HCV Product Bulletin VF

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Simultaneous genotyping and
RAS-calling with the Sentosa® SQ HCV
Genotyping Assay
www.veladx.com

Delivers genotyping & RAS identification accuracy in
a single, efficient workflow
	

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Reliable, automated solution – full workflow
automation combined with built-in controls give you
confidence in your results

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 peeds time to next steps – simultaneously analyze
S
genotyping and RAS-calling with minimal hands-ontime using our automated solution

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Expert assay design – multiplexed sequencing design
targets key regions indicated in AASLD guidelines
including genotypes 1 through 6 as well as 70 subtypes
enabling discovery of potential new relationships
between subtypes and novel therapeutics

Overview
Prior to 2011, therapies for treatment of the Hepatitis C Virus
(HCV) focused on interferon-based therapy. In 2011 it was
found that the genotype of the virus is clinically important,
both in response of interferon-based therapy and to other
potential therapies. Current AASLD guidelines recommend
specific combination therapies for each genotype as well
as subtypes 1a & 1b1 . The introduction of these targeted
therapies has reduced adverse effects and increased cure
rates from 45% to 93% in genotype 1, for example2. Further
research to develop additional genotype-specific targeted
therapies will continue to improve outcomes for this disease.
This research makes accurate determination of HCV genotype
critical in the development of novel therapies for HCV.

Accurate HCV Genotyping and RAS analysis
The Sentosa® SQ HCV Genotyping Assay uses nextgeneration sequencing technology to deliver accuracy
in the detection of Hepatitis C Virus genotypes 1-6, and
their associated subtypes as well as resistance-associated
substitutions (RASs). Compared with traditional methods
for HCV genotyping such as line-probe assays (LiPA),
this next-generation sequencing-based assay eliminates
genotyping errors (Table 1) while simultaneously delivering
additional information about RASs found in the sample. As
shown in an external study comparing HCV genotyping
results using NGS, Sanger Sequencing, and LiPA, the
Sentosa SQ HCV Genotyping assay delivered 100% accurate
genotyping assignment while the LiPA approach yielded
11% inaccurate genotyping assignments, potentially
leading to incorrect future action (Table 1).3

Speeds Time to Next Steps
Reliable and reproducible, this assay is designed for routine
use with the Sentosa SQ workflow. By integrating genotyping
and RAS identification into a single automated workflow, you
eliminate running multiple assays and are able to move to the
next steps more quickly. This fully automated solution requires
less than 2.5 hours of hands-on-time, delivering results from
sample to answer in just 2 days. Both Laboratory Information
System (LIS) connectivity and system and extraction controls
are integrated into the workflow, giving you confidence in the
results while removing many user interactions with the system
to further streamline the workflow (Figure 1).
This ready-to-use platform can be running your lab in less than
two weeks. Vela Diagnostics offers a reagent rental model as
an alternative to capital equipment purchase. Please contact
your sales representative for more information.

Sample
number

Viral Load
(IU/mL)

Genotype by
VERSANT HCV
Genotype 2.0
Assay (LiPA)

