aug2014 acrometrix

1

The world leader in serving science

Mona Shahbazian, Staff Scientist

Kara Norman, Head of R&D

AcroMetrix® Brand

Building on the Genome in a Bottle for Oncology

2

NGS Quality Control

CAP Checklist4

• Daily run controls needed

NY State2

• “Low positive control

… containing multiple known somatic

alterations of each kind”

CDC (Nex-StoCT)3

• Reference material

characteristics:

• Complexity and different

mutation types

• Most / all of variants

• “Synthetic constructs could

be used for routine QC of

each NGS run”

• What is needed for robust QC?

1. Dutch Society for Clinical Genetic Laboratory Diagnostics (VKGL): Humu 10.1001/22.368 (2013) 2. New York State Clinical Laboratory Evaluation Program (CLEP) Laboratory Standards. 3. Gargis AS, et al. Nature Biotechnology. 30, 1033–1036 (2012). 4. College of American Pathologists (CAP) Laboratory Accreditation Checklist

VKGL1

• “…a separate SNP control”

3

Reference material for oncology

• Customer needs

• Clinically relevant variants

• Various lengths of indels up to ~40 nt

• Minimize real-estate on chip/flow cell

• High quality/robustness for clinical use

4

Overlapping Variants Problematic for Variant Calling

• 7 KRAS mutant cell lines + 1 WT cell line

• Extracted genomic DNA and mixed at equal conc.

6 or 12%

Expected

Observed

275 280 281 282 283 284 285

T G C C A C C

T A A

G G

T T

A,G (5%) 8 MNVs (5%) T,A,G (3.85%) 7 MNVs (20%) 14 MNVs (19%) G,A,T (55%)

T G C C A C C

A A A C A A

G C G G G G

T T T T T

7 SNVs

29 MNVs

3 SNVs

3.9-55%

Ref

Alt

Ref

Alt

5

AcroMetrix® Oncology Hotspot Control

1 8 I N S

• MegaMix Technology

• Built on Genome in a Bottle material

• GM24385 cell line

• ~550 Common variants

• Low frequencies

• Confirmed by Sanger

• Multiple NGS platforms

• CE-IVD, US-IVD

Length

(bp)

0

10

20

30

40

10

0

20

30

40

5 0 4 S N V 2 M N V 2 9 D E L E T I O N S

1 C O M P L E X I N D E L

1 9 I N S

For In Vitro Diagnostic Use

6

Includes Important Cancer Variants

EGFR

L858R

L861Q

E746del

BRAF

V600E

HRAS

Q61R

KRAS

Q61H

G12D

NRAS

Q61R

G12D

PIK3CA

H1047R

E542K

E545K

PTEN

Y68H

AKT1

E17K

ALK

F1174L

R1275Q

CTNNB1

T41A

S45F

KIT

R804W

W853*

IDH1

R132H

IDH2

R140Q

R172K

VHL

G93R

FLT3

D835Y

FGFR3

S249C

FGFR2

S252W

JAK2

V617F

MPL

W515L

NOTCH1

L1600P

PDGFRA

D842V

GNA11

Q209L

GNAS

R201C

GNAQ

T175S

MET

Y1253D

NPM1

W288fs*

RET

M918T

TP53

R273H

R248Q

7

Performance tested at >30 external sites

2 control lots

Ion AmpliSeq™ Cancer Hotspot Panel v2 (CHPv2)

TruSeq® Amplicon Cancer Panel (TSACP)

TruSight® Tumor Panel (TSTP)

