haloplex hs get to know your dna. every single fragment

HaloPlexHS

Get to Know Your DNA. Every Single Fragment.

Agenda

Introduction

How HaloPlexHS works

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A flexible and accelerated solution

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3 Performance data

For Research Use Only. Not for use in diagnostic procedures.

From Discovery to Clinical Research

DISCOVERY FOLLOW-UP

Whole Exome

Whole Genome

GWAS

Follow-up Exome

Follow-Up WGAS

Follow-up GWAS

Clinical Research Panels

CLINICAL RESEARCH


Requirements of clinical research

FOLLOW-UP CLINICAL RESEACH

Clinical research applications require:Fast turnaround time

Flexibility in capture sizeSimple workflowHigh coverage

High accuracy in variant detectionData analysis solution


The need for sensitivity and accuracy


What are low allele frequency variants implicated in?

• Clonal evolution and pathogenesis• Tumor subclonal heterogeneity • Immunological diversity

What are low allele frequency variants?

• Variants present at a frequency below 3%

Low allele frequency variants

Adapted from Stead et al (2013) Human Mutation 34: 1432-1438


• Low allele frequency variants are difficult to detect by conventional NGS methods

• Relatively high error rate of sequencers (1 wrong base call in 100-1000 sequenced bases)

Kennedy et al (2014) Nature Protocols 9: 2586 - 2606

Requires molecular barcodes for increased sensitivity and accuracy

Low allele frequency variants


Basic molecular barcode analysis

1. Align reads

2. Group read pairs to designed probes based on read start-stop position

3. For each probe: group reads with identical molecular barcode sequence

4. Consolidate read information to one read per molecule (remove PCR duplicates)

T

T

T

A

A

C

G

Group reads with the same molecular

barcode

Barcode family

TConsensus

read

True variant

Random error

Molecular barcode

Sample Index


1. Ability to identify unique progenitor DNA fragments (de-duplication)

2. Biases and errors from PCR amplification or sequencing steps can be detected.

3. Decreased error rate, increased accuracy for variant calling (low-input DNA)

4. Low allele frequency variant detection

5. CNV detection

Benefits of molecular barcode analysis


Agenda

Introduction


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3 Performance data


Introducing HaloPlexHS – High Sensitivity Next Gen PCR

Key Features :

o More than a million unique 10nt molecular barcodes are incorporated into DNA library fragments

o Requires only 50ng starting DNA inputo Rapid workflow : From sample to sequencing-ready libraries in <6hro Compatible with FFPE sampleso More sensitive and accurate than other conventional NGS TE methods


Key Benefits

• Uniquely tag DNA fragments with more than a million 10-nt molecular barcodes

• Confidently detect mutations present at below 1% frequency in genetically heterogeneous samples

Unparalleled Sensitivity

• Differentiation of true variants from PCR or formalin fixation artifacts by targeting both DNA strands

Superior Accuracy

• Complete target enrichment in less than 6hr from only 50ng of gDNA

• From raw data to categorized mutations in 3 steps using SureCall data analysis software

Accelerated Solution


1. Select the HaloPlexHS design workflow

2. Input gene ID/name/coordinate

3. Define regions of interest (eg. Exons, UTRs, etc)

4. Click “Start Design”5. Design report in 10 minutes

www.agilent.com/genomics/suredesign

SureDesign – Create a custom design in minutes


The HaloPlexHS Workflow

Improves design coverage Redundancy reduces risk of

allele dropout if a probe fails; protects against primer site mutations

Specificity of the restriction enzymes add specificity to the capture

Amplicon tiling

Each 50ng DNA sample is fragmented in eight double-digest reactions

1


HaloPlex Others

TARGET TARGET

1. With HaloPlex each target base is covered by up to eight amplicons (different start and stop sites)!

2. If an unknown mutation appears in a restriction site, it may affect one or two fragments but all others will be present

3. If a variant occurs – it can be checked by multiple amplicons with HaloPlex

1. With other multiplex PCR based technologies, each target base is covered by only one amplicon (same start and stop sites)

2. If an unknown mutation appears in a primer site it causes a complete dropout in the target region

3. If a variant occurs, it is hard to know if it is a real mutations and not a PCR artifact

DNA variant

DNA variant

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Basics of HaloPlex technology – amplicon redundancy


Genomic region Target

0001111122322222333455422221111222111110000 Read coverage

Increased Confidence in Mutation Calling

Amplicon redundancy

provides excellent

coverage.


DNA fragments are mixed with custom HaloPlex probes and primer cassettes containing the molecular barcodes.

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Same dual hybridization requirement as regular PCR for high specificity

More than a million unique molecular barcodes are available for incorporation, ensuring unique coverage

Both primers incorporated on the probe avoiding cross reactivity

Hybridization


Probe/fragment hybrids are ligated and retrieved with streptavidin magnetic beads, followed by high stringency wash.

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Ligation, capture and wash

Only perfectly hybridized fragments will be ligated

Ligated fragments are directly captured using streptavidin


Only fully circularized DNA targets are amplified on-bead.

