SureSelectQXT SureCall 2.1 · • Same day workflow, 90-min hyb, 3.5 hrs hands-on time ... 0 5 10 15 20 25 30 35 40 Competitor I SureSelectXT SureSelectQXT . AGENDA Introducing SureSelectQXT

For Research Use Only.

Not for use in diagnostic procedures

SureSelectQXT SureCall 2.1:

Breaking Barriers in

Clinical Research Sequencing

Maria Celeste M. Ramirez, Ph.D.

Global Product Manager, NGS

Michael Borns

R&D Scientist, Agilent

AGENDA

Introducing SureSelectQXT

Benefits of SureSelectQXT

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The SureSelectQXT Transposase

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4 SureSelectQXT for Whole Genome Sequencing

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SureCall 2.0: Faster time to results 6

Accelerated Target Enrichment with SureSelectQXT



Addressing bottlenecks to ENABLE clinical research

What is SureSelectQXT?

50ng sample input

Fast turn-around time:

Streamlined workflow

Scalability

Sensitivity and Accuracy

The FASTEST capture solution in the market



Addressing bottlenecks to ENABLE clinical research

What is SureSelectQXT?

50ng sample input

Fast turn-around time: 90 minute hyb, 3.5x faster than competition

Streamlined workflow Covaris-free, 30% less hands-on-time

Scalability

Sensitivity and Accuracy

The FASTEST capture solution in the market



AGENDA



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SureSelectQXT

The Leader in Target Enrichment, now FASTER

COMPLETE and FLEXIBLE Solution

Superior PERFORMANCE

FASTEST Workflow



SureSelectQXT



FASTEST Workflow




SureSelectQXT Sample to data in as little as 24 to 36 hours

*MiSeq, HiSeq systems

< 7 hours ~12.5*-27** hours

DATA in

24-36 hrs Transposase-

based

Library Prep

90-minute Hybridization

Sequence *MiSeq and **HiSeq

systems

• FAST, EASY and CONVENIENT:

30 min hands-on time for library prep

3.5h overall hands-on time

No special equipment required for fragmentation

• 50ng SAMPLE INPUT

NO MECHANICAL SHEARING



Library Prep workflow

SPRI purification = 20min

200ng input



Fragmentation PCR

~ 1.5 hours

SPRI purification = 20min

Hybridization Capture PCR

~ 5.5 hours

Library

QC

Library prep SureSelectQXT Target Enrichment

Library

QC

50 ng gDNA input

1.5 hours library prep workflow

90-minute hyb

SureSelectQXT Target Enrichment Workflow



Library Prep Enrichment Sequence

SureSelectQXT The Fastest Enrichment Workflow

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Competitor I

Competitor N

WORK DAYS

Agilent

SureSelectQXT

• ~4 day worfklow, 72h hyb

• 1ug input

• 1.5 day workflow, 5 hrs hands-on time: 2x capture and wash

• 50ng input

• Same day workflow, 90-min hyb, 3.5 hrs hands-on time

• 50ng input



SureSelectQXT



Superior

PERFORMANCE

FASTEST Workflow



SureSelect Target Enrichment: High-level performance enables accurate variant profiling

• high % mappable reads

• unparalleled % on-target

• deep and uniform coverage of ROIs

Mapping

Metrics

• high sensitivity enables accurate detection of SNVs,

InDels and CNVs

• excellent allelic balance

Variant

Calling

Metrics



8 HapMaps

(ave)

V5 exome,

4Gb

Competitor I,

4Gb

Higher coverage of targets for the same amount of sequencing



SureSelectQXT

Competitor I

60

65

70

75

80

85

90

95

100

bases at 1x coverage bases at 20x coverage % on target (+/- 100bp)

High performance from an accelerated workflow

Pe

rce

nta

ge

(%

) Mapping

Metrics

Variant

Calling

Metrics

Comprehensive Variant Detection

More SNPs and Indels identified

• Longer baits: tolerate larger regions of mismatch

• RNA baits: stronger interaction with DNA targets

NA18507

V5 exome, 4Gb

Competitor I, 4Gb



SureSelectQXT + V5 Competitor I

SNPs

evaluated sites 52174 33823

sites that overlap with

dbSNP 50779 (97%) 32205 (95%)

concordant SNPs 50705 (100%) 32163 (99.8%)

novel SNPs 1395 1618

InDels



dbSNP 3295 (88%) 1481 (85%)

concordant SNPs 3120 (95%) 1381 (93%)

novel SNPs 461 257

SureSelectQXT



FASTEST Workflow




SureSelectQXT

SureSelectQXT - Not Just for Exomes Custom capability through SureDesign

CUSTOM



SureCall

SureSelectQXT Faster sample to data with SureCall

For SureSelect



SureSelectQXT

• Whole genome sequencing

(library prep only)

• Small or large genomes

Library Prep

Target Enrichment

Standalone Barcodes

SureSelectQXT Complete and flexible solution for WGS and target enrichment

• Targeted resequencing

(library prep + enrichment)

• Catalog (exomes, kinome)

• Custom (up to 24Mb)



AGENDA



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PCR-ready template

Fragmentation and

Tag Addition

Gap Repair Template DNA



Library Prep

Baits + Hyb and

Wash Buffers

Target Enrichment

Library

Prep

Whole

Genome

Sequencing

SureSelectQXT Complete and flexible solution for WGS and target enrichment



AGENDA



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Reference: Poster #P045 presented at Plant and Animal Genome Asia 2014 (Singapore)

Notes: - 50ng bacterial gDNA was used for each experiment.