Each laboratory is required to establish its own performance specifications

8

CHPv2 – 13 sites Panel

Site 24 22 9 21 11 1

Lot Lot 1 Lot 2 Lot 2 Lot 1 Lot 1 Lot 1 Lot 1 RLX

Run 1 2 1 2 1 1 1 1 1 1 1 2 1 2 1 2 1 2 1 1 2 3 4 1 2 3 1 1 222.5 22.1 23 23.2 22.7 21.7 24.6 23.6 26.65 25.33 21.57 22.96 23.56 22.76 22.5 21.5 23.2 22 25 22.16 22.45 21.95 20.4 24.11 22.37 22.25 21.81 24.6523.1 23.6 21.7 23.8 22.7 21.8 23.5 23 26.15 25.45 22.91 22.1 21.09 22.85 20 21.4 19.6 22 29.2 23.61 24.86 21.05 20.76 23.75 23.51 22.12 22 23.8224.4 23.6 22.3 24.1 22.1 21.3 24 22.7 26.36 25.24 22.42 21.65 21.81 22.13 21.2 21.7 22 20.9 28.2 22.36 23.01 20.37 20.61 23.31 22.76 21.45 22.63 2622.4 22.3 22.4 24.2 23.1 20.5 22.4 23.1 24.25 23.58 20.71 22.15 22.45 23.6 22.6 24.3 21.3 23.2 26.2 21.6 23.16 19.75 20.25 22.1 19.76 20.55 20.96 23.923.4 23.3 22.5 23.2 23 20.4 22.2 23.3 24.22 23.59 20.67 22.31 22.28 23.9 23 23.9 23.2 22.2 24.5 22.32 22.31 20.77 18.82 22.76 21.4 21.91 20.92 23.7424 23.1 21.7 20.5 22.1 22.1 22.6 23.4 22.95 24.96 21.42 21.01 22.34 21.83 17.6 18.9 18.6 19.4 24.8 20.11 23.11 20.2 20.65 22.2 21.16 21.78 21.43 25.5525.1 22.2 21.9 20.4 22.7 22.5 22.6 24 22.72 25.04 21.72 20.99 22.43 22.18 18.8 20.1 18.1 19.3 26 20.27 23.28 20.46 20.63 21.7 21 22.04 21.4 25.624.6 22.5 21.8 19.8 22 21.9 22.3 23.4 22.37 24.88 20.88 20.86 20.82 21.34 18.9 18.4 17.2 18.1 22.8 20.06 22.83 20.4 20.27 21.37 21.21 21.83 21.5 25.4124.7 24.3 25.5 25.2 24.4 23.1 25.8 24.5 25.63 26.9 24.87 24.43 24.64 25.34 23 21.7 22.1 24.3 27.5 23.31 24.19 23.69 25.58 23.89 21.98 22.9 21.72 24.9925.4 23.1 25.9 25.2 24.5 23.3 24.9 24.6 26.65 26.87 24.85 24.3 24.35 25.07 22.2 23.4 21.9 23.9 25.8 21.2 23.86 22.95 24.45 24.45 23.35 23.3 21.76 26.2524.3 24.2 24.8 25.4 24.2 23 26 24.8 26.5 26.78 24.53 24.35 24.84 24.84 23.2 24.6 21.1 23.4 26.2 22.86 24.71 24.15 23.47 25.5 23.36 23.76 21.66 25.2325.4 24.6 26.6 24.6 24 23 25.6 23.1 25.5 26.75 24.42 24.31 24.85 25 23 24 21.6 22.6 26.2 22.4 24.96 23.85 23.96 24.56 22.1 23 21.59 25.228.9 27.8 27.7 27.8 25.5 27.2 30.9 31.81 28.79 27.72 24.18 29.46 24.2 26.9 29.7 30.1 27.2 30.4 25.23 24.32 22.49 21.89 25.79 27.57 24.81 26.11 28.3223.6 23.5 22.9 23.8 24.4 20.7 23.2 20.2 24.86 22.86 19.99 22.28 21.93 20.43 19.6 20.4 19.5 22 20.5 21.37 22.28 21.09 19.11 22.75 22.53 20.22 22.1 22.8518.6 16.8 18.2 12.1 20 16.2 21.8 18.1 17.3 22.58 18.88 18.11 18.23 18.84 15.3 13.9 11.5 13.9 22.3 16.53 17.1 19.84 19.57 19.39 19.22 19.98 20.61 20.8218.5 17 18.2 12.1 20.7 17 22.3 18.6 17.29 22.53 18.58 18.41 18.41 19.05 14.8 13.7 12.3 14 21.5 16.92 17.67 19.62 19.65 19.38 19.28 20.41 20.5 21.1123.4 21.2 20.6 22.4 23.6 19.4 22.86 23.51 21.53 24.74 21.25 21.09 21.3 20.8 20.7 19.5 22 21.8 22.55 22.15 21 21.31 20.56 20.74 21.76 23.323.2 21.3 20.6 22.3 24 19.7 23.41 23.55 21.57 24.59 21.2 21.64 20.5 19.8 22 19.2 25 21.99 22.58 21.65 19.83 21.37 20.76 20.77 21.78 24.1619.5 22.9 20 19.4 24.9 22.2 22.4 23.4 23.01 26.24 20.2 19.58 21.83 21.93 21.1 21.4 24.6 19.7 27.5 22.32 20.45 21.6 20.5 23.56 22.26 22.43 20.15 21.0119.2 22.8 22.7 19.7 24.9 22.2 22.3 23.3 23.46 26.36 20.68 19.95 20.73 22.22 19.5 22.2 22.4 20.9 20.5 22.48 19.93 21.48 20.02 22.62 22.8 22.88 20.15 21.225.1 22.6 24.6 20.8 24.5 21.6 24.8 24.5 27.66 24.46 23.82 22.19 25.25 25 22.4 25.7 25.3 24 30.8 24.9 23.55 24.4 23 23.9 24.31 22.57 21.81 22.725.5 22.8 25.9 21.5 24.4 21.6 23.6 24.4 27.38 24.6 23.97 21.99 25.31 24.89 23.9 24.7 25.8 23.3 36.2 24.15 24.64 23.26 21.46 24.77 23.12 23.1 21.77 24.2421 20.8 22.3 22.9 22.7 20.7 22.8 24.3 22.85 23.16 20.9 23.62 22.51 22.12 20.9 22.8 22.2 20.3 22.2 20.65 23.01 23.72 21.35 23.61 21.66 22.82 20.66 20.3523.9 24.5 23.2 24.7 25.1 21.6 27.7 26.4 29.35 26.56 21.5 24.76 24.72 26.17 21.9 24.8 23.8 24.6 30 24.41 26.91 25.86 23.8 26.07 25.51 24.85 23.41 25.91 24.2122.5 25.1 24.2 24.9 25 21.8 27.6 27 28.39 26.53 21.82 25.16 24.82 26.02 22.5 26.5 24.1 26.2 29.2 24.39 26.8 24.9 23.23 25.96 23.86 25.8 23.29 26.13 24.8923.4 24.6 24.4 25.1 25 21.7 27.6 27.1 29.5 26.45 22.26 25.11 24.5 25.71 24.4 26.2 27.1 23.6 28.5 25.76 27 26.05 24.3 26.81 24.9 25.55 23.47 25.76 25.7322.4 23.1 23.4 25.4 23.9 22.2 20.4 20.9 27.6 22.79 24.15 25.01 25.1 24.32 21.8 20.5 23.8 19.6 27.2 22.5 22.71 23.31 22.8 20.95 20.75 18.97 25.61 24.7121.1 21.8 22.7 25.6 23.3 21.8 20.8 20.8 27.99 22.76 23.96 24.93 25.11 24.22 22.9 20.4 22.2 20.3 24 21.2 23.99 21.5 23.06 20.71 20.67 19.25 26.25 25.522 21.6 24.2 24.4 23.9 22.3 20.5 19.8 27.35 22.84 23.71 24.94 24.4 24.04 22.7 21.8 21.8 20.7 27.8 21.75 23.52 22.55 23.45 20.15 20.8 19.95 25.11 24.821 23.2 23.7 21.8 23.3 22.6 23.6 24.7 22.99 22.08 19.04 23 22.11 21.73 23.4 24.7 23.9 24.8 23.8 23.13 23.41 23.11 19.28 24.17 23.84 21.53 21.54 24.05 25.7621 23.6 23.5 22.1 23.5 22.7 23 24.1 22.48 22.02 18.32 22.64 22.24 21.96 23.3 23.8 24.2 23.8 24 23.27 22.94 23.38 19.14 24.53 24.17 20.88 21.5 24.3 24.6221.9 24.6 21.7 21.6 29.6 19.6 27.47 25.79 22.94 25.91 28.11 20.1 22.7 23.7 19.6 30.6 24.36 26.08 23.91 25.55 24.9 28.76 22.52 18.96 26.9622.1 28.1 23 22.4 32.6 19.8 28.04 25.25 28.08 29.39 23.7 21.9 23.9 21 31.5 27.34 28.59 26.29 28.13 26.9 29.05 24.5 18.91 29.3923.4 21.2 23.6 22.6 23.4 22.9 19.2 22.2 24.44 24.04 21.11 21.65 20.93 23.13 24 23.8 20.1 20.6 21.8 21.36 23.74 21.83 20.37 21.26 20.24 22.75 22.81 26.04 24.0522.4 21.3 24 22.6 23.4 22.9 19.1 23.3 24.07 24.12 21.32 21.46 21.62 23.24 22 23.3 23.9 20.9 21.4 21.36 22.32 21.72 19.7 21.3 21.51 22.82 22.81 25.88 22.2723.7 22.1 24.2 22.6 23.5 22.8 19.8 22.6 24.45 24.16 21.46 21.59 22.51 23.19 22.1 22.9 21.7 23 21.2 21.35 22.15 22.3 20.85 23.2 22.22 22.3 22.9 25.45 24.8523.5 21.4 22.5 22.4 23.5 23.1 19.5 23.4 24.32 24.2 21.54 21.65 22.75 23.14 24.3 24.3 23 21.4 24.1 22.07 22.85 23.8 19.28 22.54 21.93 22.71 22.82 26.18 23.7121.5 22.6 23.3 23.9 21.7 20.1 22.6 22.8 23.18 23.64 21.5 23.76 22.18 22.61 21.1 23 22.4 21.7 23.1 22.78 22.19 21.56 19.98 22.98 21.55 23.24 21.18 23.56 23.3721.9 23.4 22.3 20.1 22 20.5 22.4 19.1 21.56 24.25 21.5 22.05 22.27 22.62 21.9 22.6 22.5 20.8 21.8 22.44 23.47 22.11 19.13 22.88 22.24 23.28 21.88 22.97 23.822.6 24.1 22.4 25.2 23.6 23.4 24 23.19 21.37 24.83 21.68 24.32 23.7 22.4 24.3 22.5 23.1 22.52 23.31 21.45 20.88 25.14 25.05 21.13 21.42 25.26 23.0222.4 23.9 21.9 24.8 21.7 20.8 24 23.4 23.9 23.19 21.63 25.02 22.82 24.03 25.8 22 22.4 21.4 20.5 21.96 23.22 21.38 20.97 25.54 25.38 21.59 21.28 24.76 23.421 24.4 22 24.1 24.9 23.2 26.2 21.8 23.11 23.75 22.38 22.05 23.35 22.18 22.9 22.3 21.4 23.3 23.8 22.97 22.81 24.74 21.69 21.62 23.79 22.76 22.76 21.91 22.821.8 23.2 21.2 25.1 25 23.3 26.3 21.9 23.4 23.64 21.88 22.15 23.27 22.12 21.2 22.7 23.2 22.3 21.5 23.65 23.47 26.38 22.61 22.92 23.36 22.57 22.75 20.71 25.0120.6 23.7 22 24.9 25.1 23.5 26.1 22.1 23.8 23.89 22.05 22.01 21.2 21.98 21.6 21 21.9 20.5 22 23.9 23.81 24.6 22.1 22.55 24.11 21.06 22.97 21.21 23.7621.7 22.7 22.3 23.9 25 23.3 26.4 21.6 25.96 23.83 21.88 22.17 22.49 22.48 22.1 21.8 21.3 19.8 25.2 23.92 23.59 25.66 22.41 21.11 24.29 22.21 22.89 23.11 24.1623.9 22.7 22.3 21.4 24.6 21.7 22.7 23.9 26.13 26.27 23.89 21.57 23.16 23.3 23.2 22.6 22.5 21.3 28.2 23.02 23.37 24.89 21.86 25.11 23.77 24.2 21.94 24.3522.3 20.7 21.3 24.3 22.8 20.9 22.4 22.1 21.4 20.47 21.47 22.28 22.23 20.16 20.5 21.5 20.8 19.8 29.8 22.61 23.87 23.82 22.47 21.66 21.07 21.03 20.75 22.6824.5 23.2 25.4 21.5 25.1 22.2 24.8 26.3 27.3 25.93 23.88 24.43 23.2 26.53 23.1 22.9 24.7 24.7 28.5 26.2 24.26 25.6 23.94 23.97 25.25 24.54 22.32 25.7320.6 18.2 21.2 17 22.4 22.1 25 22.7 24.86 11.05 16.71 23.1 19.92 24.58 9.8 9.7 8.9 9.1 30.8 24.07 24.04 21.2 21.06 26.84 24.16 21.42 20.44 23.3420.6 17.3 19.5 18.7 19 16.7 19.2 19.3 19.51 20.31 18.27 19.92 16.24 21.1 20.1 18.2 21.7 21 17.9 22.61 20.83 21.73 21.01 19.99 18.95 17.31 19.26 19.9421.9 21 21.7 23.2 24.2 22.4 24.7 21.7 23.15 24.02 22.82 22.96 19.95 21.79 25.9 25.3 24.8 23.6 24.8 21.43 22.09 21.78 20.19 23.37 22.22 21.1 22.46 24.1621.4 21.3 21 23.3 24.2 22.4 24.3 21.4 22.62 24 22.6 23.01 20 21.72 25.9 24.3 26.2 26.5 26.1 21.51 22.07 21 19.4 23.34 22.5 20.94 22.52 24.3521.3 21.8 21.2 23.2 24.4 22.4 24.5 22.6 22.91 23.97 22.78 22.73 19.67 21.61 25.4 24.3 25.3 25.1 27 20.83 21.74 21.53 20.18 23.94 22.02 20.85 22.55 22.8722.1 22.1 20.6 23.5 24.5 22.7 24.4 22.3 23.02 24.05 23.05 22.82 20.43 22.1 24.1 25.1 24.7 25.9 24.3 21.63 22.23 21.8 19.76 23.25 21.87 21.07 22.55 24.9222.6 24.8 22.8 23.6 24 23.2 23.45 22.26 21.17 22.58 22.12 24.17 23.3 20.7 19.4 23.5 25.2 22.55 23.04 19.8 19.69 21.8 21.87 21.16 20 25.15 24.4722.8 24.8 23.2 23.6 24.1 23.5 23.84 22.26 21.39 22.75 22.23 24.34 23.2 20.8 19.1 23.2 25.8 22.72 23.16 19.86 19.63 21.86 21.86 21.49 20.11 25.1 24.5122.9 25 23.2 23.6 24.2 23.7 24.01 22.33 21.15 22.64 22.34 25.02 23.1 20.7 19.1 23 25.5 22.7 23.07 20.02 20.05 21.78 22.07 21.18 20.18 25.14 24.5722.3 24.1 23.1 23.7 23.5 24.8 23.01 22.25 21.66 22.74 22.32 24.84 22.7 22.9 22.7 21.9 25.2 22.71 23.01 19.84 20.1 