologies | Page 3
Topics for Today’s Presentation
Library Prep (Strand-Specific) 2
1
3 How RNA- and DNA-Seq Differ
4
What is RNA-Seq?
Targeted vs. Whole Transcriptome
5
Not approved for use in diagnostic
procedures
Summary & Upcoming 101 eSeminars
CAG EMEAI | Agilent Restricted | Page 4 Life Sciences & Diagnostics Group | Agilent Technologies | Page 4
What is RNA-Seq?
Application of massively parallel next generation sequencing to
detect and quantify the RNA from a sample
seqwright.com
Isolate RNA
from samples
Prepare library
Obtain sequences;
Analyze data
CAG EMEAI | Agilent Restricted | Page 5 Life Sciences & Diagnostics Group | Agilent Technologies | Page 5
Many Technologies for Transcriptome Studies
CAG EMEAI | Agilent Restricted | Page 6 Life Sciences & Diagnostics Group | Agilent Technologies | Page 6
Research goals in transcriptome studies
• Novel transcript discovery
• Novel gene fusions
• Novel exons/alt. start sites
• Alternative splicing
• Differential expression
analysis
• Development of a gene
signature
• SNP detection
Profiling Discovery
Gene Expr.
Alt. Splicing
SNP
CAG EMEAI | Agilent Restricted | Page 7 Life Sciences & Diagnostics Group | Agilent Technologies | Page 7
Agilent Microarrays vs. RNA-Seq
Microarrays complement RNA-Seq
Agilent’s (free) customization
capabilities allow you to
incorporate RNA-Seq
discoveries into microarray
experiment(s)
CAG EMEAI | Agilent Restricted | Page 8 Life Sciences & Diagnostics Group | Agilent Technologies | Page 8
Topics for Today’s Presentation
Library Prep (Strand-Specific) 2
1
3 How RNA- and DNA-Seq Differ
4
What is RNA-Seq?
Targeted vs. Whole Transcriptome
5
Not approved for use in diagnostic
procedures
Summary & Upcoming 101 eSeminars
CAG EMEAI | Agilent Restricted | Page 9 Life Sciences & Diagnostics Group | Agilent Technologies | Page 9
Basic RNA-Seq Library Prep Steps
Isolate Total RNA PolyA selection or
ribosomal and/or global
depletion
1st strand cDNA synthesis
2nd strand cDNA synthesis
Sequence
Chemical or enzymatic
fragmentation
Repair ends & adaptor
ligation
PCR Amplification and
optional barcoding
CAG EMEAI | Agilent Restricted | Page 10 Life Sciences & Diagnostics Group | Agilent Technologies | Page 10
5’ 3’
3’ 5’
Why Strand Specific RNA Sequencing?
• To discover antisense transcripts with potential regulatory roles
• To identify overlapping transcripts
• To accurately measure the expression level of a given transcript
Sultan et al, 2012 Biochem Biophys Res Commun 15;422(4):643-6
5’ 3’
3’ 5’
CAG EMEAI | Agilent Restricted | Page 11 Life Sciences & Diagnostics Group | Agilent Technologies | Page 11
ILM
InPE1.0
primer
SureSelect
Pre-Capture
Primer Prepared Library
PCR
ATGC TACG
ATGC TACG
ATGC
TACG
GCG
CGC
GCG CGC
GCG CGC
Prepared Library
PCR
ATGC TACG
ATGC
UACG
GCG
CGC
GCG CGC
Non-Strand-Specific
Library Prep
Directional
(strand-specific)
Library prep
How is Strand Specificity Preserved?
SureSelect
primer
RNA Seq ILM
Reverse PCR primer
Brown = P5 Adapter (drives forward read)
Green = P7 Adapter (drives reverse read)
UDG treatment removes the
dU-containing strand so it isn’t amplified
CAG EMEAI | Agilent Restricted | Page 12 Life Sciences & Diagnostics Group | Agilent Technologies | Page 12
Strand-specificity RNA-Seq Identified
Cis-NATs (Natural Anti-sense Transcripts) in Rice
CAG EMEAI | Agilent Restricted | Page 13 Life Sciences & Diagnostics Group | Agilent Technologies | Page 13
Topics for Today’s Presentation
Library Prep (Strand-Specific) 2
1
3 How RNA- and DNA-Seq Differ
4
What is RNA-Seq?
