H02

Small Customizable Next-Genera5on Sequencing Based Target-Capture Panels in aClinical Environment Can Detect Variant Muta5ons at Frequencies as Low as 0.5%
Lisa M. Chamberlain, Zhiyi Xie, Andrew R. Carson, Bradley Patay, Valerie McClain, Ogeen Kiya, Wenli Huang, Jeﬀrey E. Miller, and Tim Stenzel
Invivoscribe Technologies, Inc, San Diego, CA

Introduc5onResults: Precision and Reproducibility

In clinical trials, large scale target-capture panels provide cri5cal informa5on for the selec5on oftarget biomarkers. However, as biomarker targets are further characterized large target-capture
panels can elicit a glut of excess informa5on, complica5ng analyses. Alterna5vely, once biomarker
targets have been iden5ﬁed, the use of smaller, focused next-genera5on sequencing (NGS) basedtarget-capture assays facilitate speciﬁc variant detec5on by sequencing genomic regions of interest
with greater breadth than classic PCR-based assays. Moreover, laboratory methods for larger
target-capture panels require extensive modiﬁca5on and op5miza5on when applied to smallerscale target-capture panels to maintain op5mal analy5c quality. Here, we u5lize smaller targetcapture panels (~10kb) focusing on a few genes, allowing for high-mul5plexing of samples on NGS
plaXorms reducing cost per sample and decreasing processing 5me.
Here, we demonstrate excellent linearity, a Limit of Detec5on (LoD) of 0.5%, a loca5on speciﬁc
Limit of Blank (LoB), and good precision and reproducibility from small target-capture assays.

Libraries were generated by 2 operators on 2 days and were run on 2 diﬀerent instruments. In
total, 24 replicates each of 5%, 2%, 1%, and 0.5% (DNA:DNA) contrived samples were run.

Materials and Methods
Library Prepara+on: Whole-genome libraries were prepared using the KAPA Hyper Prep kit (KAPA
Biosystems®).
Library Hybridiza+on, Capture, and Washing: Whole-genome libraries were hybridized to probe
sets (Integrated DNA Technologies®, Coralville, IA, USA) at 65°C overnight. Buﬀers from the SeqCap
EZ Hybridiza5on and Wash Kit (Roche®, Pleasanton, CA) were used for hybridiza5on and washing.
Contrived Samples: 5 cell lines were used to generate contrived mixes of DNA with 4 expectedvariants against background DNA. Contrived mixes containing 25%, 10%, 5%, 2%, 1%, and 0.5%
variant DNA were diluted in background DNA (DNA:DNA). Prior to dilu5on, input cell lines had
expected muta5ons with variant allele frequencies (VAF) of 1 (Inser5on 1), 0.5 (SNV 2), 0.5 (SNV 3),
and 0.5 (SNV 4).
LoD, LoB, and Linearity: 3 replicates of 25% and 10% DNA:DNA dilu5ons, 4 replicates of 5%, 2%,1%, and 0.5% DNA:DNA dilu5ons, and 2 replicates of background DNA were sequenced.
Precision and Reproducibility: 24 replicates of 5%, 2%, 1%, and 0.5% DNA:DNA dilu5on contrived
samples were run through the 3-gene assay by 2 operators on 2 diﬀerent days on 2 diﬀerentinstruments.

For reproducibility, the acceptance criterion was set so no more than two 1% contrived samples
were undetected. All contrived samples ≥1% DNA:DNA test posi5ve for all 4 expected
muta5ons, demonstra5ng excellent reproducibility of this assay.
Precision analysis observed %CV (CV/ x ) of variant allele frequency (VAF), with the goal of thismetric to be ≤30%. Data is presented in Table 2, and all expected variants pass this criterion.
Day-to-day, operator-to-operator, and instrument-to-instrument varia5on analysis is presented
in Figure 2. Overall there is very liqle variance, most of which is from varia5ons in DNA:DNA%and random (residual) sources.
Table 2: %CV of VAFs for all expected variants are below the cutoﬀ of 30% for precision valida5on.
Contrived Sample(DNA:DNA%)
5%2%1%0.5%% CV of detected VAF (≤30%)
Expected VAF (%)

Expected Inser+on
1510.182.520.25

Expected
SNV 3

13.52

10.19

12.44

12.31

13.55

9.56

18.35

16.90

22.13

16.28

20.46

21.93

21.97

0.5
0.5

Expected
SNV 4

8.36

11

Expected
SNV 2

28.48

Figure 2: Variance analysis of P/R data indicates very liqle variance stemming from random sources.