22.06 22.36 21.47 20.17 24.09 24.7522.5 25.9 25.4 26 21.4 23.4 26.5 22.4 27.18 24.75 22.68 24.4 24.84 23.74 24.3 25.1 25.6 22.1 33.2 26.66 25.88 26.02 25.89 29.95 29.44 27.73 21.45 28.3318.9 22.2 23.5 26.1 23.8 20 24.16 21.52 23.26 24.3 21.9 23.55 20.1 18.5 24.2 17.9 39.6 22.37 24.37 25.23 23.05 27.99 25.04 26.96 18.98 25.01 24.9929.8 36.7 32 33 20.6 29.3 25.1 36.48 23.61 23.89 31.47 25.56 26.86 23.5 26.1 25.5 23.4 38.5 31.8 33.73 33.73 32.82 32.6 34.3 32.98 20.94 24.59 25.6924.1 27.4 25.1 25.5 22.8 23.4 26.7 25.7 37.86 21.77 26.03 28.65 25.71 24.82 22.7 26.2 25.7 23 36.1 26.45 27.74 26.94 25.96 26.86 28.11 31.22 20.85 24.87 27.1120.1 23.9 22.4 24 23.2 19.6 28.62 20.29 20.07 26.89 22.12 23.43 19.6 22.1 20.9 19 26 22.91 25.01 22.57 22.09 28.11 27.04 25.18 20.72 25.64 24.2618.7 19.8 21.2 20.2 17.9 19.8 23.4 21.3 24.24 22.09 19.2 19.16 19.94 18.92 19 21.1 20.2 21.8 27.2 20.29 23.11 18.09 18.84 24.15 25.46 24.4 19.08 22.9420.3 19.9 22.9 20.3 17.9 19.9 22.9 21.8 24.5 22.2 19.35 19.33 19.9 18.77 20.5 22.4 20.6 21.5 27.1 21.41 23.65 18.69 18.53 24.17 25 24.85 19.07 22.9526.5 27.8 28.4 28.6 26.9 27.7 27.3 29.61 25.48 27.32 27.37 25.75 25.17 41.2 42.6 44.3 46.6 51.8 22.15 27.95 29.9 34.97 36.2 36.89 20.82 25.9326.3 27.5 28.2 28.6 26.9 28.1 27.6 29.25 25.33 27.05 27.53 25.82 25.33 41.3 40.4 43.9 45.5 52 22.26 27.89 30.09 34.96 35.96 36 20.53 25.226.7 28.1 28.4 28.6 27.1 27.9 27.1 29.64 25.43 26.32 27.53 25.14 25.17 42.1 42.7 44.3 43.9 52.2 22.19 27.86 29.64 34.91 36.13 36.86 20.74 24.7526.9 28.1 28.6 28.7 27.3 28 27.3 29.33 25.57 26.79 27.53 25 25.17 40.1 41.7 43.6 46 52.5 22.15 27.8 29.92 35.05 36.45 35.56 20.67 24.1125.6 25.5 28.1 26 25.3 42.8 29.55 25.58 25.55 25.96 28.61 28.32 32.5 35.9 35.8 32.2 38 25.3 30.85 26.36 25.95 30.95 33.3 33.11 18.8 24.7125.4 25.3 27.1 25.8 25.5 42.6 29.35 25.41 25.24 25.58 28.34 28.53 33.8 36.3 35.7 30.9 37.6 24.46 30.57 26.06 25.7 30.44 33.43 33.14 17.98 26.0925.7 25.3 28.6 25.6 26 43.5 29.47 25.86 25.34 25.29 29.02 28.19 29 32.1 31 29.7 37.1 24.39 30.81 26.59 25.59 29.49 33.4 33.3 15.07 24.4424.7 25.2 27 25.3 26.2 38.7 28.2 25.83 24.55 25.44 26.67 28.57 42.5 42.6 44.1 36.3 41.5 23.94 29.5 25.03 24.91 30.11 32 33.26 18.8 24.5218.9 20.1 21.5 21.5 20.1 20 20.1 19.6 26.04 19.82 17.36 19.97 20.29 18.66 18.2 20.8 18.6 21.3 32.8 20.27 22.46 20.31 21.48 22.47 21.7 23.03 18.5 21.1519.5 20.3 19.6 20.7 19.8 19.8 20.6 19.4 25.08 19.8 16.7 19.28 19.55 18.66 17.4 21 19.2 21.9 28.5 19.26 21.25 19.75 20.9 22.65 21.02 22.86 17.99 20.9618.9 20.1 20.4 21 19.9 20 19.8 19.2 24.41 19.91 16.92 20.15 20.17 19.32 17.7 20.9 20.7 21.8 29.2 18.16 21.22 21.95 20.96 23 23.05 22.5 18.45 20.0119.8 20.8 21.1 21.6 19.9 20 19.9 19.4 24.4 19.73 17.9 18.95 19.51 19.4 18.8 19.6 21.2 20.3 30.5 20.05 21.4 20.35 20.5 22.85 21.75 24.75 18.13 21.3517.9 20.4 18.4 18.2 17.1 18 17.9 17.2 20.78 17.72 15.09 17.23 18.12 20.31 24.5 26.8 25.1 30.3 26.6 18.6 18.91 18.66 18.8 20.45 20.86 20.97 16.31 21.217.6 18.9 17 19.4 17.6 17.2 18.8 15.1 20.37 14.85 14.89 15.06 18.1 13.08 14.8 16.4 16.4 15.6 27.1 16.21 18.76 19.04 18.11 20.51 19.93 20.06 16.69 20.2717.7 18.8 17.2 19.5 17.6 17.2 19 14.9 19.81 14.89 15.16 14.79 18.45 13.27 14.6 15.2 16.2 16.4 25.5 15.52 20.07 19.68 18.21 20.51 19.77 20.09 16.88 19.6917.2 18.4 16 18.6 18.7 12.4 19.25 14.63 14.24 14.23 15.31 12.21 14.5 16.1 14.6 15.1 26.8 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10.9 8.559 10.37 10.98 9.3149.2 8 8.2 8.8 9.3 8.7 9.2 10 9.5 8.995 8.907 9.975 8.45 10.21 8.1 8.6 9 8.6 9.228 9.75 9.5 7.854 10.51 9.55 8.8 9.862 9.0059.6 8.1 8.2 9.2 9.3 8.9 9 10.3 9.605 9.113 9.05 10.08 8.158 10.22 7.7 8.6 8.9 10.1 9.297 9.8 9.55 7.95 9.955 10.22 8.857 9.928 9.158.4 7.8 8 9.1 9.2 8.8 9.2 10 9.405 8.943 8.988 9.836 8.3 10.2 8.6 8.3 8.9 8.2 9.322 9.505 9.805 7.558 10.81 9.76 8.834 9.915 8.89.1 8.7 8.2 9.2 9.4 9 9 10 9.309 9.053 8.988 9.951 8.704 10.24 9.5 8.4 8 9.1 9.333 9.805 9.55 7.708 10.1 8.954 9.033 9.953 9.0648.1 6.6 7 7.6 7.3 8.6 8.9 6.8 8.972 8.708 7.558 7.307 7.211 8.759 9.1 7.2 7.1 7.6 7.057 8.417 7.754 7.433 8.517 8.317 8.601 7.6548.4 7.4 9.2 8.2 7.7 9.3 7.2 7.6 9.514 7.683 7.061 7.652 7.554 6.651 6.5 8 8 8.4 8.341 7.838 6.936 7.377 8.796 9.243 10.04 8.716 8.4 8.38.5 7.3 9.1 7.9 7.6 9.2 6.9 7.7 9.533 7.566 7.129 7.76 7.5 7.212 7.7 8.1 7.3 7.9 8.357 7.671 6.94 7.401 8.535 9.265 9.948 8.692 8.308 8.8138.5 7.4 9.3 8.2 7.7 9.1 7.2 7.8 9.691 7.898 7.212 7.748 7.701 7.264 7.9 8.6 8.1 8.1 8.341 7.776 7.433 7.533 8.57 9.42 10.34 8.775 8.6 7.48.4 7.4 9.2 8.2 7.7 9.2 7.3 7.9 10.05 7.246 7.582 7.763 7.784 7.595 7.8 8.8 7.7 8.8 8.193 7.875 7.129 7.576 8.793 9.524 10.53 8.674 8.154 8.1088.2 7.3 8.4 7.9 8.6 7 8.848 10.8 6.777 7.26 8.324 7.099 9.9 8.7 10.3 9 7.855 7.61 7.835 6.74 9.205 8.6 9.67 8.592 8.78.4 7.7 8.2 7.7 8.4 7.6 8.981 10.77 6.649 7.26 8.367 6.742 8.2 9.8 8.2 9.2 7.808 7.595 7.813 6.839 9.724 8.5 9.24 8.554 8.6768.4 7.9 7.2 6.5 8.1 9.6 9.6 6.9 9.789 11.28 8.345 7.789 10.37 9.309 6.4 7.1 8.5 7.3 7.177 8.126 8.798 8.984 10.3 9.352 8.198 9.284 9.358.9 7.9 7.2 6.6 8.2 9.9 9.8 7.6 9.718 11.81 8.311 7.836 10.65 9.172 8.2 7.7 9 7.5 7.559 8.288 8.535 9.12 10.68 9.703 8.597 9.231 9.7558.6 7.8 7.9 6.3 8.2 9.8 9.7 7.8 9.816 11.64 8.123 7.797 10.77 9.238 9 8.7 9 8.9 7.321 8.191 8.646 9.244 10.28 9.661 8.488 9.303 9.5558.2 6.9 7.4 3.8 7.9 9 7.7 7.9 10.78 11.12 8.79 7.279 7.415 8.635 7.8 8.1 7 8 8.761 8.707 9.168 8.166 8.547 9.369 8.279 8.949 7.7198.2 7.8 7 7.7 8 10.6 11.19 8.25 7.354 7.75 8.8 7.1 7.9 7.8 8.3 9.05 8.4 9.15 7.2 8.65 9.05 8.75 8.98 8.9548.6 7.5 7.1 3.5 8.1 9 7.7 7.9 9.774 11.01 8.554 7.419 7.469 8.329 8.2 7.4 7.9 6.9 8.759 8.254 8.267 7.608 9.218 8.659 8.162 8.901 8.2627.7 8.7 8.5 9.3 8.4 9.5 9.1 8.5 9.188 9.03 7.448 7.244 5.895 9.474 9.7 10.8 9.3 8.4 9.192 8.822 8.597 8.316 8.05 9.5 8.232 9.536 9.112.6 11.4 14.9 14.4 10.7 9.6 10.2 8.74 10.98 7.225 8.463 8.314 10.4 11.9 11.7 10.2 11.4 9.896 14.37 9.258 11.79 8.926 8.673 8.333 8.556 13.388.8 9 9.2 12 9 8.9 8.1 8.3 10.4 10.09 9.615 9.589 6.479 8.895 12 9.4 10.1 9.8 9.354 10.75 7.954 9.109 10.7 8.804 8.753 9.156 11.468.8 8.9 8.1 6.7 7.6 9.7 10.86 9.891 10.58 10.15 10.21 9.359 6.8 13.9 5.8 5.5 7.878 9.955 11.14 7.057 8.85 9.95 8.013 9.552 9.164 7.7548.6 8.8 8 6.7 7.5 9.7 10.86 9.886 10.33 10.14 10.12 9.234 6.6 13.7 5.9 5.4 7.862 10.01 11.22 7.054 8.763 9.81 7.93 9.582 9.355 7.7158.8 8.9 8.1 6.7 7.6 9.6 10.85 9.967 10.72 10.1 10.06 9.31 6.8 14.1 5.8 5.5 7.862 9.955 11.09 7.15 8.85 9.9 8.11 9.617 9.2 7.78.2 9.1 9.6 7.1 7.7 9.9 10.81 10.09 10.59 9.846 10.12 9.339 7.5 12.7 6.7 6 7.97 10.22 11.27 7.4 9.105 10.05 8.215 9.725 8.904 8.3048.2 9.2 8.8 9.1 9.6 10.1 9.783 10 8.815 10.58 7.772 10.42 10.9 9.5 9.1 8.3 7.773 8.215 9.364 8.513 10.48 9.777 8.854 9.044 10.14 9.7849 10.4 7.8 7.7 9.4 9 8 8.8 9.86 9.692 7.615 7.411 7.615 8.404 8.4 8.2 7.4 7.8 9.464 9.1 8.304 7.558 10.46 10.26 11.01 9.812 8.9098.4 9.2 8.9 8.4 9.4 9.1 8.2 8.6 9.5 9.811 8.254 7.304 8.877 8.909 8.7 8.4 8.2 7.8 8.154 7.804 9.205 6.453 10.9 10.41 10.85 10.12 10.268.9 9.2 7.6 8.5 9.4 10.1 9.7 7.8 10.83 9.507 8.297 9.29 8.505 7.041 9 8 7.9 8.7 8.004 8.672 7.912 7.457 10.01 10.47 8.659 8.881 9.533 9.0738.6 8.7 8.5 8.6 9.8 10.5 9.8 7.8 11.71 9.406 8.54 9.306 9.255 7.274 9.7 9.9 9.2 9.1 7.604 8.5 7.654 7.304 9.65 10.51 8.77 8.935 10.55 9.557.7 8.2 20 8 7.7 10.8 9.207 10.01 6.271 5.602 7.364 5.676 8 7.9 8.859 9.414 8.363 7.754 8.532 8.419 8.924 6.866 8.162 7.5117.8 8.3 20.4 8.2 8.9 11.2 9.284 10.12 6.257 5.694 7.788 5.911 9.4 9.1 9.85 8.8 8.804 8.581 8.293 9.802 7.063 7.85 6.9539.5 8.1 22.2 8.8 8.7 8.5 10.9 11.9 11.66 12.3 10.13 8.967 10.94 10.74 10.6 9.2 10 9.2 12.8 11.09 9.796 8.496 9.198 9.385 8.403 10.03 7.057 7.954 9.4059.5 7.8 22.5 9.2 8.8 8.6 10.8 11.9 12.01 12.43 10.06 9.393 10.64 10.4 10.1 9.9 10.4 8.3 11.09 9.868 8.482 9.144 9.381 8.75 10.45 7.17 7.658 9.559.5 7.9 22.5 8.9 8.8 8.6 10.9 12 12.01 12.33 10.19 9.646 10.96 10.87 9.4 8.8 9.3 8.5 13 11.22 9.969 8.482 8.884 9.578 8.655 10.83 7.352 8.258 9.2599.4 7.8 22.7 8.7 8.7 8.6 11 12.1 11.87 12.38 9.936 9.449 10.92 10.87 9.3 9.3 10.5 10 11.05 9.645 8.406 8.897 9.578 8.723 10.51 7.134 7.665 9.47413.5 7.396 5.863 5.039 4.245 7 8.9 9 7.6 5.514 4.965 4.387 5.044 6.464.7 13.5 3.6 5.039 7.404 5.511 5.078 5.603 4.245 7.4 9.3 8.2 6.9 5.593 5.158 4.462 5.126 4.274 4.72 6.4397.3 8.5 18.8 7.3 7.9 7.6 9 7.4 9.223 10.95 7.967 8.877 8.772 9.515 7.4 8.7 8.4 7.5 9.82 7.561 6.757 7.87 9.269 8.475 9.096 7.031 6.456 8.0546.5 6.6 19.7 5.7 8.3 7.4 8.749 8.285 6.25 6.183 5.375 6.954 6 4.6 5.6 5 8.945 9.242 6.814 5.477 6.91 6.332 5.934 6.761 6.429 5.4695.8 7.4 16.3 6.7 8.4 6.8 10.36 10.91 5.875 8.991 6.241 10.04 15.3 10.2 11.4 9.9 8.393 7.182 8.058 7.013 8.35 8.65 6.065 8.6138.5 7.7 19.6 8.1 7.7 7.5 9.7 9.2 8.904 10.74 7.7 7.556 7.004 7.88 8.4 8.4 8.7 6.7 9.205 8.513 7.854 6.753 7.958 9.305 9.055 7.208 6.3538.6 8 19.9 7.6 9.6 9.1 8.363 10.66 7.669 7.517 6.332 8.065 8.6 8.5 9.7 8.7 9.555 8.772 8.768 7.422 7.904 