- Sequencing reads were normalized to half the size of each bacterial genome for comparison purposes.

Whole genome sequencing using SureSelectQXT

and bacterial genomes of varying GC content:



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Reference: Poster #P045 presented at Plant and Animal Genome Asia 2014 (Singapore)

SureSelectQXT versus competitor kit C.jejuni DNA (30% GC)



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Notes: - 50ng human gDNA (HapMap NA10831) was used for each experiment.

- Sequencing reads were normalized 600 million (2X100) for comparison purposes.

Whole genome sequencing using human gDNA

% duplication

SureSelectXT 2.74

SureSelectQXT 4.21

Competitor I 4.37



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Competitor I

SureSelectXT

SureSelectQXT

AGENDA



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Optimal Transposase Fragmentation Profile

Notes: - 50ng human gDNA input, NA12878

- 45°C, 10 minute incubation

- Post-PCR



High Performing Exome Captures with SureSelectQXT

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30

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80

90

100

NA 18507 NA 18997 NA 12878 NA 12155 NA 12891 NA 12156 NA 18953 NA 10831

Percent duplicate reads:

Percentage reads in targeted regions:

Percentage reads in targeted regions +/- 100bp:

...at least 1 read:

...at least 5 reads:



Average Std. Dev. %CV High Low

Percent duplicate reads: 9.51 0.475 4.99 10.26 8.74

Percentage reads in targeted regions: 71.35 1.474 2.07 74.01 68.43

Percentage reads in targeted regions +/- 100bp: 88.44 1.532 1.73 90.58 85.43

Percentage of targeted bases covered by…

...at least 1 read: 98.97 0.058 0.06 99.06 98.86

...at least 5 reads: 96.72 0.158 0.16 97.04 96.51

...at least 10 reads: 92.52 0.325 0.35 93.02 92.04

...at least 20 reads: 81.95 0.547 0.67 82.92 81.08

8 HapMaps in duplicate

V5 exome, 4Gb

2x100bp



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0

20

40

60

80

100

120


8 HapMaps

(ave)

V5 exome,

4Gb

Competitor I,

4Gb

Pe

rce

nta

ge

(%

)

SureSelectQXT

SureSelectXT

High Performing Exome Captures with SureSelectQXT



8 HapMaps

(ave)

V5 exome,

4Gb

Competitor I,

4Gb

Higher coverage of targets for the same amount of sequencing



SureSelectQXT

Competitor I

60

65

70

75

80

85

90

95

100


High performance from an accelerated workflow

Pe

rce

nta

ge

(%

)

Confidence in variant calling with SureSelectQXT

Hom/Het for HapMap

Hapmap NA18507 NA18997 NA12878 NA12155 NA12891 NA12156 NA18953 NA10831

CONCORDANCE

GT is REF 99.96% 99.95% 99.97% 99.97% 99.98% 99.96% 99.97% 99.97%

GT is variant HOM 98.97% 99.06% 98.94% 98.95% 98.89% 98.86% 99.02% 98.96%

GT is variant HET 99.49% 99.44% 99.51% 99.71% 99.37% 98.48% 99.60% 99.56%

SENSITIVITY

GT is REF 99.95% 99.92% 99.92% 99.93% 99.89% 99.93% 99.89% 99.91%

GT is variant HOM 99.77% 99.79% 99.78% 99.75% 99.61% 99.73% 99.79% 99.68%

GT is variant HET 99.36% 99.37% 99.29% 99.36% 98.61% 98.88% 99.10% 99.54%

Hom/Het for dbSNP


n_eval_sites 52174 42880 42871 42724 41458 42482 42090 42418

concordance_rate 99.86% 99.87% 99.88% 99.87% 99.90% 99.91% 99.86% 99.91%

Indels for dbSNP


n_eval_sites 3756 3184 3234 3162 2966 3046 3001 3056

concordance_rate 94.69% 95.66% 95.74% 94.92% 96.14% 96.36% 95.59% 95.93%

Notes: - Average of 2 replicate libraries for each hapmap.

- All libraries were normalized to 40 million reads (2X100).

8 HapMaps

V5 exome,

4Gb For Research Use Only.