Genotype
by Sentosa
SQ HCV
Genotyping

Genotype
by Sanger
Sequencing

1

364,100

1b

6

6

2

397,650

1b

6

6

3

3,130,050

1b

6

6

4

1,978,350

1b

6

6

5

1,997,600

1b

6

6

6

73,150

1b

6

6

7

96,800

1b

6

6

8

821,150

1b

6

6

9

44,000

4

3

3

10

2,253,900

4

3

3

11

78,650

4

3

3

12

111,100

4

3

3

13

5,529,700

4

3

3

14

155,650

4

3

3

15

550

6

3

3

16

1,042,800

6

3

3

Table 1: Genotyping accuracy with NGS compared with LiPA analysis.
150 samples were analyzed using both next-generation sequencing
(Sentosa® SQ HCV Genotyping Assay) and line probe assay analysis
(VERSANT® HCV Genotype 2.0 Assay). These samples were randomly
selected archived serum or plasma samples from 143 Asian and 7 African
patients with chronic HCV infection, viral loads ranging from 5.50x102 to
1.04x108 IU/mL (median 6.10x106). The genotype (GT) distribution was as
follows: 11 GT1a, 14 GT1b, 12 GT2, 58 GT3, 9 GT4, 7 GT5, and 39 GT6. In
16/150 (11%) of samples, discordant results between the two methods
were obtained. Confirmation testing by Sanger sequencing indicated that
the ability to discriminate at the major GT level was 89.3% (95%CI: 83.4
– 93.3) for LiPA and 100% (95%CI: 97.5-100) for Sentosa NGS. Correct GT
subtype calls were found to be 89% for LiPA and 100% for NGS. Among the
16 discordant samples, 8 GT6 were wrongly classified as GT1b with LiPA, 6

Expert sequence design
The Sentosa® SQ HCV Genotyping Assay employs a
multiplexed sequencing design targeting 3 therapeutically
important regions of the Hepatitis C Virus genome: NS3,
NS5A, and NS5B . This sequencing strategy thus maximizes
sequencing reads on the most informative regions of the
HCV genome. By targeting the NS5B region of the HCV
genome instead of the 5’UTR, typically targeted by traditional
HCV genotyping assays, even recombinant strains can
be identified. This approach enables correct genotyping
of recombinant viruses while simultaneously identifying
clinically relevant RASs(Fig. 2). Additionally, this approach
overcomes many of the uninterpretable results seen with
LiPA (6.7% uninterpretable results with LiPA) by providing
direct sequencing results.7
Furthermore, this assay design enables you to identify
subtypes beyond 1a and 1b, delivering subtypes for 70 of
the subtypes for Hepatitis C with accuracy. This advances
your research, allowing you to discover potential new
relationships between subtypes and therapeutics under
investigation. Additionally, DNA contigs are readily available
for further sequence analysis enabling assessment of
additional mutations specific to the specimen under
investigation. This may prove useful for further bioinformatics
analyses in conjunction with monitoring resistance to novel
drug treatments in your research.

GT3 as GT4, and another 2 GT3 as GT6.4

Sentosa®
Link

Sample ID
download

Samples

Plasma or
serum
samples

Sentosa®
SX101

Lysis
Extraction
Library
preparation

Sentosa®
SQ301

Sequencing
Primary data
analysis

Sentosa®
SQ Reporter

Sentosa®
Link

Secondary
data analysis
Automated
reporting

Result
upload to
LIS

Figure 1: Single, automated NGS workflow for HCV Genotyping & RAS
analysis. Workflow diagram details the steps involved from sample through
analysis using the Sentosa® SQ HCV Genotyping Assay. This workflow requires
less than 2.5 hours of hands on time and delivers results in 2 days.

Figure 2: Schematic shows recombinant HCV strains identified to date.
This depicts recombinant sites for the identified species, demonstrating that
in these cases the recombination occurs prior to NS3, leading to accurate
genotyping with the Sentosa SQ HCV Genotyping Assay while still covering
all 3 drug target regions for RAS testing.6

For more information, visit VelaDX.com/HCV

Hepatitis C Virus RNA
5’ UTR

C

E1

E2

Target
Region

Codon 1 to 267

Detection

RAV Detection
for GT 1a & 1b

Baseline
RAVs

NS1 NS2

NS3 NS4A NS4B NS5A

NS5B

3’ UTR

Codon 14 to 201

Codon 346 to 559

RAV Detection
for GT 1a & 1b

RAV Detection for
GT 1a & 1b
and Detection for
GT 1, 2, 3, 4, 5, 6

EASL
EASL
NS3 RAVs Detected AASLD
NS3 RAVs Detected AASLD
Guidelines
Guidelines
by
by
Codon
VelaDx GT 1a & 3 GT 1a GT 1b Codon
VelaDx GT 1a & 3 GT 1a GT 1b
Q80

Q80H
Q80K
Q80R

M28

Q30

L31
P32
H58
Y93

M28A
M28G
M28T
Q30D
Q30E
Q30G
Q30H
Q30K
Q30L
Q30R
L31F
L31M
L31V
P32L
P32S
H58D
Y93C
Y93H
Y93N
Y93S

Figure 3: Illustration of Hepatitis C Virus RNA genes and regions
targeted by the Sentosa SQ HCV Genotyping Assay design. Major RASs
are identified and reported through the Sentosa SQ Reporter Software
in accordance with the guidelines as well as targeted gene regions
corresponding to the genotyping information.