8.859 9.259 7.277 7.38.2 8.6 22.6 7.5 8.3 8.4 10 9.5 9.459 11.23 8.872 8.738 8.35 8.473 9.6 9.1 8.5 7.8 8.654 9.705 8.6 8.6 10.1 9.9 10.35 7.092 8.68.8 7.1 22.7 7 10.6 10.9 9.925 10.91 8.025 8.453 7.7 7.457 8.1 7.5 7.5 8.2 8.759 8.559 8.667 8.3 9.218 10.73 8.868 6.855 8.0088.3 6.6 21.2 5.6 7.6 7.7 9.3 8.8 9.718 10.39 6.53 7.212 6.386 5.639 7.8 7.8 7.3 8 7.519 9.233 8.069 7.533 7.606 8.647 7.591 6.878 8.5268.5 7.8 20.8 7 8.1 8.1 10.2 10.8 10.51 11.06 8.254 8.602 6.566 7.516 9 6.8 7.2 7.9 7.654 8.809 9.109 7.958 9.865 9.95 8.768 6.872 7.9127.5 8.8 20.6 8.5 7.5 7.1 10.3 8.8 10.76 10.5 7.6 6.903 8 8.104 8.9 8.1 7.4 7.55 8.3 8.25 7.75 8.85 8.5 8.6 7.349 8.47.8 8.6 21 8.4 7.5 7 10.5 9.3 9.75 10.43 7.854 6.653 7.15 8.65 9.3 9.8 7.9 8.3 8.158 8.7 8.559 6.35 9.25 7.35 7.95 7.259 8.5546.9 7.7 19.6 11.5 7.1 7.1 10.4 9 9.55 10.78 6.9 8.176 7.654 8.134 9 9.5 8.9 8.1 9.955 8.45 7.204 6.657 9.405 9.264 8.162 6.974 7.9047.6 7.6 20.1 11.5 10.3 9.5 9 10.85 6.46 7.955 7.407 8.096 9.6 9.8 8.9 7.9 10.4 9.524 8.208 6.015 9.505 8.922 8.1 6.785 9.058.4 8.2 20.4 11.5 8 7.6 10.4 9.6 9.55 10.8 6.953 8.118 7.054 8.212 9.1 8.9 8.4 7.2 9.8 8.95 8.35 6.85 9.7 9.95 8.2 6.908 8.67.9 9.1 18.1 7.1 8.1 7.1 9.6 10.2 10.6 10.22 6.803 8.93 7.5 7.419 8.2 8.4 9.7 7.2 9.15 7.6 7.75 7.35 9.55 9.65 10.41 6.799 9.48.9 7.1 19.1 6.8 11.1 9.4 9.5 9.692 7.357 6.903 7.1 7.307 9 8.3 9.1 8.2 9.159 7.361 9.005 6.15 9.105 9.25 9.705 6.928 8.8599 7.5 19 6.7 6.9 6.4 10.7 9.1 9.009 9.635 7.85 6.707 7 7.4 8.8 7.3 8 8.308 7.407 9.255 6.603 8.504 8.404 9.9 6.995 7.96.7 6.8 18.9 6.5 10.8 9.2 9.655 9.617 7.704 7 7.204 6.957 8.9 8.6 7.9 7.3 9.105 7.75 9.105 6.4 9.5 9.9 9.05 6.808 7.36.8 7.9 19.8 7.7 7.7 8.1 8 7.757 9.156 8.851 7.972 6.962 9.416 7.7 8.1 8 8.3 10.08 7.38 9.091 5.675 8.462 8.833 8.323 7.239 8.957.1 7.9 19.3 7.5 7.1 8 8.1 8.2 7.664 8.787 9.091 8.383 7.397 9.354 9.1 10 7.4 7.7 10.01 7.652 8.984 5.714 8.385 8.642 7.97 7.197 8.2047 6.9 20.1 7 10.5 6.8 9.359 9.125 7.504 6.45 8.05 7.264 8.2 7.2 7 8.2 7.95 9.7 7.954 6.853 7.25 7.3 7.254 7.114 7.58.8 8.3 20.5 6.8 8.4 8.5 10.3 10 11.16 9.979 7.411 8.669 8.004 7.095 7.5 8.7 8.4 7.9 8.754 8.45 8.959 7.5 11.11 10.46 11.35 7.834 7.8514.6 16.6 34 14.3 15 13.7 19.4 14.6 17.35 18.26 13.16 14.01 16.2 14.37 14.9 14.9 15 16.2 14.5 17.85 15.56 15.65 14.36 16.46 15.15 18.12 12.66 15.359.6 9.2 24.6 9.1 18.4 11.7 14.03 15.27 11.39 9.788 10.85 8.592 11.1 11.2 12.4 12.9 15.8 14.72 18.31 22.9 20.29 12.16 11.76 9.568 9.289 12.416.7 8.7 19.1 6.8 7 7.7 10.3 10 10.06 10.2 8.25 7.708 8.304 8.05 6.9 7.5 7.2 8 8.804 8.809 9.6 8.15 9.45 8.104 8.85 7.111 9.6058.1 9.2 21.4 7.3 7.9 7.5 9.7 9.7 9.605 10.79 7.704 9.414 7.057 7.368 9.6 7.8 8.3 14 8.759 7.958 8.504 6.85 8.868 9.705 9.6 7.198 8.557.1 7.4 17.2 5.9 7 7.8 8.016 7.764 7.161 7.118 6.021 7.769 8.3 7.6 8.6 8.6 7.758 7.322 7.808 5.205 7.257 8.604 8.467 6.82 6.8537.9 8.7 23.6 7.1 7.6 7.4 10.4 8.8 10.52 12.02 8.701 7.603 8.8 10.05 9.2 8.9 8.8 8.1 8.143 8.693 9.989 7.201 8.517 8.397 10.59 7.898 9.8157.4 8.2 20.4 6.9 7.9 7.1 8.7 8 8.904 10.29 6.953 7.419 7.104 7.05 7.6 8.8 8.2 8.3 7.6 7.254 6.853 6.703 8.663 8.918 9.009 7.402 8.2 6.2097.7 8.2 19.9 6.8 8.7 7.6 9.109 10.33 7.3 7.4 7.3 7.004 8.7 9 8.2 8.2 7.7 7.7 7.654 6.7 7.758 9.109 8.35 7.399 8.15 7.457.1 8.8 18.4 6 8.4 6.9 8.4 8.3 8.659 9.257 7.45 7.904 7.708 7.454 7.9 6.9 9.1 8.8 7.35 8.35 8.15 7.45 8.15 8.4 8.854 7.152 8.904 8.5047.6 8.9 19.1 5.7 8.4 7 8.6 8.7 9.25 9.172 7.45 7.9 6.8 7.454 7 7.7 7.8 7.8 7.254 9.05 7.5 6.907 9.35 8.804 8.759 7.211 8.804 7.58 8.6 18.5 6.1 8.4 6.9 8.4 8.6 8.563 9.14 7.018 8.07 6.96 7.404 8.3 6.9 9.5 7.954 8.754 7.611 7.554 8.617 8.212 8.55 7.202 9.459 8.5177.9 8.5 20.1 8.2 9 8.9 9.4 8.5 10.4 9.866 7.608 7.528 7.75 8.552 7.3 8.3 8.2 6.4 8.028 8.5 8.95 8 9.5 9.65 7.781 7.845 6.603 8.46.6 7.4 17.8 7.9 8.2 6.8 9.7 7.8 8.772 9.115 7.321 8.05 6.19 7.647 7.3 7.6 8.5 7.8 7.811 9.355 7.9 8.763 8.567 8.1 9.7 7.192 7.307 6.8539 9.7 10.7 9.6 9.6 8.2 10.4 9.8 11.92 9.437 9.584 9.445 8.927 9.532 10.5 9.9 9.2 10.4 10.68 8.375 8.735 10.59 11.62 10.64 9.97 9.021 11.68 11.118.9 9.8 11 11.5 9.8 9.1 8.9 9 10.59 8.401 9.828 8.35 8.6 10.15 9.6 10.8 10.3 9.4 8.759 9.355 7.604 9.66 10.31 11.46 12.07 9.176 11.36 9.868.9 9.2 11 11.3 8.9 9 8.8 8.9 10.42 8.206 9.114 8.415 8.454 9.994 11.5 9.9 10.2 10.9 8.354 8.713 7.9 9.196 10.51 10.21 11.8 9.131 10.41 9.98.7 9.3 10.9 11.3 9.1 8.8 8.7 8.6 10.36 8.013 9.736 8.197 8.4 9.883 10.1 9.1 9.7 10.3 8.9 9.455 8.104 9.455 9.955 10.86 11.44 9.13 10.51 9.7557.6 7.5 9.5 8.7 8.1 7.1 8.8 9.1 9.1 8.903 7.356 8.692 7.204 9.754 9.1 7.9 8.1 8.3 7.958 8.3 6.35 9.605 9.059 7.7 8.408 7.169 9.705 8.4547.4 7.5 8.7 9.2 8.1 7.2 8.2 8.2 9.3 8.87 7.08 8.546 7.354 9.528 8.9 8.3 8.4 9.2 8.154 8.304 5.95 9 8.35 7.45 8.95 7.199 8.3 8.358 9.8 10.3 9.8 9 7.3 8.6 8.5 10.22 8.981 5.761 9.367 9.698 10.6 9.8 8.9 8.8 8.1 9.684 8.25 7.8 11.01 8.604 10.06 10.4 8.421 9.96 8.7098.9 9.8 9.7 7.9 8.9 8.2 8 9.2 11.97 10.28 8.735 9.051 10.27 10.1 9.5 8.7 9.5 11.5 9.605 8.6 8.254 9.655 10.41 11.81 10.84 9.195 10.938.7 9.7 9.8 7.9 8.7 8.2 7.9 9.1 12.02 10.42 8.511 9.409 10.14 9.42 9.9 9 9 9.5 9.605 8.559 8.254 9.355 10.21 11.45 10.74 9.183 11.968.2 9.8 9.5 9.5 9.6 8.4 7.8 9.3 10.41 8.896 8.38 8.405 9.498 8.929 8.5 9.1 8.9 9.6 9.177 8.377 7.357 9.73 10.52 9.446 10.04 8.834 9.38.8 9.6 9.8 9.9 8.2 10.1 8.704 9.853 7.458 9.817 9.788 9.253 9.3 8.7 9.4 8.6 8.427 9.67 8.794 8.564 10.22 11.77 9.922 9.736 10.5210.2 13.9 11.9 16.6 13.8 11.5 11 12 12.73 11.53 7.621 10.81 9.55 9.488 14.2 11.4 11.8 13.4 9.808 11.6 9.899 11.88 12.1 10.7 10.96 9.962 15.318.8 9.9 9.3 8.6 10.4 8.1 9.2 9.7 10.65 9.237 9.845 8.016 7.789 9.531 8.9 7.8 7.3 9.5 9.764 8.826 8.318 10.33 11.22 10.01 11.19 8.663 10.238.3 9.8 10.2 11.8 13 11.2 9.9 9.2 10.45 10.09 10.05 7.904 9.955 9.309 10.1 11.3 10.3 10.3 10 8.904 7.858 10.46 11.76 12.61 11.93 9.558 10.468.6 10.6 10.9 10.8 10.8 9.6 9.8 10.1 11.21 11.31 8.898 10.14 9.75 9.796 9.1 9 7.8 9.7 9 9.65 9.209 11.05 11.05 12.51 12.47 9.311 11.68.6 9.7 9.8 8.7 9 8.1 9.6 11.1 8.659 10.54 7.948 10.14 8.704 8.297 10.1 8 8.2 9.9 8.75 8.7 7.407 10.01 10.66 11.25 10.76 9.06 9.557.9 10.3 8.6 9.8 8.1 8.4 9.9 9.6 10.87 9.233 8.689 9.97 9.674 9.264 8.5 8.1 7.1 9.7 8.221 9.259 8.066 9.469 9.2 9.915 10.06 8.931 9.9058 10.2 9.9 10.3 8.4 8.8 9.7 9.7 10.3 9.306 8.7 9.905 8.954 10.4 8.2 8.2 8 7.5 8.404 8.85 8.158 9.45 9.305 9.65 9.755 8.978 10.88.7 10 9.6 10.1 8.4 8.8 10.6 10.3 10.42 9.251 9.018 9.519 9.679 10.02 9.7 7.5 7.6 9.3 9.018 9.118 8.804 9.205 10.31 9.82 9.98 8.947 10.168.6 9.6 8.1 7.1 9.2 10.4 11.4 9.005 7.789 9.214 8.396 8.054 7.7 7.4 7.7 7.7 7.958 8.008 9.273 10.72 11.8 12.58 12.72 8.834 10.518.6 11.7 9.5 10.4 14.9 11.3 10.7 13.5 11.74 9.579 10.14 9.774 11.62 10.57 11.3 9.6 9.7 11.7 9.677 12.28 8.656 13.02 14.44 14.22 11.75 9.159 10.327.4 8.1 9.2 9 8.7 7.9 9.5 8.9 8.801 9.702 6.628 8.846 9.269 9.309 9.7 9.8 9.4 8.6 7.591 9.314 7.277 9.015 9.95 10.85 9.638 8.364 8.82615.8 22.8 21.1 31.9 21.5 20.3 13.8 18 18.04 14.99 11.8 16.17 12.98 17.07 17 15.5 14.1 16.6 17.1 20.69 19.94 13.78 19.32 13.8 16.68 13.53 23.727.6 9.4 7.5 8.8 9 8.1 8.8 8.2 9.5 8.566 7.715 8.6 7.357 8.65 8.4 7.2 7.5 8.1 8.204 7.804 9.56 9.555 10.6 10.01 10.8 8.468 10.817.6 8.9 7.5 8.4 8.9 8 8.3 8 8.517 8.604 7.304 9.123 7.708 8.854 8.4 8.3 8.6 9.6 8.859 8.768 9.059 9.059 10.98 10.06 10.21 8.466 10.348.7 9 10.4 10.6 8.6 8.2 11 9.7 10.53 9.567 8.406 10.31 8.726 8.876 9.4 8.7 10.3 9.3 9.459 8.957 7.39 9.96 9.488 9.765 9.994 9.12 10.599.1 10.4 11.9 12.6 11.4 10.7 8.1 9.6 11.2 9.444 8.659 10.2 10.05 7.874 10.7 9.8 10.8 11 9.633 13 9.748 10.68 11.35 11.2 10.92 9.368 10.719.1 9.9 10.7 12.6 10.8 10.9 8.6 9.3 10.96 9.224 8.889 10.16 10.07 8.055 11.8 9.9 9.9 10.6 9.722 12.84 9.949 10.93 11.52 10.4 10.66 9.217 10.28.6 9.4 8.9 8.9 11 10.2 10.1 10.1 10.68 10.64 9.266 9.78 9.754 10.45 9.9 9.9 11.2 11.2 8.276 9.014 9.122 12.56 10.94 13.18 11.47 8.216 10.427.5 9.8 10.8 10.6 8.9 8.3 9.7 9.6 9.905 9.577 9.832 8.024 9.409 8.918 10.2 8.6 9.2 9.2 9.555 7.904 7.2 9.9 9.255 9.855 10.1 8.556 9.6057.6 10 9.6 10.6 9.8 9.8 9.405 9.533 10.06 7.976 10.11 9.003 11.1 9.7 7.9 10.5 9.955 8.354 7.25 9.155 11.65 9.805 10.11 8.877 10.369.3 9.9 8.6 9.7 9.8 8.1 9.5 10.1 11.9 10.81 8.71 8.824 9.659 7.958 9.9 10.3 9.7 11.8 7.904 8.304 6.45 9.705 11.45 11.8 10.21 8.691 9.610.2 8.2 9.9 10 9.1 9.2 10.63 11.32 10.44 11.37 8.388 11.49 10.5 8.4 9.6 11.5 8.889 9.86 9.445 9.931 11.32 11.16 10.94 9.085 10.82 8.2629 9.9 10.4 9 8.7 7.8 10.4 9.2 11.28 9.336 8.772 8.959 8.909 10.03 9.7 9.6 9.3 10.3 9.273 8.471 7.758 9.073 9.95 9.86 10.57 8.635 8.809 9.869.1 9.8 9.4 8.9 8.7 7.9 10.5 9.2 10.91 9.408 8.8 9.091 8.704 10.1 10.3 9.9 9 9.3 9.305 8.75 7.8 10.11 10.06 10.81 10.3 8.705 10.57 98.7 9 10.3 9.6 9.3 8.5 8.8 10.6 11.37 9.854 8.136 8.814 9.8 8.38 8.7 8.6 6.9 8.9 6.65 7.769 6.053 7.7 9.96 9.5 11.41 8.233 10.85 9.37.6 8.4 9.3 9.7 9.3 8.6 8.9 10.7 11.26 9.884 8.404 8.86 8.116 8.567 8 8.2 7.8 9.5 7 8.559 6.256 