Webinar, 9th June 2015

Exiqon NGS Services

- microRNA-Seq in serum and plasma

Dr Hazel Pinheiro
The highlights of this webinar

● NGS for discovery of novel microRNAs

● NGS and PCR together – improve the biomarker discovery workflow
● NGS in biofluids is challenging
● Exiqon’s pioneering work with biofluids enables robust NGS results
● Unique Quality Control – >50 spike-ins and PCR QC
● Ensure reads are reliable – normalize to read number (TPM)
● Validate using miRCURY LNA™ microRNA PCR system
– assays for any microRNA

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Agenda

• Introduction to Exiqon and microRNA biomarkers in biofluids

• Why do NGS?
• Challenges of working with clinical Biofluid samples
• NGS Biofluids workflow at Exiqon
• Sample requirements for Biofluid samples

• Quality control at every step

• Superior data analysis and biological interpretation

• Reliable Biofluids NGS data

• Validation of your NGS data
3
• How to get started
Broad product and service portfolio for RNA analysis

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Exiqon – your trusted and experienced Service Partner

● Partnership
● Consultation from start to finish
● Close communication with dedicated project
management team
● Experience from in-house microRNA biomarker
programs in biofluids
● Expertise
● 9 years’ experience with microRNA profiling
● SOPs, QC checks, LIMS
● Industry leading data analysis
● Speed
● Short turn-around-times
● Flexibility
● Exploratory pilots to very large projects 5
 Exiqon – Biofluid microRNA pioneers

● 7 Years’ experience profiling microRNAs in biofluids

● In-house clinical microRNA diagnostic biomarker programs in biofluids
● >1 million data points from healthy and diseased individuals

miRSign Colorectal Cancer test (Cancer cells versus normal cells)

Colorectal Cancer (Recurrence stage II test)

Colorectal Cancer (Early detection diagnostic test)

Colon Cancer (treatment)

Biofluid based programs
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Prostate Cancer (treatment)
Exiqon – Biofluid microRNA pioneers

● 7 Years’ experience profiling microRNAs in biofluids

● In-house clinical microRNA diagnostic biomarker programs in biofluids
● >1 million data points from healthy and diseased individuals
● Knowledge of biofluid pre-analytical variables
● Optimized protocols and QC procedures for biofluids
● First commercial microRNA service provider
● >30,000 samples for >1,800 customers

Perform microRNA-Seq in biofluids with the experts

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MicroRNAs in biofluids make excellent biomarkers

● Highly stable in clinical

samples
● Minimally invasive
● Biomarkers for:
● Diagnosis
● Prognosis
● Treatment response
● Safety

Adapted from Guire et al. Clinical Biochemistry 2013

Agenda

• Introduction to Exiqon and microRNA biomarkers in biofluids

• Why do NGS?
• Challenges of working with clinical Biofluid samples
• NGS Biofluids workflow at Exiqon
• Sample requirements for Biofluid samples

• Quality control at every step

• Superior data analysis and biological interpretation

• Reliable Biofluids NGS data

• Validation of your NGS data
9
• How to get started
NGS enables discovery of novel biomarkers

● Hypothesis-free
Variation in hsa-let-7b-5p (isomiRs)

● Novel microRNAs GUGAGGUAGUAGGUUGU

GUGAGGUAGUAGGUUG
15
14
GUGAGGUAGUAGGUUGUGU 13
GUGAGGUAGUAGGUUGUGUGG 12

● IsomiRs GUGAGGUAGUAGGUU 11
UGAGGUAGUAGGUUGUGUGGUUUC 10
● Length variants UGAGGUAGUAGGUUGUG 9
8
UGAGGUAGUAGGUUGUGUGGU
UGAGGUAGUAGGUUGUGUGG 7
UGAGGUAGUAGGUUGUGUGGUUU 6
● RNA editing UGAGGUAGUAGGUUGUGUGGUU 5
miRBase
sequence
UGAGGUAGUAGGUUGUGUGGUUUCA 4
● May alter seed sequence UGAGGUAGUAGGUUGUGUG 3
UGAGGUAGUAGGUUGUGUGGUUUCAG 2
UGAGGUAGUAGGUUGU 1
0 100 200 300 400
● Novel biomarkers Reads per million (RPM)
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Which platform should I choose to profile
microRNAs in serum or plasma?