Ordering Information

Sentosa® SQ HCV Genotyping Assay Specifications
Analytical
sensitivity

>1,000 HCV IU/mL for genotypes 1a, 1b, 2, 3
&4
>2,000 HCV IU/mL for genotypes 5 & 6

Analytical
specificity

No cross-reactivity with HAV, HBV, HIV, CMV,
EBV, BKV, Dengue virus or genomic DNA

Reproducibility

99.2% (95% confidence interval: 97.20%99.79%)

Controls

1 system control, 1 extraction control

Amplicons
targeted

NS5B, NS3, & NS5A;

Automated result
calling

GT 1 through 6 with RAS reporting for GT1a,
1b & 3. Sequence information for NS5B, NS3 &
NS5A accessible in BAM files.

Coverage/target

>200x for genotyping, >500x for RAS calling

Sample types
supported

Plasma & serum

Sample input
required

530 uL

Sample
throughput

15 samples/run, 80 samples/week

Product Name

Pack Size

Item Number

Sentosa® SQ HCV Genotyping Assay

4x16 tests

690019

Time to results

2 days

Sentosa® ST Template Kit

8 runs

690007

Hands on time

Less than 2.5 hours

Sentosa® SQ Sequencing Kit

8 runs

690005

Sentosa® SQ 318 Chip Kit

8 runs

300301

4x16 tests

300352

1. A
 ASLD/IDSA HCV Guidance, Panel (September 2015). “Hepatitis C guidance: AASLD-IDSA
recommendations for testing, managing, and treating adults infected with hepatitis C virus.”.
Hepatology (Baltimore, Md.). 62 (3): 932–54. doi:10.1002/hep.27950. PMID 26111063.

Sentosa® SX101

1

400089

2. L iang, TJ; Ghany, MG (May 16, 2013). “Current and future therapies for hepatitis C virus infection.”.
The New England Journal of Medicine. 368 (20): 1907–17. doi:10.1056/NEJMra1213651. PMID
23675659.

Sentosa® NGS Starter Kit

1

400105

Sentosa® SQ301 (120V)
with SQ301 Minicentrifuge

1

690026

Sentosa® ST401

1

690027

4. D
 ata from “Next Generation Sequencing (NGS) for HCV genotyping and optional identification of
resistance-associated variants” . Presented at AASLD Annual Meeting 2015 (Kok Siong Poon, Evelyn
S. Koay, Cui Wen Chua, Mui Joo Khoo,**Zhang Rui,**Elian Rakhmanaliev,**Wen Huang and **Gerd
Michel
*Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital,
Singapore; Department of Pathology, Na=onal University of Singapore, Singapore;**Vela Research
Pte Ltd., Singapore)

Sentosa® Link

1

400045

5. S immonds P, Bukh J, et al. (2005). “Consensus proposals for a unified system of nomenclature of
hepatitis C virus genotypes”. Hepatology. 42 (4): 962–73. doi:10.1002/hep.20819. PMID 16149085

Sentosa® SQ Reporter
(software, server and perpetual license)

1

690014

Sentosa® SX Virus Total Nucleic Acid Plus
II Kit

References

6. R
 eference: Bhattacharya et al. Virology Journal 2011, 8:458
7. R
 eference: Siemens VERSANT HCV Genotype Assay 2.0 Package Insert. 26017. Rev 5. 2012-08.

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

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Suite 250
Fairfield, NJ 07004
T: +1 877 593 7528 (Toll-Free)
E: infoUSA@veladx.com

For more information, kindly contact your Vela Diagnostics representative. All rights reserved. Vela is a trademark of Vela Diagnostics Holding Pte Ltd.
Sentosa® is a registered trademark of Vela Diagnostics Holding Pte Ltd in several markets including the US and the European Union.

NHCV-20170320-PB-US-EN-001-RUO

Vela Diagnostics USA Inc.