Successful gene expression studies using validated qPCR ...€¦ · Assay design & validation design • amplicon length • primer positions (exonic or intron-spanning) • transcript

Successful gene expression studies using validated qPCR assays

Jan Hellemans, CEO Biogazelle

webinar

October 28th, 2015

Agenda

• Requirements for high quality qPCR assays

• Approaches for qPCR assay validation

• How good a result can be achieved with highly optimized design tools

• How to easily discover additional genes of interest

• Best practice in qPCR

Online poll

How would you rank your current knowledge about gene expression assay design?

a) basic

b) advanced

c) expert

qPCR is reference technology for nucleic acid quantification

• sensitivity and specificity

• wide dynamic range

• speed

• relatively low cost

• conceptual and practical simplicity

qPCR is easy to perform ≠ easy to do it right

• many steps involved

• all need to be right

Introduction

R

relative quantification

quality control

statistical analysis

C

Prepare – cycle – report

P

experiment design

samples

assays

prepare cycle report

R

relative quantification

quality control

statistical analysis

C

Prepare – cycle – report

P

experiment design

samples

assays

prepare cycle report

Assay design & validation

design

• amplicon length

• primer positions (exonic or intron-spanning)

Considerations & best practices


design

• amplicon length



gene

exonic

intron-spanning


design

• amplicon length


• transcript coverage


gene

transcript 1

transcript 2

transcript 3

2 3 12 3 2coverage


design

• amplicon length



in silico verification

• specificity prediction (retropseudogenes and other homologues)

• secondary structure analysis

wet lab validation (experimental)

• specificity assessment (gel, melt, amplicon sequencing)

• Cq of NTC (for SYBR assays)

• amplification efficiency determination (slope, E, SE(E), r2)



design

• amplicon length



in silico verification

• specificity prediction (retropseudogenes and other homologues)

• secondary structure analysis

wet lab validation (experimental)

• specificity assessment (gel, melt, amplicon sequencing)

• Cq of NTC (for SYBR assays)

• amplification efficiency determination (slope, E, SE(E), r2)


Properties of the perfect assay

• specific for the gene of interest => no off-target amplification

• detection of all transcript variants

• detection not affected by polymorphisms => no allelic bias or drop out

• amplification efficiency ~100%

• no gDNA co-amplification

• no primer dimer formation

The perfect assay

Online poll

How do you currently obtain your ‘perfect’ qPCR assay?

a) using your own home brewed assays

b) buying pre-designed assays (commercial)

c) currently not designing any gene expression assays

The perfect assay

• For some genes, there is no perfect assay

• no unique sequence (homology with other genes –pseudogenes)

... or the best possible

Gene homology in olfactory receptor genes prevents perfect designs

1532 / 2043 (75%) of genes without perfect design have homologous genes that differ less than 12.5% (2 variations per 16 bases)

0%5%

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distances (clustalW) between all genes without perfect design

The perfect assay

• For some genes, there is no perfect assay

• no unique sequence (homology with other genes –pseudogenes)

• no common sequence among all transcripts

• regions are excluded because of repeats, secondary structures, SNPs, homology, ...

• Make the best possible compromise and report potential issues

• Design in silico quality control wet lab validation

... or the best possible

Assay design using primerXL

• database of genomic information (transcripts, SNPs, ...)

• tools for target region selection (maximize transcript coverage)

• primer3 design engine

• analysis of secondary structures and SNPs in primer & probeannealing regions

• specificity prediction (BiSearch, bowtie)

• relaxation cascade (from perfect to best possible)

Impact of primer mismatches on qPCR assay performance

Lefever, Clin Chem 2013

BiSearch specificity prediction

• BiSearch loose• 1222222222222222

• BiSearch strict• 1233333333333

BiSearch specificity prediction

• BiSearch loose• 1222222222222222

• only the gene of interest (FFAR2)

• BiSearch strict• 1233333333333

reads seq gene_list official_symbol location

2843 CATGGCAGTCACCATCTTCTGCTACTGGCGTTTTGTGTGGATCATGCTCTCCCAGCCCCTTGTGGGGGCCCAGAGGCGGCGCCGAGCCGTGGGGCTGGCTGTGGTGACGCTGCTCAATTTCCTGGTGTGCTTCGGACCTTACAGATCGGAA

ENSG00000126262 FFAR2 19:35940617-35942667

1897 GTAAGGTCCGAAGCACACCAGGAAATTGAGCAGCGTCACCACAGCCAGCCCCACGGCTCGGCGCCGCCTCTGGGCCCCCACAAGGGGCTGGGAGAGCATGATCCACACAAAACGCCAGTAGCAGAAGATGGTGACTGCCATGAGATCGGAA

ENSG00000126262 FFAR2 19:35940617-35942667

1535 GTAAGGTCCGAAGCACACCGAGAGCTGGGAGCAGGAGCTACACAGTCTGCTGGCCTCACTGCACACCCTGCTGGGGGCCCTGTACGAGGGAGCAGAGACTGCTCCTGTGCAGAATGAAGGCCCTGGGGTGGAGATGCTGCTGTCCTCAGAA