Targeted vs. Whole Transcriptome
5
Not approved for use in diagnostic
procedures
Summary & Upcoming 101 eSeminars
CAG EMEAI | Agilent Restricted | Page 14 Life Sciences & Diagnostics Group | Agilent Technologies | Page 14
How RNA-Seq and DNA-Seq Differ
DNA-Seq
• Mechanical shearing (Covaris)
• Total DNA used in library prep
• Quality assessed by fragment size and concentration
• Replicates not required; some analysis use paired samples
RNA-Seq
• Enzymatic/chemical fragmentation
• More RNA species: requires initial
selection from total RNA (poly-A,
ribosomal depletion, globin
depletion)
• RIN or RINe (RNA-Integrity
Number) to assess integrity of
initial RNA isolation; also size
distribution and concentration
• Analysis more complex (biological
replicates for gene expression ,
normalization)
CAG EMEAI | Agilent Restricted | Page 15 Life Sciences & Diagnostics Group | Agilent Technologies | Page 15
Quality Metrics of RNA-Seq Libraries
Levin et al. (2010), Nature Methods 7(9):709-15
• Library Complexity
• Strand-Specificity
• Coverage (reads/locus)
(10X, 20X, 100x)
• Sequencing Depth (number
of reads/sample)
• Mapping Efficiency (how
many reads can be aligned
to genome/transcriptome)
CAG EMEAI | Agilent Restricted | Page 16 Life Sciences & Diagnostics Group | Agilent Technologies | Page 16
Topics for Today’s Presentation
Library Prep (Strand-Specific) 2
1
3 How RNA- and DNA-Seq Differ
4
What is RNA-Seq?
Targeted vs. Whole Transcriptome
5
Not approved for use in diagnostic
procedures
Summary & Upcoming 101 eSeminars
CAG EMEAI | Agilent Restricted | Page 17 Life Sciences & Diagnostics Group | Agilent Technologies | Page 17
More Sequencing Means Greater Diversity of RNA
Types
Tarazona et al., Genome Res. 2011 Dec;21(12):2213-23
Need more reads to
detect transcripts with
lower abundance like
regulatory RNA than
for protein coding
genes
Protein Coding RNA
CAG EMEAI | Agilent Restricted | Page 18 Life Sciences & Diagnostics Group | Agilent Technologies | Page 18
How Much Sequencing is Needed?
Depends on experimental goal
Sequencing Depth
• Expressed as numbers of reads
per sample
• No set recommendation
30M-80M reads/sample
(Gene Expression)
100-200M reads/sample
(alt splice, novel transcripts)
Coverage
• Number of reads/locus
30X for Common SNPs
>50X Rare SNPS
Lander/Waterman Equation
Illumina HiSeq 2000: 300M-400M
paired end reads/lane; ~11 days
MiSeq: 30M-34M reads/run; 6-39 hours
CAG EMEAI | Agilent Restricted | Page 19 Life Sciences & Diagnostics Group | Agilent Technologies | Page 19
How much sequencing for whole transcriptome RNA-
Seq equivalent to an Agilent Gene Expression array?
Similar dynamic range to Agilent’s Microarrays
only achieved after 80M reads!
# reads
1M
10M
40M
80M
Myth: NGS has
much better
dynamic range
than microarrays
CAG EMEAI | Agilent Restricted | Page 20 Life Sciences & Diagnostics Group | Agilent Technologies | Page 20
Target Enrichment: It’s just like fishing…
Why perform target enrichment?
1. Sequence only your desired regions of interest (exons, gene panels, etc.)
2. Sequence more samples per lane/run (i.e., multiplex)
3. Save time and money
4. Faster time to data = Smaller datasets
5. Identify variants in samples with increased reliability and accuracy = Higher
Coverage
What is the basic concept?
