integrated dna technologiesnmgroup.ca/document/2014/2014_03.pdf · • next generation sequencing...

Detecting Rare Genomic Copies or Events

Integrated DNA Technologies

Nick Downey, PhD

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INTEGRATED DNA TECHNOLOGIES

Common Diagnostics Challenges

• Detecting targets early with accuracy

• Detecting different strains (e.g., antibiotic resistance)

• Compiling multiplex assays to detect targets

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MGB Eclipse® Probes Are Now Available From IDT

• GMP production of minor groove binder (MGB) probes and primers

• For use in human in vitro diagnostic (IVD) applications

• FAM, HEX™, or TET™ dyes

• MGB stabilizes hybridization, which raises the Tm of the probe

• Short functional probes 13–20 bases can be useful for: • Allelic discrimination • Designing probes in limited unique sequence space • Low complexity AT rich regions

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MGB Eclipse vs. ZEN™ Fluorescent Signal

No Baseline Subtraction With Baseline Subtraction

IDT FAM/ZEN

IDT MGB

Competitor MGB

IDT FAM/ZEN

IDT MGB

Competitor MGB

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RNase H2–Dependent PCR (rhPCR)

• Primer deblocking is required for PCR, which in turn requires that primers be annealed to the target DNA sequence.

• The enzymatic deblocking cleavage event is sensitive to base mismatch and confers added specificity to the ensuing PCR reaction.

• Primer-dimer formation is highly reduced.

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Biased Amplification vs. Biased Signal Generation As PCR occurs, signal is generated from the degradation of the probe. If multiple targets are present (which occurs in rare allele reactions) signal generation occurs only from the targeted allele, and intensity is lowered proportionally.

F

F Q SNP rddddmx

SNP

In biased amplification, only the sequence of interest (red) is amplified, meaning that a single target can be identified in a background containing a high amount of nearly identical DNA (blue). Using this technique, rare alleles can be detected in a background of >1000-fold higher untargeted DNA.

Biased signal generation (PrimeTime) Biased amplification (rhPCR)

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2 rhPrimer Designs Are Possible

• 1st generation (GEN1): rDDDDMx, good for general purpose

• GEN1 primers are most appropriate for standard genotyping applications and for multiplexed amplification

• 2nd generation (GEN2): rDxxDM – best specificity, but at a cost—higher enzyme amounts required, and will need specific titration to application

• GEN2 primers are most appropriate for rare-allele detection or for applications where extremely high fidelity of template amplification is desired

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GEN1: For General Purpose Use and Minimizing Primer-Dimer Formation

• GEN1 primers are most appropriate for standard genotyping applications and multiplexed amplification.

• This primer design is robust and works well with low levels of RNase H2 enzyme.

• Mismatch guideline: M base should be the same base as the template strand.

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GEN2: For Rare Allele Genotyping

• Most appropriate for rare-allele detection or for applications where extremely high fidelity of template amplification is desired

• May require use of higher amounts of RNase H2 enzyme (range is 1–100X that needed for GEN1 primers); titration and optimization need to be performed for each GEN2 primer set

• For simplicity, we recommend use of GEN1 primers for most needs

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103 bp synthetic amplicon, run using 2 x 106 copies template For AGCTCTGCCCAAAGATTACCCTG

Rev CTGAGCTTCATGCCTTTACTGT

Blocked-For AGCTCTGCCCAAAGATTACCCTGaCAGC-x Blocked-Rev CTGAGCTTCATGCCTTTACTGTuCCCC-x Probe: FAM-TTCTGAGGCCAACTTCCACTGCCACTTA-FQ

Blocked Primers Provide Specificity and Sensitivity

No RNase inhibitor was present in these reactions. Even though these primers have an RNA residue, non-specific cleavage by single-stranded RNases (such as RNase A) or alkaline hydrolysis will not cause background signal or false positive amplification because cleavage by these routes leaves a 3′-phos, which blocks primer function.

Two-step PCR 60°C anneal/extend: 30 sec 95°C melt: 10 sec 2.6 mU RNase H2 Anneal/cleavage/dissociation/polymerase extension can take place in the same timeframe as using unmodified primers and with comparable kinetics. PCR Cq values are unchanged.