NGS qPCR

Hypothesis free Hypothesis driven

Discovery, novel microRNAs Differential expression

“All microRNAs”? Profiling & Validation

Larger amount of sample Smaller amount of sample

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Combining NGS and PCR
– the ideal Biomarker discovery workflow

DISCOVERY PHASE VALIDATION PHASE

Genome wide Discovery screening Validation set

screening on subset miRNA signature

Limited sample size: Larger sample size: Large sample size:

Hypothesis free NGS Custom Pick-&-Mix Custom Pick-&-Mix

profiling PCR panels PCR panels or
individual assays

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NGS and PCR: a powerful combination

● NGS followed by qPCR profiling of candidate microRNAs

● Include more samples without increasing budget
● Increased confidence in results

NGS + qPCR panels (46 miRs)

Statistical significance
20 140

# of microRNAs screened
qPCR panels (752 miRs) 65

NGS
NGS 50
qPCR panels
0 20 40 60 80 100 120 140 160
Pick-&-Mix Number of samples
qPCR panels Number of samples analyzed 13
for a fixed budget
Agenda

• Introduction to Exiqon and microRNA biomarkers in biofluids

• Why do NGS?
• Challenges of working with clinical Biofluid samples
• NGS Biofluids workflow at Exiqon
• Sample requirements for Biofluid samples

• Quality control at every step

• Superior data analysis and biological interpretation

• Reliable Biofluids NGS data

• Validation of your NGS data
14
• How to get started
Exiqon has solutions to challenges of NGS in Biofluids

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For further information:

Blondal et al., Methods 2012
Agenda

• Introduction to Exiqon and microRNA biomarkers in biofluids

• Why do NGS?
• Challenges of working with clinical Biofluid samples
• NGS Biofluids workflow at Exiqon
• Sample requirements for Biofluid samples

• Quality control at every step

• Superior data analysis and biological interpretation

• Reliable Biofluids NGS data

• Validation of your NGS data
16
• How to get started
Exiqon NGS Services workflow: microRNA Biofluids

● Consultation with Exiqon NGS and

microRNA experts

● Experimental design
● We provide protocols for sample
collection and storage
● Avoid heparin tubes
● Avoid hemolysis

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Exiqon NGS Services workflow: microRNA Biofluids

● Human serum or plasma

● Optimized protocol for biofluids

● Unique to Exiqon services

- not a commercial kit

● > 50 RNA spike-ins for

comprehensive QC

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Exiqon NGS Services workflow: microRNA Biofluids

● Unique Exiqon qPCR based QC

● Specifically developed for biofluids
● Hemolysis indicator
● Use Spike-ins to monitor:
● RNases
● RNA isolation efficiency
● Inhibition
● Outlier detection

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Exiqon NGS Services workflow: microRNA Biofluids

● Specialized protocols for

challenging biofluid samples

● Size selection
● 1 x 50bp reads

● 10M raw reads per sample

● Illumina NextSeq or HiSeq

NextSeq 500 HiSeq 2500 20

400 million reads 2000 million reads
Exiqon NGS Services workflow: microRNA Biofluids

● Complete sample-to-answer
solution
● QC of sequencing data
(including >50 spike-ins)
● Mapping
● Unsupervised and
supervised analysis
● Differential expression
analysis including statistics
● Biological interpretation
● Personal scientific discussion 21
Exiqon’s NGS Data analysis provides
unique microRNA information
● IsomiRs

● Novel microRNAs using miRPara

● Differential expression analysis
● Group comparisons
● Statistical analysis (t-test, ANOVA)

● Biological interpretation (XploreRNA)

● validated and predicted target genes
● microRNAs sharing target genes
● tissue expression & disease information

● Gene Ontology (GO) enrichment analysis 22

Impact on
biological pathways
Exiqon offers the most comprehensive yet easy-to-read
NGS data report on the market

Personal follow-up call:

Discuss your results 23

with Exiqon’s NGS and
microRNA experts
Agenda

• Introduction to Exiqon and microRNA biomarkers in biofluids

• Why do NGS?
• Challenges of working with clinical Biofluid samples
• NGS Biofluids workflow at Exiqon
• Sample requirements for Biofluid samples

• Quality control at every step

• Superior data analysis and biological interpretation

• Reliable Biofluids NGS data

• Validation of your NGS data
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• How to get started
Excellent Reproducibility of NGS in Biofluids

Exiqon’s rigorous QC including > 50 RNA Spike-ins demonstrates

highly reproducible Biofluids workflow from RNA isolation to NGS
How reliable is an NGS read?

● TPM = Tags per million mapped reads

● TPM adjusts for different numbers of reads between samples
● TPM threshold will affect number of microRNAs detected
1200

10M raw reads

1000
60M raw reads
800

600

400

200

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0
no filtering 1 TPM 5 TPM 10 TPM

Threshold TPM (tags per million mapped reads)

How reliable is a sequence detected at ≤ 1 TPM?

● Real lowly expressed microRNA?