7.754 9.8 8.909 11.6 8.272 10.52 10.118.3 8.4 10.2 10.3 9 8.4 8.5 10.4 10.9 9.638 8.333 8.816 8.3 8.144 7.6 7.6 7 8.4 6.8 8.15 7.354 7.5 10.11 9.205 10.7 8.194 10.05 10.47.7 9.6 9.7 9.8 7.9 10.5 10.51 9.91 8.295 8.685 8.413 8.155 6.8 7.5 7 7.5 6.907 8.408 6.95 6.953 10.55 9.664 10.83 8.187 10.4 9.867.9 11.4 9.7 8.8 10 10.1 10.9 10.07 8.25 10.11 9.519 9.51 9.8 8.7 7.9 11.4 8.454 7.95 8.563 9.255 12.61 9.86 11.12 8.641 11.018.7 11.6 10.4 8.5 10.9 10 11.9 10.03 9 11.52 10.31 11.41 9.6 8.7 8.7 10.3 7.7 8.1 9.005 9.4 12.61 9.6 11.4 8.577 10.69.4 11.8 10.2 8.9 11.2 10.1 12.61 10.29 9 10.66 10.22 10.4 10.4 9.3 8.7 9.6 8.709 7.65 7.954 10.15 11.5 10.52 10.46 8.662 11.168.8 10.6 9.2 9.3 10.9 9.4 12.11 10.19 9 10.41 10.11 11.15 10.1 8.5 9.4 10.9 8 8.65 8.6 10.15 11.6 10.5 11.1 8.571 11.058.6 9.2 9.9 10.2 9.3 10.5 10.6 9.652 7.762 9.076 9 9.161 8.9 10.5 9.5 11.3 9.241 8.456 8.204 9.828 10.8 11 10.85 8.441 11.258.7 9.3 9.7 10.1 9 9.2 10.75 9.519 7.083 8.961 8.454 8.798 9.6 10.2 8.9 11.6 9.305 8.446 7.9 9.747 11.16 11.41 11.01 8.443 11.368.9 9.5 10.7 9.7 9.2 9.4 10.86 9.678 7.012 9.227 8.55 8.839 9.7 10.1 11.2 10.7 9.96 8.454 7.9 10.04 10.21 11.76 10.8 8.374 12.568.2 9.5 9.2 9.9 9.1 9.4 10.71 9.467 7.072 8.991 8.354 8.635 8.9 10.6 8.7 11.4 9.393 8.463 8.104 9.701 10.91 11.62 10.48 8.354 11.068.7 8.8 10.5 9.7 9.2 10.4 7.5 9.86 9.58 8.24 7.962 8.968 8.521 8.3 8.9 7.5 9.7 7.719 7.804 9.359 9.109 10.05 10.97 10.01 7.661 9.0098.7 8.5 10.6 9.6 9.2 7.1 10.3 7.5 10.29 9.609 8.521 7.934 9.56 8.768 7.3 7.9 7.1 9.6 7.712 8.384 8.304 9.77 10.43 10.1 10.18 7.699 9.888.4 7.7 9.1 8.2 8.3 6.8 8 9.9 10.21 8.802 7.854 8.324 9.309 8.171 9.1 9.3 9.7 8.7 8.2 8.258 8.2 9.4 8.9 9.205 8.767 7.683 9.6058.4 8.3 9.3 8.6 8.3 6.8 8.1 10 9.96 8.743 7.958 8.496 9.56 8.318 10.5 8.9 8.8 9.4 8.05 8.85 7.5 8.954 9.3 9.555 8.724 7.722 8.18.6 8.6 7.7 9.6 8.2 8.5 8 7.8 8.909 8.819 8.473 8.108 7.157 8.624 7.2 8.3 6.7 8.7 8.25 7.8 7.254 6.903 8.3 8.859 7.09 7.201 8.78.3 8.7 7.4 9.6 8 8.3 7.9 7.7 8.754 8.75 8.537 8.03 7.457 8.415 6.8 7.6 6.2 8 8.008 8.4 7.65 6.803 8.05 8.604 6.938 7.084 8.857.8 8.7 7.3 9.8 7.9 8.3 7.9 7.6 8.659 8.901 8.384 8.105 7.1 8.59 6.7 8.1 6.8 8.7 7.754 8.354 8.212 6.353 7.511 8.654 6.851 7.119 8.458.2 8.3 7.3 10.3 8.5 9.3 7.3 6.7 9.557 9.568 8.853 8.207 7.754 6.862 7.8 7.6 6.7 8.4 9.073 8.822 7.361 7.318 7.275 7.437 6.526 7.195 8.9446.8 6.4 7.5 7.8 7.9 7 6.356 7.599 4.48 6.499 4.712 4.86 3.4 4.5 3.8 4.1 9.356 6.576 6.228 6.191 7.054 7.658 7.633 6.481 7.77.1 8.1 7.4 9.1 7.9 7.3 7.204 7.697 7.55 5.95 6.95 7 5.7 6.9 6 6.6 7.954 7.004 6.203 6.5 5.85 7.407 7.104 6.915 7.67.7 6.8 5.4 7.3 8.3 10.1 7 9.1 9.363 9.702 8.309 8.547 6.685 6.917 5.7 7.9 5.7 7.5 8.021 9.347 8.939 7.473 9.478 10.68 8.102 7.299 7.9487.3 7.6 7.8 14 8.8 11.2 8.5 8 7.941 8.569 6.171 7.89 8.885 5.014 6 8.9 6.8 7.7 9.339 8.925 7.128 5.686 7.95 8.07 8.413 7.282 8.1698.1 8.3 6.3 9.9 8 6.7 5.7 7.007 7.244 6.371 7.206 6.098 8.257 8.7 9.8 10.7 9.7 6.518 6.91 6.753 7.45 8.212 8.124 6.406 7.633 8.5598.3 8.2 6.9 10.2 7.6 10 7.2 6.5 8.554 7.556 7.24 7.404 8.031 8.467 8.8 8.8 9.9 8.5 7.344 7.454 7.85 7.754 7.7 8.65 7.257 7.692 9.158.1 8.9 7.7 10.5 7.9 10.4 7.4 7.1 7.85 7.652 7.761 7.283 8.324 8.676 8.7 8.2 10.1 8.8 7.504 7.8 7.9 7.6 8.517 8.954 6.85 7.694 9.258.8 8.2 7.7 10.8 8.2 10.6 6.9 7.3 8.2 7.638 7.679 7.298 8.387 8.391 8.7 8.7 11.1 9.3 7.5 7.654 7.7 7.85 8.304 9.56 7.254 7.927 9.257.3 8.5 8.5 7.5 5.5 6.7 8.8 7.2 8.354 7.96 6.899 8.778 6.71 7.597 7.1 5.6 8.7 8.112 8.358 7.465 7.758 8.754 8.417 8.304 6.977 9.1597.8 8.5 8.1 10.5 6.7 8.7 8.5 7.2 7.407 7.632 6.618 7.288 8.059 6.667 7.1 6.2 6.9 8.7 8.75 7.548 6.953 6.85 7.5 8.6 8.523 6.657 8.457.6 8.7 7.3 7.8 7.8 8.4 8.6 6.7 8.354 9.727 7.55 7.833 7.304 7.33 6.7 7.4 6.1 6.6 10.59 7.799 8.091 10.13 9.971 8.373 10.2 7.858 9.1057 8.3 7.8 8 8 8.5 8.7 6.8 8.25 9.664 7.561 7.785 7.054 7.361 5.7 7.1 7.3 7.2 10.73 7.757 8.217 10.12 9.942 8.447 10.21 7.878 9.77.6 8.6 8 8.2 8 8.5 8.6 6.7 8.659 10.04 7.823 7.852 7.3 7.394 6.7 6.7 6.1 7.3 10.73 7.942 8.07 10.17 9.913 8.629 10.33 7.792 9.2095.1 7.8 7.1 4.5 7.2 8.5 7.1 6.8 8.221 7.139 6.623 6.377 7.204 6.509 5.7 5 5.996 4.155 8.084 6.273 10.05 9.5 9.121 8.799 5.254.9 5.3 5.85 3.641 4.917 4.613 5.3 3.8 6.6 3.94 3.341 4.838 5.495 4.913 5.0855.432 5.918 3.532 5.034 5 4.4 4.7 5.9 4.069 5.452 4.938 4.79220 19.6 16.9 14.5 13.5 19.6 11.6 11.8 15.84 13.81 12.09 11.83 10.63 12.44 10.5 9.5 9.8 10.3 13.27 15.23 11.18 13.49 12.6 14.74 11.22 7.567 15.9619.5 18.7 17 14.7 13.2 18.9 11.5 11.8 15.63 13.37 11.75 11.75 10.6 11.74 10 9.3 8.7 8.8 12.76 15.5 10.58 13.89 12.33 14.29 10.77 7.466 15.8719.7 18.8 17 14.8 13.4 19.2 11.3 11.8 15.7 13.68 11.99 12.13 10.75 12.06 9.6 9.6 10.8 10.5 13.13 15.58 10.93 13.89 12.45 14.49 10.9 7.46 15.619.8 18.7 17 14.7 13.5 19.2 11.4 11.8 16.11 13.72 12.01 12.34 11.19 12.24 9.6 9.2 11.2 10.4 14.07 15.5 10.86 14.29 12.71 14.8 11.24 7.602 16.1311.8 15 13 18.5 10.9 14.2 8.8 10.8 12.3 8.716 9.05 8.864 8.8 9.698 8.8 8.6 8.2 11 10.85 10.46 8.008 9.705 8.75 10.1 10.76 7.653 13.4111.8 15 13.1 18.4 10.8 14.2 8.7 10.8 11.31 8.742 9.027 8.785 9.214 9.772 8.2 8.7 9 9 10.81 10.52 8.108 9.514 9.064 9.164 10.56 7.583 12.3111.8 14.5 13.2 18.6 10.9 14.3 8.7 11 11.91 8.692 9.2 8.929 9.655 9.853 8.7 8.3 8 10.6 10.81 10.6 8.4 9.75 9.35 9.105 10.76 7.752 13.7611.8 14.6 13 18.7 10.7 14.1 8.7 10.7 11.1 8.627 8.877 8.877 10.43 9.672 7.5 9.3 9.2 9.7 10.76 10.58 9.004 9.421 9.524 8.986 10.57 7.705 13.76.9 8.7 7.8 7.7 6.3 7.9 6 6 6.443 6.551 7.457 7.029 6.221 5.778 7 6.2 4.6 5.6 7.908 6.7 5.903 5.706 7.863 6.792 8.461 6.95 6.553 8.6916.2 7 7 8 7.3 9.3 6.6 6.9 7.65 7.566 6.44 7.074 6.45 5.435 6 7.1 5.7 7.3 6.85 5.803 6.253 6.303 8.2 7.454 6.106 6.66 6.1536.2 6.8 6.6 7.8 6 6 7.758 7.674 6.41 7.207 6.51 5.594 6.5 6.4 6.1 6.9 7.254 6.563 6.453 6.85 8.863 7.758 5.956 6.736 8.055.2 7.1 6.7 7.7 7 9 6.2 6.5 7.257 7.756 6.402 7.154 6.703 5.564 6.5 6.8 6.6 8.2 7.15 7.1 7 6.25 7.55 7.508 6.507 6.728 6.257.7 8.5 6.6 6.3 5.3 6.4 7.6 5.8 6.439 6.591 5.624 7.542 6.613 7.81 0.2 6.4 6.713 6.009 6.269 6.313 8.425 8.024 8.438 6.118 7.3877.9 8.3 6.3 6.2 5.3 6.2 6.8 4.8 6.456 6.607 5.314 7.62 5.906 7.431 5.1 4.7 5.1 5 6.403 5.7 5.803 6.153 7.954 7.665 8.074 5.847 8.2548.9 8.8 7.4 8 7.2 8 5.5 7.6 7.4 8.696 6.208 7.716 7.057 6.233 8.3 7.8 7 7.1 7.604 5.703 6.009 6.55 7.704 8.4 7.9 6.709 7.5117.3 9.2 7.7 7.3 6.8 8.8 7.3 8.2 8.258 6.815 6.784 7.022 6.6 6.576 8 6.8 7.3 9.2 5.85 7.154 6.009 6.1 10.15 9.45 8.552 6.008 6.953 5.857.2 9.4 7.6 7.1 6.7 8.6 7.3 8.2 8.351 6.68 6.637 7.105 6.507 6.695 8.3 6.5 8.2 9 5.903 7.004 6.053 6.053 10.21 9.414 8.584 6.019 7.65 6.0597.7 9.5 7.8 7.2 6.8 8.8 7.2 8.3 8.404 6.688 6.738 7.105 6.753 6.709 8.6 6.1 7.3 8.2 6.45 6.703 6.1 5.2 10.15 9.255 8.46 6.081 8.05 6.77.2 9.2 7.3 7.2 6.8 8.6 7.3 8.2 8.249 6.682 6.589 6.971 6.7 6.526 7.5 7.4 8.2 9.1 6.5 6.75 5.7 6.003 9.965 9.059 8.408 6.033 7.254 6.58.6 8.4 6.8 9.9 5.6 7.2 7.418 7.232 7.834 8.464 6.655 6.61 7.6 7.4 6.9 7.7 6.802 7.692 6.6 6.201 8.5 7.55 6.536 6.419 6.607 6.058.5 8.3 6.7 9.7 6 8 5.8 7.3 7.169 6.948 7.492 8.398 6.714 6.383 7 7 7.3 6.768 7.61 6.4 6.309 8.204 7.415 6.346 6.421 7.35 5.8158.5 8.3 6.9 9.6 5.9 7.9 5.7 7.4 7.106 7.165 8.41 8.176 6.601 6.438 7.1 7.1 7.5 8 6.901 7.618 6.253 6.435 8.308 7.404 5.981 6.456 7.6 5.858.4 8.2 7.3 9.7 6 8 5.6 7.3 7.54 7.355 7.951 8.019 6.524 6.867 6.5 7.3 6.9 7.1 6.842 7.457 6.2 6.348 8.2 7.6 5.846 6.477 7.7 5.957.2 9.2 8.1 7.4 8 9.4 6.4 6.9 8.354 7.019 6.3 6.85 5.959 7.204 7.1 6.5 7.8 6.5 7.357 8 5.9 6 8.004 6.55 7.6 7.323 8.5 6.358 9.1 8.1 7.7 8 9.3 7.4 7.1 8.95 7.047 6.653 7.25 7.304 7.75 5.7 6.9 6.8 5.9 7.35 6.5 5.7 8.1 8.95 6.55 8 7.288 8.1 6.76.9 8.8 8.3 7.6 8 9.4 7.5 6.7 9.61 7.033 6.75 7.104 6.557 7 6 6.8 5.6 6 6.45 6.95 7 6.65 7.754 6.5 7.35 7.28 8.4 6.6037.2 8.4 7.3 7.7 8.1 9.4 8 7 9.059 7.324 6.55 7.3 7.25 7.004 6.4 7.5 7 6.6 7.2 7.2 7.05 6.4 8.5 7.05 8.3 7.358 8.2 7.37.2 8.4 8.2 7.6 8.9 11 7.3 7.3 8.759 7.231 5.965 8.785 7.6 6.471 7.7 7.3 6.2 7.4 7.608 6.1 6.507 6.153 9.3 7.254 9.355 7.167 8.504 7.7087.5 8.4 8 7.6 8.9 10.9 7.3 7.2 8.667 7.103 5.886 8.858 7.465 6.471 7.4 7.3 6.6 7.1 7.519 6.118 6.212 6.118 8.074 7.175 9.114 7.125 8.417 7.4617.8 8.2 8 7.6 8.9 11 6.8 7.2 8.654 7.115 5.875 8.905 7.2 6.54 7.6 8.5 7.8 7.55 6.95 6.45 6.75 8.7 7.2 9.1 7.17 8.5 7.6048.1 7.5 8 7.6 9.1 11.1 6.9 7.3 8.804 7.29 6.106 8.746 7.508 6.466 8.7 7.9 7.2 8 6.75 7.1 7.004 6.203 8.312 7.254 9.15 7.245 8.008 8.358.1 7.9 8.6 8.7 8.4 9.9 8.3 8.4 8.953 8.238 8.394 8.415 6.5 9.375 6.3 6.8 6.4 6.6 6.557 6.853 5.503 6.9 7.9 8.463 8.904 7.168 8.504 78.2 8.4 8.8 8.4 8.4 9.8 8.3 8.3 9.045 8.229 8.309 8.578 6.45 9.121 6.3 7.2 7.604 6.9 6.603 6.25 7.05 7.704 8.95 7.209 8.354 6.9078.4 8.2 8.7 8 8.3 9.8 8.7 8.3 8.849 8.259 8.407 8.589 6.557 9.145 6.9 6.4 6.2 7.4 6.953 7 5.6 7.354 7 8.454 9.405 7.22 8.504 6.858.1 8.6 8.7 7.4 8 9.5 8.5 7.9 8.95 8.25 8.072 8.612 6 9.121 6.1 7.6 7.4 6.2 6.757 6.853 5.7 6.51 