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rhPrimers Reduce Primer Dimer Events in HCV Assay

• Typically, “intelligent” primer design can reduce the incidence of primer-dimers and false priming events. Nevertheless, these unwanted events still occur.

• Sometimes, it is necessary to design primers to specific sequences as dictated by the target sequence available.

• This HCV assay was cited in a Roche patent (US06001611) relating to the problem of primer-dimer formation.

• Blocked primers and RNase H2 solved the problem of primer-dimer formation.

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ST280A:

GCAGAAAGCGTCTAGCCATGGCGTTA

ST280A rGD4 SpC3:

GCAGAAAGCGTCTAGCCATGGCGTTAgTATG-x

ST778AA:

GCAAGCACCCTATCAGGCAGTACCACAA

ST778AA rGD4 SpC3:

GCAAGCACCCTATCAGGCAGTACCACAAgGCCT-x

Hepatitis C virus subtype 1b amplicon (242 bp): gcagaaagcgtctagccatggcgttagtatgagtgtcgtgcagcctccaggaccccccctcccgggagagccatagtggtctgcgga

accggtgagtacaccggaattgccaggacgaccgggtcctttcttggactaaacccgctcaatgcctggagatttgggcgtgccccc

gcgagactgctagccgagtagtgttgggtcgcgaaaggccttgtggtactgcctgatagggtgcttgc

Cloned synthetic target 0 or 2.6 mU RNase H2 +/– Rat cDNA

HCV Subtype 1b qPCR Assay

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25

50

100

150

250

Primers alone U B U B U B U B U B U B

Primer dimers

Primers

True Positive

Plus RNase H2 Minus RNase H2 - - - - + + - - - - + + Rat cDNA

- - + + + + - - + + + + Target

• Unmodified primers gave the same false products with or without target. • Only blocked primers + RNase H2 correctly gave true positive products.

rhPCR Provides Specific Amplification Product in HCV Subtype 1b qPCR Assay

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Second Generation (GEN2) Cleavable Primer Designs Improve Specificity for SNP Assays

First generation (GEN1) primer design:

CAGCCTCATCCAAAAGAGGAAAcAGGAM-x “DDDDMx” primers

Second generation (GEN2) primer design:

CAGCCTCATCCAAAAGAGGAAAcAxxAM “DxxDM” primers

Non-nucleotide groups make cleavage less efficient, but more specific.

Mismatch guideline: M base should be the same base as the template strand

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rhPCR SNP Assay for SMAD7 (GEN1 vs. GEN2)

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Detection of Rare Variants in a Mixed Sample

Genomic DNAs homozygous for the 2 SMAD7 alleles were mixed at different ratios to determine the limit of detection of the “rare” allele in a mixed sample.

Input “mismatch” SNP Input “match” SNP

0 +666

0 +66

0 +6

0 +0

66000 +666

66000 +66

66000 +6

66000 +0

Control (unmodified)

28.7 32.2 35.9 - 21.1 21.1 21.2 21.0

rC-AGGAx 28.1 31.4 35.4 - 28.4 30.7 31.3 31.3

rC-AxxA 27.9 31.4 34.6 - 28.8 31.9 34.8 37.9

rU-AGGAx 28.1 31.3 35.7 - 27.9 30.4 31.1 31.2

rU-AxxA 28.2 31.9 36.1 - 28.9 32.4 36.3 39.0

1:1000 1:10,000 1:100

New, “second generation” cleavable primers are able to distinguish the presence of 1:10,000 of the “match” allele in a large background of the “mismatch” allele.

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Looking Deeper ...

• Sometimes, looking at individual SNPs does not provide enough information about the sample.