• Isolate the transcripts of interest
• Regions that are captured/amplified from initial library (pre-capture library) undergo additional amplification and processing creating a post-capture library
CAG EMEAI | Agilent Restricted | Page 21 Life Sciences & Diagnostics Group | Agilent Technologies | Page 21
Benefit of Target Enrichment RNA Target Enrichment
An enriched library provides more reads/transcript and better sequence coverage for
specific targets for the same sequencing depth
CAG EMEAI | Agilent Restricted | Page 22 Life Sciences & Diagnostics Group | Agilent Technologies | Page 22
RNA-Seq Target Enrichment in Cancer
Aim: RNA Target Enrichment of 467 Cancer Genes (~all Tyr Kinases
+ Genes from Cancer Gene Census); Overall >800 target transcripts
Method: Enrich cDNA from K-562 CML cell line cDNA libraries and
compare results from before and after target enrichment
No bias
CAG EMEAI | Agilent Restricted | Page 23 Life Sciences & Diagnostics Group | Agilent Technologies | Page 23
Benefits of RNA-Seq Target Enrichment in Cancer
• Increased coverage improves ability to find rare transcripts and novel fusions
• Whole transcriptome analysis requires >40x more sequencing to achieve
same depth for targeted regions
CAG EMEAI | Agilent Restricted | Page 24 Life Sciences & Diagnostics Group | Agilent Technologies | Page 24
How SureSelect RNA Target Enrichment Works
CAG EMEAI | Agilent Restricted | Page 25 Life Sciences & Diagnostics Group | Agilent Technologies | Page 25
Indexing (barcoding): More Samples/Lane
Used with Target-Enriched Libraries • Index is a nucleotide barcode, usually 6 bp in length
• Links reads to samples
• Included with sequencing adapter
• Allows multiple samples to be mixed on single lane to take full advantage of sequencing capacity
• Also known as multiplexing
CAG EMEAI | Agilent Restricted | Page 26 Life Sciences & Diagnostics Group | Agilent Technologies | Page 26
Topics for Today’s Presentation
Library Prep (Strand-Specific) 2
1
3 How RNA- and DNA-Seq Differ
4
What is RNA-Seq?
Targeted vs. Whole Transcriptome
5
Not approved for use in diagnostic
procedures
Summary & Upcoming 101 eSeminars
CAG EMEAI | Agilent Restricted | Page 27 Life Sciences & Diagnostics Group | Agilent Technologies | Page 27
Which technology is best suited for your project?
RNA-Seq
• Discovery of transcripts, fusions, splice variants
• Higher cost (2-3x more than a microarray)
• Complicated data analysis
Microarrays
• Differential expression studies
• Lower cost with higher sample throughput
• Well-established data analysis
Profiling? Discovery?
What scientific question am I trying to address?
How much sample material do I have?
How fast do I need/want the results?
How much money do I have?
How many replicates will I need?
CAG EMEAI | Agilent Restricted | Page 28 Life Sciences & Diagnostics Group | Agilent Technologies | Page 28
Key Considerations for RNA-Seq
• Choose the approach most suited for your research
question (whole transcriptome or targeted enrichment)
• Think outside “the box”: sequencing costs + library prep
costs + reagents + time + analysis
• Consult your friendly statistician/bioinformatician to
determine how many total reads/sample, how many
reads per locus, and how many (biological) replicates are
needed to address the biological question
• Develop a plan for data analysis; practice with NGS data
deposited in GEO, SRA, ArrayExpress
CAG EMEAI | Agilent Restricted | Page 29 Life Sciences & Diagnostics Group | Agilent Technologies | Page 29
Key Considerations for RNA-Seq
CAG EMEAI | Agilent Restricted | Page 30 Life Sciences & Diagnostics Group | Agilent Technologies | Page 30
Why we know NGS 101 & more…
Agilent Technologies Solutions for the NGS workflow
The Gold Standard for Sample QC
2100 Bioanalyzer Instrument & Kits
2200 TapeStation Instrument & Kits
NGS Analysis Software
GeneSpring NGS
SureCall
Validation Technologies
qPCR- Mx system & Brilliant reagents
Microarrays- CGH, CGH+SNP,
Gene Expression & miRNA
The Leader in NGS Target Enrichment
SureDesign
SureSelect
HaloPlex
Bravo Automation
CAG EMEAI | Agilent Restricted | Page 31 Life Sciences & Diagnostics Group | Agilent Technologies | Page 31
The NGS 101 eSeminar Series continues…
Event Date & Time Speaker Topics
Methyl-Seq 101 Wed, Oct 9
4 pm ET
Alex Siebold, PhD
Field Application
Scientist
• Methylation Mechanisms and
Significance
• Review of Comparative
Technologies
• Introduction to Methyl-Seq
NGS Data Analysis 101 Thu, Oct 10
1 pm ET
Jean Jasinski, PhD
Field Application
Scientist
• Analysis Workflows, File Formats,
and Data Filtering
• DNA-Seq vs. RNA-Seq
Considerations
• Integrating Disparate Data Sets to
Create a More Complete Story
NGS Panels 101 Fri, Oct 11
1 pm ET
Adam Hauge,
University of
Minnesota
• Panel Design Process
• Quality at the Bench: Tips, Tricks,
and Lessons Learned
• Considerations for Future Panels
CAG EMEAI | Agilent Restricted | Page 32 Life Sciences & Diagnostics Group | Agilent Technologies | Page 32
Contact Us
800.227.9770
Agilent_inquiries@agilent.com
www.agilent.com/genomics
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