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PCR amplification

Thousands of different amplicons, one primer pair

On-bead PCR of ligated fragments simplifies workflow

Ready for sequencing in <6hr!


Agenda

Introduction


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5

3 Performance data


HaloPlexHS Performance - High Uniformity and Specificity

Uniform coverage of targeted bases: >95% covered at 10% of average depth

High Specificity: >80% on-target specificity

• important since deep sequencing is required for low frequency variant detection

9.9kb 48.1kb 142.7kb 260kb 1.8Mb 4.8Mb0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

HaloPlexHS sequencing performance

On-target specificity 10% of ave. depth Coverage at 10X Coverage at 20x


HaloPlexHS Performance – Excellent coverage even with FFPE samples

5.8 3.8 3.6 1.9 2.1 8.2FFPE Sample

1FFPE Sample

2FFPE Sample

3FFPE Sample

4FFPE Sample

5Cell line

NA18507

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0

100

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HaloPlexHS performance with FFPE samples

Specificity bp (%) Coverage at 20x (%)Coverage at 100x (%) Average depth Seq Region

Cove

rage

Sequ

enci

ng D

epth

DIN

Excellent coverage of target bases (>90% covered at 100x) even with poor quality FFPE DNA. A custom cancer panel was used to enrich FFPE DNA of varying qualities as indicated by the DNA Integrity Number (DIN) provided by the 2200 Tapestation System, where a DIN of 10 and 1 indicate intact gDNA and completely degraded gDNA respectively.


HaloPlexHS Performance – Detection down to 0.5% variant allele frequency

• Detection of down to 0.5% allele frequency in HapMap dilutionsExpect

ed a

llele

fr

equency

HapMap cell lines, NA18507 and NA10831, were mixed to generate allelic fractions ranging from 0.5% - 5%. The close agreement between expected and observed frequency at various chromosomal positions demonstrates the high sensitivity of HaloPlexHS for low frequency variant detection. Data shown is representative of replicates (sequencing depth = 2000x – 4000x)

1789

1700

5

5515

2040

1521

2907

7

5522

9255

5523

8268

5524

0461

5524

2609

9224

4422

5342

42

5649

4991

6923

3215

6923

3716

6886

3314

3616

5041

chr3 chr4 chr6 chr7 chr7 chr7 chr7 chr7 chr11 chr12 chr12 chr12 chr16 chr21

0.0%

1.0%

2.0%

3.0%

4.0%

5.0%

6.0%

7.0%

8.0%

Detection down to 0.5% allele frequency

5% 2.5% 1% 0.5%


HaloPlexHS Performance – Detection down to 0.5% variant allele frequency Chr. Pos. 5% 2.5% 1% 0.5%

chr3:178917005 58 18 18 10

1062 746 1052 1280

chr4:55152040 113 27 30 12

2102 1391 1912 2602

chr6:152129077 219 69 43 26

3989 2682 3816 5143

chr7:55229255 60 27 7 7

883 757 998 1285

chr7:55238268 170 61 35 17

3155 2194 3142 4132

chr7:55240461 102 45 34 14

2337 1766 2444 3028

chr7:55242609 33 9 4 10

592 400 554 666

chr7:92244422 78 31 29 14

1562 1306 1738 2264

chr11:534242 201 63 32 14

3629 2391 3656 4952

chr12:56494991 74 26 13 12

1584 1136 1508 1802

chr12:69233215 120 50 36 12

2518 1885 2666 3309

chr12:69233716 31 16 8 3

734 637 818 979

chr16:68863314 14 8 6 2

233 265 365 394

chr21:36165041 8 5 1 2

117 88 128 175

Figure 1B. Number of total unique reads covering the target regions. The unique reads covering each allele fraction at the various chromosomal positions are highlighted (green)

Number of total unique reads covering the target regions. The unique reads covering each allele fraction at the variouschromosomal positions are highlighted (green)


Simplify Data Analysis with SureCall


Agenda

Introduction


1

2


5

3 Performance data


HaloPlexHS - a flexible solution

Compatible with both ILM and ION PGM platforms

Create custom designs up to 5Mb (2.5Mb for ION)

NGS Disease Research Panels are available in catalog or made-to-order format

Multiplex up to 96 samples for ILM and 16 samples for ION


Accelerate Time to Results

1 2

Prepare DNA libraries in

<6hr

Begin sequencing on

a desktop sequencer

Analyze your data

Day 1

Prepare Sequence Analyze

Day 2


Summary

• HaloPlexHS Target Enrichment System is a high sensitivity method for the accurate identification of low allele frequency variants

• HaloPlexHS incorporates unique molecular barcodes into each DNA library fragment

• HaloPlexHS performs with high coverage, on-target specificity and with a sensitivity that allow detection of alleles down to below 1% allele frequency (at least 0.5%)

• It is ideally suited for cancer research and studies that involve the detection of somatic variants in heterogenous samples


haloplex hs get to know your dna. every single fragment

Documents

diagnostic procedures

haloplexhs high sensitivity

haloplexhs customer

nt molecular barcodes

variants present

increased accuracy

increased sensitivity

dna library fragmentsrequires