7.061 7.554 8.868 7.062 8.317 6.7177.7 7.9 8.2 8.9 8.7 9.6 10.2 7.6 9.624 8.913 8.778 7.157 6.824 9.575 8 7.1 6.7 7.4 6.871 6.971 7.018 7.365 7.766 7.924 8.329 6.898 8.082 6.7647.6 7.7 8.5 8.9 8.6 9.5 9.8 7.7 9.85 9.127 9.122 7.325 7.257 9.74 6.2 7.5 6.9 8.2 7.4 7.054 7.104 7.55 8.204 8.1 8.754 6.986 8.254 6.8077.5 7.9 8.3 8.9 8.3 9.3 9.9 7.6 9.679 8.915 9.065 7.306 7.179 9.78 8 7 5.6 7.3 6.907 7.6 7.007 6.75 8.141 8.8 8.707 6.726 8.371 5.9537.2 7.8 8 8.8 8.1 9.2 9.6 7.4 9.014 8.701 8.46 7.24 6.627 9.465 7 6.3 5.6 7.2 7.257 6.853 6.703 7.257 8.262 7.804 8.517 6.588 8.35 6.35.2 4.6 5.2 6.8 5.725 11.14 6.579 7.071 4 7.143 5.9 5 5.479 5.154 4.602 3.782 6.458 6.884 5.823 6.914 5.503 4.7074.8 5.3 4.6 5.2 7.2 5.584 11.14 6.614 7.018 4.106 7.418 5.4 6.1 5.4 5.524 5.355 4.659 3.814 6.448 6.911 5.931 6.957 5.45 4.8525.4 5.7 5 6.9 7.8 8.9 6.994 12.13 6.527 7.552 5.17 7.808 5.3 4.9 4.3 4.7 7.518 6.679 5.733 5.571 7.454 8.017 7.699 7.064 6.589 5.1677.7 7 7.8 9.2 8.3 10.9 7.747 11.77 9.559 7.323 6.303 7.164 6.2 6.8 5.8 7.8 12.8 7.848 6.914 6.7 6.688 7.85 8.64 8.009 6.965 7.65 5.9067.3 7.2 7 7.7 7.3 8.7 6.2 6.8 8.704 9.264 8.7 7.504 7.057 7.454 7.5 7.7 6.8 8.5 7.55 8.963 6.45 8.1 8.6 9 7.257 7.582 7.904 6.257.2 6.9 8.2 7.8 7.3 8.7 6.9 8.2 8.5 9.186 8.404 7.5 7.104 7.6 7.9 8 8.6 7.7 8.05 6.203 8.804 7.45 8.85 7.55 7.491 8.7 5.7037.5 9.9 7.5 7.4 5.4 7.7 6.8 6.9 6.432 6.279 5.839 6.051 4.949 5.898 7.2 6.8 7.3 7 5.8 6.15 3.65 3.95 4 4.6 3.6 6.956 8.05 6.457.3 9.5 7.7 7 5.6 7.8 6.1 7.1 6.234 6.419 5.87 6.287 5.278 5.67 7.1 6.6 7 5.7 5.005 5.8 4.8 4.752 4.602 3.5 6.93 7.15 5.1057.7 9.8 7.1 6.9 5.3 7.6 6.9 7 6.25 6.361 5.709 6.061 5.404 5.371 6.8 7.5 8.1 6 5.5 5.2 3.152 5 4.4 4.55 4.25 6.939 7.5 5.2538.8 10.1 9.2 9.4 10.2 9.8 9.7 9.858 9.055 9.75 9.9 8.8 9.3 9.1 7.908 10.41 7.104 8.054 8.229 8.229 8.484 7.315 10.61 8.2128.4 9.5 9.2 9.4 7.9 7 10.2 9.6 9.955 9.921 10.15 9.214 10.36 10.37 8.8 8.7 10.3 9.4 7.95 9.3 6.403 8.254 7.854 8.1 8.1 7.269 10.21 8.39.2 10.2 7.8 9.5 7.8 7 10.4 9.8 8.95 9.821 10.51 9.4 11.05 10.01 10.1 8 10 10.2 8.45 9.205 6.55 8.9 8.75 8.7 8.55 7.177 9.8 8.458.3 8.8 9 8.3 7.7 6.9 10.3 9.1 9.605 9.917 9.95 9.35 10.35 9.7 8.2 9.3 10.4 9.4 7.1 9.45 6.05 8.3 8.25 8.704 7.854 7.205 10.55 7.6511.3 6.5 10.7 12.9 12.8 10.9 12.48 10.31 11.98 10.42 12.02 12.62 11.6 10.7 12.3 10 10.55 8.687 8.649 10.65 9.4 8.75 10.62 7.983 9.9128.7 8.3 8.9 8.3 8.2 7.7 9.1 9 10.51 10.21 11.25 8.673 9.765 8.969 7.3 7.7 9 9.2 8.7 10.76 7.65 10.1 9.205 9.305 8.1 7.863 10.96 8.1127.5 8.1 9 7.7 8.4 8 9.2 8.9 9.114 10.26 11.31 8.751 9.218 8.984 7.6 8.2 9.6 8.4 8.85 10.58 7.866 9.844 9.414 10.51 8.659 7.832 11.24 9.7748.7 8.3 8.8 7.8 8.3 8 9.3 8.8 10.06 10.2 11.32 8.827 8.954 9.077 8 10 8.6 8.2 8.904 10.6 8.104 9.765 9.5 9.955 8.354 7.845 10.61 9.257.8 7.8 7.9 8.5 7.8 7.2 6.6 8.6 10.36 8.454 8.824 10.04 10.25 9.608 8.4 10.2 9.2 8.3 7.35 8.95 8.1 9.4 8.054 8.75 8.7 7.515 8.9 8.58.1 8.1 8.2 8.3 8.6 7.7 7.7 8.2 10.36 8.585 9.23 10.07 10.3 10.25 9.7 11.2 9.2 9.2 8 9.55 8.604 9.3 8.904 8.9 9.8 7.629 9.1 78.4 8.1 9.1 7.6 6.9 7.5 9.7 7.6 11.31 9.138 8.807 8.362 10.36 8.79 7.8 9 8.9 8.5 7.65 10.76 9.905 9.85 9.25 8.404 9.15 7.862 9.86 8.558.6 8 8.8 7.5 6.8 7.5 9.9 7.7 10.72 9.053 8.885 8.305 10 9.091 7.6 8.4 8.9 8.8 7.654 11.22 9.25 9.455 8.754 9.109 9.205 7.873 8.704 8.558.5 9.6 9.2 7.8 7.1 6.8 9.5 7.4 10.77 8.653 10.43 8.454 8.763 7.904 7.8 9 8.8 8.9 7.35 9.505 7.758 8.659 9.91 8.463 9.2 7.61 10.41 7.458.8 8.8 8.4 8.4 9.5 8.2 9.674 8.795 10.26 8.854 9.264 7.954 7.9 10 10.1 10 8.604 10.86 7.762 8.722 10.21 8.809 10.05 7.626 10.53 7.1078.1 9.2 8 8 7.1 7 9.5 8 10.16 8.66 9.705 9 9.005 7.554 7.6 8.6 8 8.2 7.55 8.404 7.3 8.1 10.15 8.5 10.05 7.456 10.5 7.87.4 8.4 8.7 8 7.4 7.1 9.7 7.9 9.419 8.607 10.16 8.75 8.704 7.87 7.6 9.2 8.1 10 8.804 8.613 8 8.5 8.754 8.254 9.164 7.465 9.96 7.1047.7 8.1 9.5 8.6 8 7.8 10.5 9.4 11.17 11.17 11.81 7.843 11.2 10.05 8.6 8 10.2 7.7 9.231 10.78 9.585 8.955 10.77 11.14 10.18 8.203 9.86 7.4047.7 8.3 9.1 8.5 7.5 7.7 10.2 8.7 11.11 11.01 10.98 7.991 10.8 10.03 8.8 8.4 10.4 9.1 9.659 10.81 9.613 8.639 10.7 11.09 9.899 8.054 9.8 7.6087.7 8.4 9.3 8.5 7.5 7.8 10.3 8.8 10.84 11.15 10.64 7.957 10.72 10 9.1 8.5 9.5 7.9 9.605 10.64 9.665 9.02 10.65 10.85 9.713 8.301 10.45 7.2577 8.8 8.4 8.3 9.8 6.8 10.95 8.941 8.677 7.834 9.3 8.712 7.3 6.5 10.7 8.3 7.05 9.25 7.654 8.15 8.4 9.05 9.6 7.245 9.5 77 7.2 6.9 7.5 7.1 6.9 7.2 8.2 10.6 7.043 8.815 7.958 7.425 7.569 9 8 8.4 8.8 7.354 9.614 6.703 8.763 10.38 9.064 9.224 6.275 8.804 9.2096.8 7.2 7.6 7.8 7.1 7 8.9 8.3 10.76 8.185 9.221 8.319 7.84 8.467 8.4 9.4 8.9 9.8 7.715 10.26 6.853 8.909 9.505 8.818 9.88 6.399 8.15 8.2087.1 7.3 7.2 7.6 7 6.8 8.5 8.1 10.76 7.925 9.221 8.021 7.933 8.124 8.1 9 9.3 8.5 7.107 9.309 6.65 8.85 9.7 8.554 9.71 6.183 8.759 7.8047.1 6.9 7 7.5 8.3 8.2 10.96 6.793 9.003 8.084 7.652 8.005 8 7.5 9.1 7.7 6.707 10.06 6.81 8.262 8.754 8.559 9.105 6.202 8.212 8.81310.7 10.9 7.8 10.4 9.5 9.4 9.7 13.7 11.5 10.24 18.62 11.73 12.9 6.5 10 9.7 9.1 8.8 8.634 10.14 9.5 8.812 9.819 8.285 8.372 9.617 10.4610.9 11.3 7.8 10.4 9.5 9.4 9.8 13.8 11.36 10.09 18.62 11.38 12.61 6.5 10 8.5 9.5 9.3 8.565 10.14 9.3 8.895 9.858 8.21 8.381 9.722 10.218.7 10.2 9.8 8.3 8.5 7.718 10.24 12.16 7.407 9.466 13.7 6.277 7.891 7.886 7.496 8.129 8.543 10.48 13.79 8.813 7.7548.1 8.7 9.2 7.1 9.3 8 7.504 9.727 12.5 6.931 9.449 6.609 10.2 12.1 13.5 12.9 6.139 7.819 7.413 8.846 7.994 8.649 10.19 13.85 9.755 8.30410.6 10.9 12.2 12.2 10.1 10.7 13.21 12.33 13.09 12.6 13.21 11.5 11.2 10.6 13.7 11.4 10.25 11.42 10.32 9.56 10.61 13 11.34 10.86 11.72 11.8110.8 10.9 12.1 12.2 9.9 11.2 9.8 10.7 12.93 12.46 13.51 12.61 14.44 11.92 10.5 11.1 10.8 11.3 10.44 12.12 10.75 8.881 11.17 12.7 11.18 10.83 12.35 12.110.5 12.2 12.6 12 10.9 11.6 10.1 11.7 10.95 9.947 12.68 11.59 13.24 12.97 11.8 12.3 12.7 12.7 11.46 12.14 10.33 9.164 12.14 12.76 13.67 12.77 12.98 11.414.6 8.2 4.5 8 7.4 10.3 7.8 5.1 8.975 8.343 8.241 7.479 9.194 11.14 5.3 6.1 5.4 6.4 7.329 10.2 8.036 7.011 9.276 9.414 6.747 8.656 5.9628.2 8.8 8.1 9.6 7.6 8 9 8.8 10.21 7.703 10.9 9.194 10.14 8.963 8.4 9.2 9.4 8.457 10.13 9.45 8.804 9.184 11.17 8.531 8.418 9.557.2 7.9 7.5 9.5 7.7 8.3 9.4 7.5 9.35 8.578 8.004 8.45 9.5 7.45 9.2 7.7 9.6 8.8 8.2 9.4 7.85 7.75 9.8 9.005 7.5 8.684 8.7598.7 8.8 9.1 8.7 7.5 8.4 8.5 5.7 10.27 9.215 11.02 6.357 9.8 9.474 8 8.7 8.6 8.5 9.385 7.505 8.841 7.559 7.687 9.968 9.459 7.597 8.37.2 8.5 9.3 9.9 7.6 8.5 8.9 8.2 9.77 8.742 8.852 8.136 8.321 10.67 7.3 8.2 8.9 8.7 9.132 11.17 7.615 7.315 9.885 9.314 9.258 8.673 8.9136.1 8.2 8.2 8.9 8 8.1 8.9 6.4 10.01 8.306 9.304 8.573 8.95 9.712 7.3 8.6 7.3 9.55 9.255 8.754 7.5 9.6 9.905 9.86 8.291 9.056 9.1 8.4 9 8 8.1 9.3 6.4 9.614 8.116 9.262 8.547 8.963 9.614 7.9 9.319 9.56 9.282 7.762 10.22 9.77 9.865 8.215 10.116.9 7.5 9.2 9.1 8.2 8.2 9.3 6.7 10.17 8.298 9.239 8.832 8.95 10.19 7.8 7.5 7.6 7.5 9.805 10.15 9.15 8.15 10.05 9.55 9.55 8.194 9.75.4 6.8 8.3 6.9 6.4 8.1 8.5 6.6 8.898 7.951 10.34 7.745 8.647 7.742 9.2 7.4 8.9 9.1 8.25 8.639 8.559 7.55 8.772 9.681 9.158 7.845 8.9044.8 4.5 5.2 4.3 8.3 4 6.59 4.9 4.3 4.95 5.65 5.7 4.2 5.1 5.15 5.25 5.403 5.65 6.05 3.904 6.103 4.454 4.8 4 4.7 8.5 4.552 6.546 4.802 4.4 4.6 5.45 5 4.8 4.4 5 5.353 4.505 5.103 5.5 6.003 4.4 5.796.4 95.5 86.3 96.2 94.9 95.9 95.12 92.56 94.48 94.87 94.18 95.25 93.1 90.1 92.5 92.8 94.2 94.49 94.45 95.46 95.05 94.79 95.05 95.16 93.53 95.49 94.58100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99.88 10047.2 46.9 38.6 47.8 47.5 44.1 43.1 45.3 45.59 45.5 48.42 44.87 48 46.73 46.6 48.3 45.1 47.6 49.6 45.49 47.22 43.74 46.14 45.41 44.17 46.97 45.39 45.546.4 46.5 44.4 45 48.9 47.6 46.5 47.9 45.42 44.07 42.97 45.57 46.04 44.55 43.5 43.1 46.9 44.4 49.1 45.09 46.87 48.27 46.54 47.7 44.37 45.97 51.11 46.72100 100 100 100 100 100 100 100 100 100 100 98.82 99 100 100 99 100 100 100 100 100 100 100 100 10045.9 21.7 23.1 62.1 32.54 37.01 28.9 69.2 60.4 55.4 55.18 64.8158.5 59 55.2 52.5 22.6 63.9 55.53 55.37 60.63 64.89 63.4 65.8 66.2 67.7 58.9 58.41 59.85 58.49 60.77 64.3 63.78 62.98 55.9841.1 38.4 38.1 38.8 40 38 43.73 40.38 46.68 39.39 39.76 36.2 38.7 37.7 37.7 36.3 36 37.6 39.15 39.15 40.91 39.05 38.98 39.71 38.87 34.4 36.5741.4 39.8 40.8 36.5 41.8 42.8 38.51 40.78 42.02 41.08 38.45 40.64 40.9 39.6 38.9 37.4 36.5 39.41 40.69 40.81 38.5 39.65 38.31 39.43 42.49 38.4753.8 54.5 53.5 57.1 54.6 46.6 53.99 50.7 55.8 51.61 52.14 54.15 55 52.8 52.9 52.9 55.8 51.04 53.09 54.78 54.1 50.97 51.44 51.55 51.27 56.2192.1 91.5 91.7 91.4 60.6 52.4 92 92.8 91.84 92.23 92.4 93.7 91.6 93.05 92.6 92.2 92.7 91.4 94.2 92.05 92.15 93.8 93.6 92.84 92.55 93.1 93 92.184.4 90.7 83.8 91.6 89 66.8 88.63 86.75 86.15 89.71 87.68 91.33 81.7 78.4 76.9 80 84.9 89.54 89.55 90.66 92.37 89.68 85.95 86.65 92.9 75.14 81.1789 88.7 91.8 89.1 89.3 89.1 89.5 85.4 87.95 89.44 80.43 87.97 87.76 93.4 91.1 90.5 90.3 89.2 88.3 91.27 89.46 90.27 90.9 90.44 91.49 90.68 88.99