● Sequencing artifact?
● Novel sequences – what is a real microRNA? tRNA fragment?
● miRPara novel microRNA prediction software

● We report all reads and advise on what makes biological sense to

follow up on

● Will increasing sequencing depth help detection of sequences with

low count numbers?

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Oversequencing does not add information

● Pool of serum and plasma sequenced with either 10M or 60M raw
reads per sample
● More reads does not mean more microRNAs
1200

10M raw reads

1000
60M raw reads
800

600

400

200

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0
no filtering 1 TPM 5 TPM 10 TPM

Threshold TPM (tags per million mapped reads)

Agenda

• Introduction to Exiqon and microRNA biomarkers in biofluids

• Why do NGS?
• Challenges of working with clinical Biofluid samples
• NGS Biofluids workflow at Exiqon
• Sample requirements for Biofluid samples

• Quality control at every step

• Superior data analysis and biological interpretation

• Reliable Biofluids NGS data

• Validation of your NGS data
29
• How to get started
Exiqon’s LNA™ microRNA PCR system is
the obvious choice for NGS data validation

• miRQC study assessed

concordance of differentially
expressed microRNAs
between 12 platforms

• Exiqon’s LNA™ PCR system

is the only one to cluster with
NGS platforms

Adapted from Mestdagh et al.

Nature Methods 2014

More info: www.exiqon.com/mirqc

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Validate your NGS results with Exiqon PCR service
- Top-performing microRNA qPCR system
● LNA™ Universal RT microRNA PCR system

● Complete sample-to-answer solution

● Consultation to Data Analysis
● Advice on reference genes for normalization

● State-of-the-art facilities
● 8 Roche Light Cyclers 480
● High throughput robotic pipetting stations
● Template-free environment for PCR setup

● Rigorous QC at every step

● Highly skilled and experienced scientists
and technicians 31
Excellent sensitivity and linearity of
miRCURY LNA™ microRNA PCR – ideal for biofluids
Plasma or serum
● >1,400 assays wet-lab
validated to strict criteria
● Serial dilution of a pool of 647
synthetic microRNAs
● Near perfect linearity at very
low concentrations as found in
plasma and serum
● No need for pre-amp
● From just 20 µl biofluid

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miRCURY LNA™ PCR assays for any microRNA

1 RT reaction per sample

● All microRNAs reverse transcribed

or
Validate any microRNA
● Any microRNA in miRBase v20
Multiple assays Individual assays ● Novel microRNAs
● IsomiRs

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Choose between Exiqon’s PCR Service or
Reagents
● Everything you need to validate your NGS results in your own lab
● cDNA synthesis kits, master mix
● GenEx software
● microRNA assays provided in:
● individual tubes
● bulk plates
● ready-to-use PCR panels: pre-defined or custom

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Agenda

• Introduction to Exiqon and microRNA biomarkers in biofluids

• Why do NGS?
• Challenges of working with clinical Biofluid samples
• NGS Biofluids workflow at Exiqon
• Sample requirements for Biofluid samples

• Quality control at every step

• Superior data analysis and biological interpretation

• Reliable Biofluids NGS data

• Validation of your NGS data
35
• How to get started
Getting started with microRNA Biofluid
NGS services at Exiqon
● Biofluid sample collection:
exiqon.com/biofluids
● Download example report:
exiqon.com/ngs-services
● Guidelines
● Replicates?
● Data analysis?
● Pre- and post support
● FAQ?
● Contact us for a free no-obligation
consultation to discuss your project with
our NGS experts 36

exiqon.com/contact
Exiqon Services Portfolio

Sample preparation
• Preparation of high quality total RNA
• Isolation of exosomes from biofluids
• miRCURY™ Exosome and RNA Isolation kits, and customized protocols
• Clinical samples, Biofluids, cell lines, tissues and custom requests

RNA-seq NGS MicroRNA qPCR MicroRNA Array

• Illumina platforms • miRCURY LNA™ profiling
• Whole transcriptome Universal RT microRNA • miRCURY LNA™
PCR microRNA Arrays
• mRNA
• Ideal for samples with
• microRNA (including limited RNA content
microRNA discovery)
• miRNome profiling or
• microRNA Biofluids validation of candidate
• Small RNA microRNAs
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 Why use Exiqon NGS Services?
● First microRNA service provider and 7+ years
experience with biofluids
● Exiqon pioneered microRNA biomarker discovery
from biofluids
● Protocols optimized for serum and plasma
● Rigorous quality control at every step
● Superior data analysis and biological interpretation
● Personalized scientific consultations
● Fast turn-around time and first class reports
● Market-leading miRCURY LNA™ qPCR validation
platform

Thank you – any questions? 38