Results: LoD, LoB, and Linearity
To establish the LoD and linearity of our small target-capture panel assays, contrived samples
were assayed using a target-capture panel covering 3 genes and 4 expected variants.
Linearity data is graphed in Figure 1. Each expected variant is graphed separately; and displays
data from DNA:DNA% with 3 replicates for 25% and 10% and 4 replicates for 5%, 2%, 1%, and0.5%. Equa5ons for line of ﬁt and R2 values are listed on each graph. R2 values are signiﬁcant,ranging from 0.994 to 0.998.
LoD was established at 0.5% (Table 1). Of par5cular interest, we note that Expected SNV 4 isreliably at detected at a lower VAF of 0.25% (due to variable representa5on in the contrivedsample).
No expected muta5ons were detected in 100% background DNA samples (N=2), LoB wasestablished for each expected variant as the background variant rate + 5*stdev.
Figure 1: Linearity for 4 selected variants
VAF = 0.060444 + 1.029597*Expected
VAF
R2 = 0.998

VAF = 0.0590254 + 0.9853445*Expected
VAF
R2 = 0.996

VAF = 0.0696287 +0.84947*Expected VAF
R2 = 0.995

VAF = -0.012821 +0.738447*Expected VAF
R2 = 0.994

Insert 1SNV 2
SNV 3
SNV 4

Conclusions
Table 1: LoD is established at 0.5%, and all expected muta5ons are observed at that VAF.
Expected Insert 1
Expected SNV 2
Expected SNV 3
Expected SNV 4
Tests
Tests
Tests
Tests
posi5veVAF
posi5veVAF
posi5veVAF
posi5ve
N Expected VAF
Expected
Expected
Expected
RangeforRange
forRange
forRange
for
VAF
VAF
VAF
VAF
(%)
muta5on(%)
muta5on(%)
muta5on(%)
muta5on(%)
(%)
(%)
(%)
325%
25-26.3 100%
12.5% 11.6-13 100%
12.5% 10.1-10.9 100%
12.5% 8.8-10
100%
3
10%
10-11.1 100%
5.0%
4.6-4.9100%
5.0%
4.1-4.5100%
5.0% 3.47-3.52 100%28
5%
4.7-5.6100%
2.5%
2.4-2.6
100%
2.5%
2-2.7
100%
2.5%
1.8-2.1
100%
282%
1.8-2.2100%
1.0%
1.1-1.2
100%
1.0%
0.7-1
100%
1.0%
0.6-0.9100%
281%
0.9-1.2100%
0.5%
0.5-0.7100%
0.5%
0.4-0.6100%
0.5%
0.3-0.4
100%
28
0.5%
0.5-0.8
100%
0.25% 0.3-0.4 67.9%
0.25% 0.2-0.3100%
0.25% 0.2-0.4
64%

Small hybridiza5on panels are cost eﬀec5ve in detec5ng low-frequency variants fromsmaller subsets of genes while using far less DNA than individual PCR-based biomarker
assays. Small hybridiza5on assays focus on the most per5nent genes for a targeted
therapy and have the poten5al to greatly assist in understanding the molecular
backgrounds of responders, super-responders, and non-responders, informa5on that canhelp improve pa5ent outcomes. Developing NGS target-capture panels withbioinforma5cs in compliance with ISO 13485 and QSR design control requirements makesthese assays suitable for pre-market submissions to worldwide regulatory authori5es.

AMP Global, April 3–5 2017, Berlin, Germany