Lot 1 Lot 2 Lot 1 Lot 1 Lot 1

33

CHPv2

SNV

15-35%

5-15%

genomic

Lot 1 Lot 2 Lot 1 Lot 2

1111 3 4 2 26 20

397

Run-to-run variation

Library prep issue?

Difficult variants?

Bioinformatics?

Detected

Not Detected

Runs

Va

ria

nts

9

Control highlights bioinformatics issues

Panel

Site 11 11

Lot Lot 1 Lot 1

Run 1 125 2729.2 26.928.2 28.126.2 25.924.5 22.624.8 24.326 24.522.8 24.127.5 26.325.8 28.126.2 26.926.2 27.830.4 28.220.5 20.322.3 19.221.5 19.722 23.225 23.127.5 22.220.5 2430.8 30.336.2 30.922.2 22.130 28.329.2 27.628.5 2827.2 27.224 26.227.8 24.823.8 23.824 23.130.6 26.131.5 27.121.8 22.321.4 22.321.2 21.824.1 23.123.1 25.521.8 22.523.1 24.920.5 24.223.8 21.921.5 22.822 21.825.2 21.428.2 26.529.8 25.128.5 28.330.8 2717.9 18.124.8 25.326.1 2627 25.324.3 25.925.2 23.325.8 23.325.5 23.625.2 24.933.2 33.539.6 37.338.5 36.636.1 34.226 28.727.2 27.827.1 27.451.8 51.352 51.852.2 5252.5 51.838 37.437.6 37.737.1 36.841.5 40.132.8 31.728.5 30.829.2 31.930.5 32.826.6 23.527.1 26.225.5 25.426.8 26.126.3 26.826.5 27.225.8 26.429.8 3233.4 31.621.2 21.921.8 21.914.2 14.813.8 14.814.5 14.915 15.223.8 23.925.2 24.225.8 25.127.2 24.628.2 26.427.8 29.526.5 26.225.2 26.528 26.125.4 28.325.7 26.127.2 27.326.4 26.923.3 2423.2 2423.5 24.323 23.925.5 25.725.5 25.124.6 26.331.8 27.217.817.8 20.723.6 27.211.61213.1 12.39.49.612.111.611.112.110.514.8 14.912.715.5 13.713 13.113.213.313.513.213.414.21514.5 15.215.5 15.11414.812.812.812.6125.75.45.87.36.66.57.57.67.17.36.14.64.74.74.76.16.55.15.66.34.95.55.36.37.25.46.36.46.46.56.95.14.75.24.75.86.512.8 11.211.313 11.511.65.961111.57.79.78.78.211.410.211.29.69.78.899.18.59.48.99.610.510.58.8914.5 16.615.8 18.310.714 12.29.511.188.89.49.69.87.37.710.88.58.68.89.89.48.96.56.45.3710.19.57.68.88.29.59.69.37.49.47.717.1 17.48.27.88.310109.79.699.99.87.77.49.18.88.88.88.78.99.48.510.810.510.210.610.410.37.27.27.57.57.56.98.16.87.59.48.58.47.68.278.86.76.86.75.13.53.812.412.112.412.39.99.8109.36.27.27.17.64.74.55.59.18.88.38.48.898.88.88.78.89.28.898.998.810.910.910.910.710.110.110.110.18.512.8 9.47.57.28.68.28.29.69.99.28.211.610.511.51111.511.18.28.49.48.79.18.58.28.79.37.98.17.67.812.312.310.111.511.812.19.39.512.61110.212.211.511.611.49.27.47.394.2 9410049.6 48.149.1 46.2100 10060.458.936 35.636.5 38.355.8 55.994.2 93.684.9 85.588.3 87.2

SNV

CHPv2

15-35%

5-15%

genomic

Germline Somatic

Lot 1 Lot 2 Lot 1 Lot 2

NextGENe TS 4.0.2

SNV

15-35%

5-15%

genomic

10

3 sites – TruSight® Tumor Panel

• Good

performance

in H0007

(custom

bioinformatics

solution)

• Library prep

differences (A

and B)