• Next generation sequencing may be a better option. • But whole sample sequencing can lead to bandwidth problems

• xGen® Lockdown® Probes provide a robust target capture system. • Each oligo individually QC’ed by ESI

• Flexibility in scale of synthesis and probe number

• Fast turnaround time

• Capture protocol available online

• Platform agnostic

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How Target Capture Works

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Probe Performance and Validation—Design of Tm Experiment

120 bp 120 bp

1, 3, or 7 bp (All T) 7 bp (All T or All C) 7 bp (All T or All C)

Top strand = 121, 123, or 127 bp respectively Top strand = 134 bp

1 bp mismatch (G-T or T-T)

120 bp

120 bp

120 bp

120 bp

Ultramer® Oligonucleotides had either 1, 3, or 7 G-T or T-T mismatches

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Probe Performance And Validation—Conclusions

• 1–7 base mismatches had <5°C ΔTm

• 1 or 2 1–7 base insertions had <4°C ΔTm

• These small changes in Tm will not affect capture

• Thus use of a 120mer capture probe is sufficient

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xGen® Lockdown® Probes Rescue Panel Dropout

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xGen® Lockdown® Probes Improve Coverage and Uniformity

# Reads

Data from Foundation Medicine comparing results of a large set of IDT xGen® Lockdown® Probes with a focused Agilent SureSelect® set

IDT xGen® Lockdown® Probes: 100% >150X coverage Agilent SureSelect® set: 80.7% >150X coverage

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Case Study: Detecting and Typing Hepatitis C

• Compare HiSeq runs to see if xGen® Lockdown® probes can enrich for viral sequences.

• Look at how robust a panel might be in analyzing different strains.

• Attempt second round of design to create a more robust panel.

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xGen® Lockdown® Probes Successfully Capture Viral DNA

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A Single Panel Can Provide Data for Different Strains

Case: Sample is identical to probes Case: Sample has same sub-genotype Case: Sample has different sub-genotype Case: Sample has different genotype Read depth from a MiSeq run Identity of the probe sequences to the sample

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Supplementing with xGen® Lockdown® Probes Improves Coverage of Low Identity Regions

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Online Order Tool Allows Custom Solutions

• We offer several input options: • For human, mouse, and rat we can process

gene symbols or RefSeq IDs

• For other species we can process FASTA format sequence

• We recommend 1X tiling and probe length of 120 bases for most designs

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Blocking Oligos—Function

Two classes of blocking oligos are needed: I. Cot1 DNA to block Alu, LINE repeat

elements

II. Oligonucleotide blocking oligos to block linkers/adapters

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Slides to help answer questions

(not shared with customers)

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Detergent is ESSENTIAL for Robust P. abyssi RNase H2 Activity

5’-CTCGTGAGGTGATGcAGGAGATGGGAGGCG-3’

3’-GAGCACTCCACTACGTCCTCTACCCTCCGC-5’

0

10

20

30

40

50

60

70

80

90

100

Triton-X-100 Tween-20 Tween-80 Ctab N-Lauroylsarcosinate

Per

cent

Cle

aved

0.0001

0.001

0.01

Percent detergent

in reaction

0.1% Triton X-100 is present in dilution buffer and also in storage buffer

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P. abyssi RNase H2 Is Only Active at Elevated Temperatures

5’ 32P-CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3’

3’ GAGCACTCCACTACGTCCTCTACCCTCCGC 5’

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P. abyssi RNase H2 is Active Across a Broad Range of Mg2+ levels

Usually 3 mM Mg2+

is recommended

5’ 32P-CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3’

3’ GAGCACTCCACTACGTCCTCTACCCTCCGC 5’

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Pyrococcus abyssi RNase H2 is Very Thermostable

• The enzyme can be incubated at 95°C for >45 minutes with little loss of activity.

• The enzyme will survive thermal cycling (e.g., PCR reactions).

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Why Do We Use Pyrococcus abyssi RNase H2

• Pyrococcus abyssi RNase H2 is very thermostable.

• The RNase H2 enzyme is inactive at low temperature. “Hot start” is achieved without need for a modified “hot start” polymerase.

• The enzyme is active across a wide range of magnesium concentrations, including concentrations commonly used in PCR.

• Most RNase enzymes cleave to leave a 3′ Phosphate. P. abyssi RNase H2 cleaves to leave a 3′ OH

integrated dna technologiesnmgroup.ca/document/2014/2014_03.pdf · • next generation sequencing...

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