ENSG00000141456 AC091153.1 17:4574680-4607632

1097 CATGGCAGTCACCATCTTCTGAGGACAGCAGCATCTCCACCCCAGGGCCTTCATTCTGCACAGGAGCAGTCTCTGCTCCCTCGTACAGGGCCCCCAGCAGGGTGTGCAGTGAGGCCAGCAGACTGTGTAGCTCCTGCTCCCAGCTCTCGG

ENSG00000141456 AC091153.1 17:4574680-4607632

1091 CATGGCAGTCACCATCTTCTGAGGACAGCAGCATCTCCACCCCAGGGCCTTCATTCTGCACAGGAGCAGTCTCTGCTCCCTCGTACAGGGCCCCCAGCAGGGTGTGCAGTGAGGCCAGCAGACTGTGTAGCTCCTGCTCCCAGCTCTCGGT

ENSG00000141456 AC091153.1 17:4574680-4607632

Wet lab validation

PCR composition• total volume: 5 μl

• instrument: Bio-Rad CFX384 (with CFX Automation System)

• mastermix: Bio-Rad SsoAdvanced SYBR

• primer conc: 250 nM each

PCR program• default cycling protocol for SsoAdvanced SYBR (Ta=60°C)

Samples• cDNA: 25 ng (total RNA equivalents – Agilent Universal human reference

RNA = MAQC A)

• gDNA: 2.5 ng (Roche)

• NTC: water + carrier (5 ng/μl yeast transfer RNA)

• synthetic template (pooled 60-mers in concentration range: 20 M – 20 copies)

setup

Wet lab validation

• lab validation of 103 053 assays (human, mouse and rat coding genes)

• 1 456 142 reactions

• 3 822 PCR plates (384-well)

• equivalent to 15 288 PCR plates (96-well)

some numbers

305 m

Amplification efficiency

• initial publication: Vermeulen et al., Nucleic Acids Research, 2009

• Biogazelle approach (easy & cost effective)

• 60-mer

• no modifications, standard desalted

• 7 points dilution series: 20 000 000 > 20 molecules

• equivalent to full length double stranded template

• limitation: behavior of first cycles amplifying from cDNA are not evaluated

synthetic templates

30 nt 3’30 nt 5’

ds template ss oligor2<0.99 1 1median E 2.00 2.01average E 2.00 2.01count E <> [1.90-2.10] 1 3paired t-test p-value 0.14

Amplification efficiencydistribution (n = 50 133)

89%

Amplification efficiencydistribution (n = 50 133)

89%

redesign

redesign

Specificity

amplicon sizing ( + melt analysis for SYBR assays)

• limited sensitivity for detecting low level non-specific coamplification

• failure to observe non-specific amplification of sequences with similar size and/or Tme.g. expressed pseudogenes or homologous genes

next level of specificity assessment

• in silico specificity predictions by BiSearch

• massively parallel sequencing of pooled PCR products

• average coverage > 1000-fold lab specificity > 99.9%

• 50 – 200 times more sensitive than size analysis and Sanger sequencing

NGS for increased sensitivity

Specificitymost assays are 100% on-target

Specificity

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2/3 of non-specific assays may go unnoticed without NGS

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0 < x < 0.10.1 < x < 0.20.2 < x < 0.30.3 < x < 0.40.4 < x < 0.50.5 < x < 0.60.6 < x < 0.70.7 < x < 0.80.8 < x < 0.9

0.9 < x < 1

Specificity

perfect 60 293 86%

acceptable(<10% non-specific) 5 866 8%

predicted non-specificity(no specific design found) 1 204 2%

failing specificity QC criteria 2 467 4%

the power of in silico verification

Online poll

How do you validate your assay’s specificity?

a) Melt curves

b) Size analysis (gel or capillary)

c) Restriction digestion with gel analysis

d) Sequencing of PCR products

Online poll

Do you know the MIQE initiative?

a) Yes

b) No

MIQE compliant PrimePCR assayvalidation data sheet for human, mouse & rat

• building on the confidence validated on > 100,000 assays

• skip wet lab validation

• 9 organisms

• assays for SYBR or with probe

27,155 19,762 19,310 25,006 15,307

20,184 19,049 6,572 21,360

PrimePCR assay for 9 extra organisms

Screening vs targeted

Transcriptome• microarray or RNA-seq• hypothesis generating• few samples (high cost per sample)

Gene panels• qPCR (high sensitivity, low cost)• selection of genes based on pathway, cell type or disease• convenient balance between blind screening and targeted

analysis

Individual gene(s)• qPCR• high flexibility, customization• low cost per sample

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ribo-depletion RNAseq -detection

poly-A RNAseq - detection

qPCR - detection

ribo-depletion RNAseq -quantification

poly-A RNAseq - quantification

qPCR - quantification

Saturation analysisMAQC A - % of PrimePCR

Questions?

Thank you for your attention!

Documents

Successful gene expression studies using validated qPCR ...€¦ · Assay design & validation design • amplicon length • primer positions (exonic or intron-spanning) • transcript