• Run-to-run

differences

Panel

Site

Lot Lot 1 Lot 2

Run A B A B 1 1 1 2 3 4 1 2 3 410.9 10.6 14.4 13.7 15.32 17.75 10.94 12.07 10.37 9.55 12.08 15.38 12.08 12.5711.1 10.6 14.2 13.6 15.63 17.89 10.94 12.18 10.51 9.63 12.31 15.57 12.26 12.7410.8 10.4 14.2 13.6 15.4 17.77 10.82 12.01 10.33 9.48 12.07 15.44 12.14 12.4814.3 16.8 18.2 15.7 18.83 17.39 12.19 13.67 11.83 13.3 16.26 14.79 15.91 14.2614.4 17 18.5 15.9 19.06 17.47 12.15 13.75 11.92 13.31 16.08 14.88 15.86 14.2616 15.4 17.3 14.9 18.2 18.32 12.5 12.93 12.31 12.65 16.85 15.66 17.48 15.2318.5 19 17.7 23.2 12.1 13.48 14.36 15.58 13.55 14.48 17.44 16.29 16.98 15.2619.6 18.7 19.3 22.4 19.02 19.62 13.59 14.59 13.5 13.68 17.34 17.16 17.35 15.919.7 18.9 19.5 22.5 19.59 19.37 13.69 14.6 13.52 13.72 17.49 17.19 17.41 15.9914.6 16.8 16 15.8 18.74 17.71 13.63 13.33 13.67 14.21 17.71 15.65 17.55 16.2414.5 16.8 15.8 15.8 15.24 15.09 12.15 12.34 11.71 13.08 15.36 12.91 15.03 13.4414.2 16.8 16 16.1 17.06 15.69 12.28 12.63 11.81 13.04 15.2 12.99 15.07 13.4723.3 18.2 24 16.8 17.68 19.73 13.29 13.08 13.2 13.33 16.92 16.79 16.92 16.2119.9 23.9 22.3 22.4 19.92 22.27 13.96 14.67 13.91 14.38 18.48 17.42 18.58 17.1321.1 25.8 20.5 22.8 17.91 18.95 14.24 15.76 14.67 15.53 19.61 16.31 19.17 16.425.2 22.2 24.6 20.3 18.39 17.66 13.25 14.85 12.89 14.35 18.95 17.13 18.62 17.0224.9 22.2 24.3 20.2 19.59 19.16 13.5 14.9 13.02 14.45 18.97 17.22 18.68 17.0982.2 74.5 97.7 74.6 20.81 22.73 13.67 15.43 14.43 15.41 17.87 16.98 20.08 16.8516.8 16.9 17.4 13.8 16.54 19.77 10.97 11.78 11.49 11.02 15.98 12.7 16.01 12.8516.5 16.7 17.2 13.7 16.28 19.45 10.75 11.4 11.35 10.7 15.61 12.62 15.8 12.7920.1 19.7 19.3 18.8 21.31 21.95 12.11 16.37 14.82 17.97 15.97 14.8920.1 19.6 19.5 19 21.31 22 12.45 16.48 12.59 15.78 18.36 16.09 18.21 15.5418.49 20.42 12.18 14.79 12.24 12.67 15.41 16.99 15.69 13.7718.21 20.23 11.55 14.56 11.63 12.05 14.87 16.79 14.88 13.0517.93 19.27 12.2 14.75 11.99 12.41 15.13 17.01 15.38 13.527.8 19.4 8 21.7 13.53 9.848 11.25 10.71 11.36 10.17 13.54 11.53 13.4 11.8826.9 18.6 23.2 19.9 13.03 13.99 14.56 17.46 16.74 14.97 17.45 19.85 18.05 15.7527.1 19.2 22.4 20.3 22.79 24.68 16.07 17.09 17.93 16.12 18.7 19.44 19.22 16.9127.5 19.4 22.7 20.4 23.06 24.78 16.92 17.52 18.41 16.62 19.82 19.89 19.55 17.3312.8 12.1 12.7 14.5 21.17 21.29 13.44 14.2812.8 12 12.6 14.4 20.56 21.1 13.67 14.3913.3 12.3 12.9 14.7 20.91 21.22 13.43 14.212.9 11.9 12.8 14.4 20.66 20.96 13.51 14.2212.9 16.5 13.4 18.2 21.18 19.99 13.97 14.6813.3 16.4 13.6 18.3 21.19 20 14.13 14.7513 16.3 13.4 18.4 21.77 20.61 13.88 14.5412.9 16.5 13.1 18.3 22.09 20.66 13.91 14.6229.8 32.3 26.9 28.2 22.4 22.02 16.08 17.74 16.75 16.55 21.33 19.84 21.65 19.8227.7 29.3 27.1 26.6 19.79 18.4 17.46 16.28 17.6 15.71 21.02 18.05 20.36 18.3325 25.4 27.1 24.5 15.16 15.92 14.77 15.73 21.6 19.23 21.05 18.9923.2 22.9 24.1 25 19.22 21 14.55 15.36 14.63 14.89 18.77 17.03 18.5 16.2323.1 22.7 24 24.8 19.13 20.98 14.81 15.28 14.69 15.03 19.28 16.9 18.89 16.4124.8 22.5 22.2 19.9 15.86 16.3 10.17 15.85 10.49 12.09 13.05 14.84 12.59 12.4325 22.4 22.3 20.1 16.34 16.68 11.61 15.89 11.52 13.37 14.13 14.9 13.91 13.7724.9 22.8 22.3 20 16.46 16.85 11.94 16.08 11.82 13.62 14.22 15.12 14.15 14.0516.6 18 20.1 19.6 17.3 15.68 10.47 11.75 10.29 11.49 16.28 15.84 16.06 15.4212 11.3 13 13.5 17.04 19.03 9.12 11.1911.9 11.4 13 13.9 17.35 19.02 9.15 11.1912.2 11.8 13.6 14.1 17.74 19.36 9.5 11.6312.3 11.4 13.3 13.8 17.48 19.28 9.27 11.3213.5 17.3 13.7 15.6 19.91 20.26 14.16 15.7113.1 17.2 13.3 15.4 19.81 20.34 13.96 15.5713.4 17.4 13.6 15.9 19.95 20.33 14.26 15.7713.2 17.4 13.4 15.7 20.49 20.617.6 8.6 10 10.7 16.09 17.25 9.42 9.73 9.36 10.16 13.03 11.35 13.15 11.927.7 9 10 11.1 15.52 16.95 9.93 11.497.6 8.9 9.9 11.1 16.84 17.69 10.32 9.9 10.1 11.15 14.05 11.44 14.13 12.867.5 8.9 10.3 10.9 17.44 17.95 11.36 9.98 11.25 12.41 15.63 11.49 15.64 14.2515.1 15.1 15.4 16.4 14.29 16.02 8.81 10.08 9.2 9.8 12.8 12.79 12.82 12.815.4 15 15.6 16.4 14.67 15.98 8.78 10.05 9.12 9.68 12.75 12.65 12.69 12.5811.4 12 9.7 11.1 14.56 11.57 8.46 9.4911.8 12.2 9.9 11.2 14.82 11.77 8.75 9.7411.4 12 9.7 11.1 14.54 11.68 8.49 9.511.8 12.2 10.3 11.1 14.76 11.76 8.57 9.612.8 13.5 14.7 14.6 13.16 13.22 7.92 8.31 7.33 8.3 10.29 11.17 10.24 10.5612.6 13.9 14.5 15.5 6.561 4.85 8.09 8.21 7.34 8.38 10.36 10.98 10.34 10.669.7 10.5 11.39 14.23 5.08 4.01 5.67 5.05 6.13 4.019.8 10.6 11.84 14.65 5.07 4.66 6.13 5.09 6.84 4.499.8 10.5 12.5 15.04 5.06 4.59 6.04 5.08 6.82 4.5512.2 18.5 14 17.1 11.76 10.17 6.27 9.45 5.89 7.68 7.91 11.06 8.14 8.512.2 18.2 14.1 16.9 12.11 10.43 6.99 9.5 6.76 8.44 8.61 11.14 9 9.5112 18.3 14 16.6 7 9.4 6.68 8.4 8.63 10.89 8.91 9.426.8 7 13.14 12.36 5.54 11.15 5.76 8.81 8.92 10.14 9.44 7.846.9 6.4 13.48 12.75 6.03 11.13 6.44 9.73 9.79 10.05 10.34 8.657.2 6.6 6.04 11.01 6.41 9.78 9.92 10.02 10.33 8.719.3 7.8 9.6 12.82 12.66 9.46 9.579.4 7.9 9.6 12.09 12.1 8.25 8.259.1 7.5 9.3 11.82 11.87 7.94 8.039.1 7.7 9.4 11.79 11.9 8.04 8.118.5 8.3 9.7 11.14 11.97 6.16 6.47 5.92 7.22 10.48 8.63 10.34 9.568.5 8.6 9.8 12 12.63 6.03 6.57 5.78 6.6 9.44 8.82 9.26 8.968.4 8.6 9.7 12.68 12.87 5.63 6.63 5.38 5.98 8.71 8.75 8.4 8.278.3 8.5 9.6 12.71 13.34 5.64 6.56 5.32 5.97 8.69 8.68 8.36 8.269.6 10.5 12.1 13.3 12.38 12.91 6.76 10.47 5.65 8.73 8.77 10.42 8.13 8.79.2 10.3 11.8 13.2 11.01 13.05 6.49 10.25 5.44 8.58 8.52 10.26 7.96 8.69.2 10.4 12 13.1 11.38 13.29 6.46 10.3 5.41 8.53 8.34 10.34 7.99 8.6210.1 13.6 12.2 14.6 15.42 12.97 5.69 9.56 5.77 8.42 8.8 11.43 9.06 9.5810.1 13.6 12.1 14.3 15.28 12.91 5.68 9.54 5.75 8.4 8.97 11.38 9.07 9.7710.2 13.6 12.5 14.5 15.06 12.98 6.08 9.82 5.99 8.53 9.23 11.64 9.28 9.9815.28 15.78 5.62 7.72 5.11 7.46 10.37 7.98 9.09 8.148 8.4 10.9 3.96 7.32 4.3 6.05 6.06 8.54 6.13 7.418 8.4 10.9 4.21 7.32 4.64 6.5 6.54 8.4 6.6 7.857.7 8.1 10.6 4.09 7.17 4.5 6.43 6.53 8.43 6.48 7.795.023 5.556 3.57 3.12 4.42 5.16 4.12 4.324.992 5.489 3.49 3.08 4.62 5.07 4.13 4.266.445 7.542 3.23 4.21 5.79 3.91 4.266.423 7.57 3.11 3.91 5.68 3.91 4.236.352 7.496 3.17 4.07 5.72 3.93 4.247.4 7.9 7.795 8.045 4.51 4.82 4.54 4.77 5.99 5.93 5.94 6.576.534 8.559 4.23 4.18 5.11 5.46 5.01 5.357.762 8.974 3.9 3.88 5.09 5.35 4.6 5.298.5 7.5 7.577 6.668 4.09 4.788.6 7.6 7.54 6.719 4.18 4.898.8 7.9 7.766 6.377 4.28 5.018.5 7.7 6.959 5.998 4.17 4.879.1 6.544 8.716 7.14 6.87 6.21 7.14 7.9 5.58 8.18 5.237.8 8.1 8.1 8.2 8.039 7.497 6.47 7.09 6.77 6.99 7.21 7.72 7.02 7.527.8 8.3 8.2 8.4 6.933 7.661 4.96 5.97 5.38 6.06 6.43 7.55 6.35 7.0510.3 7.3 7.3 7.7 7.767 9.067 5.31 6.08 5.14 5.8 6.95 6.01 7.11 5.9310.1 7.3 7.594 8.785 5.2 6.01 5.12 5.78 6.63 5.89 7 5.837.9 9.1 8.373 7.858 4.46 5.53 4.43 5.01 7.32 5.8 6.64 5.088.1 9.1 8.406 8.023 4.44 5.75 4.52 5.15 7.41 6.04 6.76 5.278.1 9.4 8.3 7.664 3.84 5.4 3.83 4.44 6.75 5.74 5.93 4.487.494 7.064 6.11 7.1 5.57 7.66 6.66 7.45 7.16 6.896.937 7.861 4.83 5.53 5.35 5.43 5.9 6.6 6.06 6.126.115 6.799 6.03 4.9 6.08 4.57 5.65 5.33 5.66 5.18.5 7.5 5.852 7.352 5.1 4.258.4 7.8 5.734 8.781 5.09 4.348.5 7.7 5.85 7.492 5.2 4.328.6 7.6 6.267 7.55 5.25 4.376.367 6.678 5.57 5.73 5.54 5.39 5.31 6.23 5.83 5.973.892 3.626 4.59 4.77.076 7.16 5.59 6.41 5.69 6.15 7.14 6.63 7.17 6.534.462 6.469 4.38 3.57 4.9 4.114.64 3.79 4.89 3.52 5.1 4.29 4.77 4.056.63 6.583 3.96 5.66 4.12 4.71 3.98 5.05 4.22 3.536.7 6.693 3.95 5.72 4.14 4.68 4.15 5.06 4.26 3.566.63 6.684 3.73 5.58 4.05 4.58 3.99 4.99 4.19 3.486.181 6.177 4.66 3.75 4.2 3.58 4.35 4.38 4.47 4.416.14 6.006 4.62 3.7 4.08 3.47 4.38 4.38 4.5 4.3513.6 25.1 34.5 19.9 6.041 5.062 4.54 4.8413.4 24.8 34.4 19.6 5.696 4.853 4.12 4.5113.5 25 34.5 19.5 5.855 5.024 4.31 4.687.5 5.888 6.198 3.85 5 4.19 3.94 4.2 5.27 4.44 3.847.5 6.609 6.45 4.51 5.26 4.84 4.72 4.91 5.49 5.09 4.576.352 6.191 4.33 5.04 4.59 4.44 4.66 5.32 4.86 4.377.8 5.985 6.106 4.29 5.65 4.71 5.47 5.02 5.3 5.02 5.167.8 5.934 6.084 4.42 5.77 4.77 5.55 5.17 5.4 5.1 5.2911 8.3 7.3 7.24 6.46 5.82 5.18 5.49 5.27 5.77 6.65 5.79 6.737.9 9.5 8.2 7.37.5 11.2 8.2 9.8 7.573 7.007 5.11 5.82 4.95 6 6.91 6.49 6.73 6.646.941 4.285 3.31 4.49 3.04 3.24 3.13 4.64 3.526.29 3.901 3.65 4.5 3.02 3.26 3.22 4.45 3.546.08 3.628 3.64 4.35 3.05 3.6 3.34 4.26 3.637.8 8.2 3.14 4.03 3.01 4.42 4.52 4.04 3.867.7 8.3 3.05 3.9 4.18 4.57 3.95 3.87.6 8 5.535 4.374 3.8 3.99 4.37 3.84 3.557.7 9.3 8.8 8.8 6.438 6.151 3.13 4.74 3.83 4.36 5.79 4.93 5.85 5.287.7 9.5 9.1 8.8 6.549 6.286 3.15 4.85 3.9 4.43 6.16 5.14 6.04 5.557.5 7.2 6.152 5.628 3.29 3.96 3.29 3.97 4.62 4.13 4.64 4.237.5 7.2 6.09 5.574 3.15 3.71 3.59 4.65 3.86 4.55 4.217.5 5.964 5.408 3.09 3.7 3.47 4.63 3.77 4.5 4.137.5 4.981 4.617 3.52 3.8 4.9 4.94 4.42 5.367.826 8.278 4.82 5.62 4.68 5.66 5.84 5.85 6.38 5.447.608 8.017 5.51 5.857.38 9.17 4.67 4.56 4.64 4.28 6.38 5.67 6.33 5.928.3 10.1 8 8.3 8.325 8.15 7.04 6.61 7.11 6.67 7.41 7.83 7.34 8.089.1 7.9 7.9 7.8 7.787 7.89 5.83 6.2 6.09 6.24 7.89 7.13 7.79 7.17.7 6.593 7.713 4.99 5.92 5.26 5.86 5.65 5.77 5.44 5.57.6 7.2 6.737 7.958 5.03 5.97 5.25 5.85 5.81 5.81 5.49 5.467.6 7.7 4.26 7.18 4.34 6.6 5.77 6.11 6.36 5.797.7 7.7 7.264 8.138 4.27 7.33 4.4 6.72 5.8 6.12 6.38 5.818.2 7.9 7.623 7.398 4.67 5.65 4.22 5.26 6.54 5.53 5.89 5.768.2 8.02 7.625 5.85 5.525.703 5.433 5.31 4.98 6.99 5.46 5.54 5.294.421 6.058 4.41 3.18 4.59 3.264.721 6.363 4.47 3.4 4.94 3.484.605 5.358 3.68 3.54 3.92 3.5 5.04 3.46 4.944.521 5.173 3.56 3.36 3.6 3.24 4.95 3.15 4.735.98 4.455 3.64 3.52 4.66 3.57 4.26 3.555.109 5.247 4.45 4.28 3.98 4.44 5.12 4.38 5.65 3.955.049 5.132 3.99 3.93 3.61 4.08 4.8 4.18 5.47 3.755.364 5.291 3.92 4.6 4.04 4 5.41 4.92 5.25 4.585.335 5.158 3.81 4.5 3.92 3.95 5.2 4.92 5.16 4.425.305 5.142 3.43 4.58 3.7 3.65 4.71 4.95 4.67 4.24.825 3.45 3.35 4.27 4.26 4.65 4.493.49 3.41 4.4 4.4 4.73 4.554.963 3.9 3.94 4.064.868 3.789 3.83 44.863 3.934.784 3.79 3.85 3.995.786 5.501 3.245.711 5.523 3.26.257 5.944 3.515.782 5.592 3.296.009 5.131 3.15 4.33 3.39 3.89 5.54 4.81 5.42 4.215.507 4.852 3.08 4.28 3.33 3.9 5.53 4.75 5.36 4.265.83 5.412 3.73 3.88 3.81 4.09 5.21 3.96 4.94 3.925.905 5.417 3.8 3.97 3.87 4.06 5.15 4.01 4.92 3.974.179 3.73 4.64 4.414.254 3.647 4.61 4.54.179 3.607 4.58 4.454.313 3.734 4.7 4.515.182 4.417 3.85 4.955.079 4.356 3.86 4.925.034 4.342 3.69 4.745.085 4.363 3.82 4.956.487 6.599 4.27 4.846.784 7.012 3.716.527 6.872 3.556.683 6.836 3.475.48 6.144 27.44 7.775.77 6.401 27.82 7.995.394 6.244 27.63 7.75.452 6.272 27.12 7.663.67 3.624.453 4.925 3.38 3.484.597 5.191 3.39 3.53.706 3.809 3.423.421 3.551 3.03 3.574.922 3.534 3.134.915 3.56 3.15.43 3.823 3.364.831 3.624 3.176.168 6.219 3.14 3.346.179 6.139 3.11 3.466.125 6.012 3.295.906 5.888 3.157.8 8.7 8.6 10.5 7.214 7.664 4.9 4.5 4.96 4.53 4.97 4.89 5.11 4.698.2 8.7 8.7 10.4 7.24 7.718 4.76 4.61 4.97 4.65 4.98 5.01 5.25 4.759 7.9 9.5 9 5.687 6.362 3.45 4.65 3.89 4.52 5.57 4.97 6.08 4.748.7 8 9 9.1 5.621 6.115 3.38 4.62 3.94 4.54 5.62 4.96 6.09 4.788.5 4.15 4.99 4.42 4.12 4.94 4.69 4.89 4.588.5 4.729 4.843 4.24 5.03 4.41 4.27 5.12 4.75 5.01 4.688.4 8.7 6.392 6.47 5.9 5.32 5.15 5.4 5.53 5.19 4.98 5.368.6 8.8 6.483 6.434 5.96 5.35 5.25 5.17 5.36 5.19 5.01 5.338.2 6.687 6.23 4.3 4.43 4.42 4.24 6 4.58 5.92 4.939.2 5.783 7.542 4.46 3.62 3.78 3.85 4.85 4.08 3.517.6 7.5 8.2 9 6.366 8.068 5.29 4.8 4.51 4.72 5.97 5.26 6.45 5.379.6 8.1 8.1 8.6 6.54 7.318 4.77 5.32 4.85 5.26 6.8 6.15 7.24 6.0747 45.4 44.4 43.648.4 46.2 47.9 45.799.6 99.7 99.8 99.9 88.96 88.25 99.74 99.7 99.77 99.69 99.86 99.78 99.74 99.847.5 43.2 47 47.845.4 40 44.6 45.2 47.81 48.52 45.42 47.59 44.63 46.49 45.54 46.68 46.11 45.9740.9 38.6 41.1 38.648.7 40.6 48.3 40 19.73 21 43.37 46.99 43.62 47.24 43.6 45.39 44.03 46.1148.8 47.1 49.5 47.1 48.23 49.81 40.49 38.9 40.18 38.93 46.48 45.89 46.25 46.5353.8 50.2 53.5 50.8 26.43 27.42 50.5 50.65 49.66 50.81 50.05 51.11 50.98 50.6837.5 42.3 39 43.298.1 96.4 98.5 96.6 96.6 96.96 96.53 96.65 95.67 96.45 95.63 96.5194.8 94.3 94.2 94.595.2 92.7 95.2 91.4

Lot 2

TSTP

H0006 H0007 H1111

Lot 1 Lot 2 Lot 1

SNV

15-35%

5-15%

genomic

244

Custom MiSeq®

system VariantStudio

No intronic variants

11

Run controls are important in QC monitoring

• High coverage does not guarantee variant detection

• Routine use of standardized material helps track run

performance

Mean Depth

9k 47k 32k 29k

12

How else can this technology assist labs with quality

assurance?

13

Massively parallel LOD study

3%

1 2 1 2 1 2 1 2 1 2 1 2

dPCR assigned value

Run #

55

0 V

aria

nts

Samples

ND 0.5% 70%

5% 11% 18% 30% 48%

Frequency

• Variants drop out

at different levels

• Multiplexing

minimizes

runs/preps

• 12 libraries

(versus

hundreds)

14

Standardized controls allow users to track performance

• Variant context affects performance

• Important to test variants in regions of interest rather than generic

variants

• Bioinformatics/post-VCF filtering impact results

15

Acknowledgments

R&D

Nakul Nataraj

Aron Lau

Kara Norman

Product Mgmt

Patty Chiang

Quality Assurance

Lorenzo Sapinoso

Program Mgmt

Susan Ewald

Regulatory

Erin Kipperman

Operations

Anjali Gupta

© 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are

the property of Thermo Fisher Scientific and its subsidiaries unless otherwise

specified. TruSight, TruSeq, and MiSeq are trademarks of Illumina, Inc.

16

Appendix

17

Stability - AcroMetrix® Oncology Hotspot Control

• -20C or below for 18 months

• Stable through five freeze-thaw cycles

• 4C for 6 days (open vial stability)

18

Hotspot Control Summary

• Variant Distribution

5-15% 15-35% genomic

Complex 0 1 0

DEL 15 14 0

INS 9 8 2*

MNV 0 2 0

SNV 317 155 32*

• Subset of variants covered by different panels

* genomic variants

supported by whole

genome sequencing data

(Complete Genomics)

19

Significant pooling and libraries needed for LOD study

pooling

10

@ 5%

10

@ 5%

10

@ 5%

40

@1.3%

20

@ 2.5%

20

@ 2.5%

10

@ 5%

40

@ 50%

40

@ 5%

40

@ 2.5%

40

@1.3%

141

LIBRARIES

12

LIBRARIES

1 1 1 1 1

1 1 1 1 1

1 1 1 1 1

1 1 1 1 1 1 1 1 1 1

1 1 1 1 1

1 1 1 1 1

1 1 1 1 1

Sensitivity study for 40 variants

50%

5%

2.5%

1.3%

40

4

2

1

# Libraries Needed

1

1

1

1

Freq.

3 reps