V5 exome data, 4Gb

8 HapMaps

High sensitivity and concordance

SureSelectQXT

SureSelectQXT

SureSelectXT



Comprehensive and balanced variant detection

More SNPs and Indels identified

• Longer baits: tolerate larger regions of mismatch

• RNA baits: stronger interaction with DNA targets

V5 exome, 4Gb

Competitor I, 4Gb



SureSelectQXT

Competitor I

SureSelectQXT + V5 Competitor I

SNPs



dbSNP 50779 (97%) 32205 (95%)

concordant SNPs 50705 (100%) 32163 (99.8%)

novel SNPs 1395 1618

InDels



dbSNP 3295 (88%) 1481 (85%)

concordant SNPs 3120 (95%) 1381 (93%)

novel SNPs 461 257

*NA18507

Excellent Reproducibility with SureSelectQXT

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10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

-1 4 9 14 19 24




...at least 1 read:








...at least 1 read: 98.66 0.030 0.03 98.73 98.48

...at least 10 reads: 95.47 0.296 0.31 96.04 94.4

...at least 20 reads: 88.3 0.971 1.1 90.04 84.03

24 samples

Kinome, 300Mb

2x100bp



Ave of 72 samples (24 samples x 3 users)

Kinome, 300Mb, 2x100bp

High performance from large or small captures

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60

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90

100

rep-1 rep-2 rep-3 rep-4 rep-5 rep-6




...at least 1 read:




8 Hapmaps

1Mb, 100Mb

2x100bp






...at least 1 read: 99.58 0.048 0.05 99.62 99.49

...at least 10 reads: 97.55 0.209 0.21 97.79 97.19

...at least 20 reads: 93.44 0.749 0.80 94.07 92.04

Notes: - Average of 6 replicate libraries.

- All libraries were normalized to 1 million reads (2X100).



Kinome Rep-1 Rep-2 Rep-3

% dups 6.05 6.09 6.73

% on target 62.86 62.17 64.09

Percentage of

targeted bases

covered by

1x 98.91 98.97 98.92

10x 96.79 96.46 96.25

20x 93.06 92.19 91.70


% dups 8.93 8.99 9.74

% on target 67.20 67.34 71.79

Percentage of

targeted bases

covered by

1x 98.69 98.66 98.69

10x 96.12 96.08 96.31

20x 91.55 91.31 92.27


% dups 7.19 7.52 7.32

% on target 65.34 66.60 66.63

Percentage of

targeted bases

covered by

1x 98.85 98.82 98.84

10x 96.52 96.53 96.67

20x 92.76 92.76 93.20

Under fragmentation

Optimal fragmentation

Over fragmentation

Variable DNA fragmentation profiles and their

effect on target-enriched sequencing results

Kinome,

300Mb

2x100bp For Research Use Only.


AGENDA



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The Analysis Pipeline Puzzle: Piecing together different solutions for different steps

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Control

Software

FASTQ

file

reads +

quality

1. Primary Analysis

Aligner

BAM file

reads

aligned to

genome

FASTQ

file

reads +

quality

2. Read Alignment

Variant

caller

VCF file

variant

call

format

BAM file

reads

aligned to

genome

3. Variant Calling

Additional filtering on read depth & quality score

Filter based on population frequencies

Non-synonymous variants

Functional prediction

Inheritance pattern

Final

report

VCF file

variant

call

format

Mutation

report

3. Tertiary analysis: interpretation

SureCall: from alignment to mutation report

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Guided data analysis from alignment to categorization of mutations

• Streamlined Workflow

• Faster Time to Results

- Alignment of FASTQs to variants

• Designed for clinical research

• Free of Charge

www.agilent.com/genomics/surecall

SureCall 2.0: Simplified Raw Data to Variants

Page 43



http://www.agilent.com/genomics/surecall

SureCall’s Simple 3-Step Analysis Workflow

1. Import Sample FASTQs

2. Describe Samples

3. Run Analysis

44



Types of Analysis

Description Result

Single Sample Analysis • For individual samples • SNPs and indels

Pair Analysis • CNV analysis using normal reference

• Paired tumor-normal samples

• SNPs and indels

• CNVs

• Somatic mutations

Trio Analysis • For trios, typically mother, father and child • SNPs and indels

• De novo mutations

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SureSelectQXT

Trimmer Yes

Remove Duplicates Yes

Region Padding Yes - 100 bp



Visualize and review categorized mutations from the entire capture…



New in SureCall 2.1: Analyze the entire capture, focus on your known variants



Visualize and review only your known variants



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Definitive Answers, FASTER

• SNPs and Indels



• Copy number variants

Enabling a Clinical Research Workflow

SEQUENCE

<1.5 day

3

DESIGN

~10 mins

1 4

ANALYZE

~1 hour

ENRICH

<1 day

2



Thank You!

SureSelectQXT

Introductory Promotion

50% discount when purchased with an

exome or a qualifying custom bait library

Contact your local rep for more information

Documents

SureSelectQXT SureCall 2.1 · • Same day workflow, 90-min hyb, 3.5 hrs hands-on time ... 0 5 10 15 20 25 30 35 40 Competitor I SureSelectXT SureSelectQXT . AGENDA Introducing SureSelectQXT