TECH NOTE

The Utility of Twist Respiratory Viral Controls for

SARS-CoV-2 and Multiplexed Pathogen Detection
INTRODUCTION F O L D E N R ICH ME N T
10,000
Emerging viral infectious diseases require rapid responses to
develop assays for detection and measurements. This is especially
5,000
true with the COVID-19 pandemic. Researchers responded by
developing rapid detection tests including quantitative PCR
0
(qPCR) and Next Generation Sequencing (NGS) for detecting the % BA S E S A T >=1X
SARS-CoV-2 viral genome. 100%
98%
The symptoms for SARS-CoV-2 are similar to other diverse 96%
viral pathogens resulting in respiratory illnesses. As influenza 94%
season approaches, there is a push to develop assays capable of 92%
detecting more than one pathogen in a single test, which would 90%

allow for rapid pathogen identification without lengthy systematic hBoV HRV D68 H1N1 H3N2 IBV MuV hPIV1
testing to rule out individual pathogens. MeV hPIV4 229E NL63 SARS SCV-2 MERS OC43

Figure 1: Fold-enrichment and percent of genome covered at 1x for viral controls

With a multiplexed assay comes the need for an increased set after target enrichment. Full names for abbreviated viruses are present in Table 1.
of diverse viral controls. In this technote we demonstrate the
compatibility of the diverse Twist Respiratory Viral controls as ME R S CO R O N A VIR U S
positive controls for the Twist Respiratory Virus Research Panel. 100%
PCT REF ALLELE

We also demonstrate that these same viral controls serve as

50%
useful negative controls for routine SARS-CoV-2 testing.
0%
RESULTS H U MA N R H IN O VIR U S
100%
PCT REF ALLELE

To determine the compatibility of the Twist Respiratory Viral

controls as positive controls for the Twist Respiratory Virus 50%
Research Panel (PN 103066, 103067 & 103068) we created
libraries for NGS target enrichment. Libraries were made with the 0%
0% 20% 40% 60% 80% 100%
indicated viral control at 1,000,000 copies spiked into 50 ng of POS IT ION ON V IRAL T E M PL AT E

human reference RNA. Captures were performed using a 16 hour Figure 2: Fraction of reads with the reference allele at all bases across the MERS
hybridization with the Twist Standard hybridization kit (PN). coronavirus synthetic template (top) or human rhinovirus synthetic template (bottom).

Capture of the viral controls at 1 million copies showed excellent

SARS CoV2

coverage, enrichment and uniformity. Every viral template was

hPIV1
hPIV4

MERS

SARS
H1N1
H3N2

hBoV

OC43
NL63

229E
MuV
HRV

MeV

enriched at more than 2500-fold above the input fraction and

D68

IBV

100
had more than 99.9% of bases covered with at least 1x coverage D68
(Figure 1). After capture, coverage was relatively even across the HRV
viral genomes, even for segmented viruses such as influenza. This H1N1 80
H3N2
uniform coverage is due to capture efficiency but also reflects the
SA M P L E G E N OME

IBV
synthesis and manufacturing excellence of these viral controls. In hPIV1
hPIV4 60
Figure 2 we show 99.9% of bases that were captured with at least MeV
20x coverage represented with at least 95% of reads showing the MuV

expected reference allele. hBoV 40

MERS
For the viral controls, hybrid captures were very specific leading NL63
OC43
to little or no contamination or cross-alignment. To measure cross- 229E
20

contamination or cross-alignment we aligned capture results SARS

SARS CoV2
against each Twist Respiratory Viral control. For every captured 0
control, at least 99.3% of reads mapping to the capture space of GE N OM E M APPE D T O

the panel mapped to the target genome (Figure 3). Figure 3: Percent of reads from each enrichment sample (rows) mapping to each
genome covered by the enrichment panel (columns). Entries on the diagonal line
represent percentages of on-target reads, while entries off the diagonal line represent
percentages of off-target reads. Full names for abbreviated viruses are present in Table 1.
TWIST BIOSCIENCE | TECH NOTE

A. PHYLOGENTIC TREE B. COVERAGE FROM A SARSCOV2 CAPTURE

SARSCoV2
MERS 2,500
1
0
1 SARS
SARS
50
SARSCoV-2

CO V ER A G E O V ER V IR A L G ENO ME
1 0
Coronavirus HKU1 MERS
1
1
Coronavirus OC43
1 0

Coronavirus 229E OC43

1 1

Coronavirus NL63 0
NL63
1

0
Figure 4: A. Phylogenetic tree constructed from a multiple alignment of whole 229E
1
reference genomes for all human-infective coronaviruses. B. Coverage from a SARS-
CoV-2 capture experiment over all coronavirus genomes synthesized as standards. 0
Purple regions represent areas of very high (>97%) homology between SARS-CoV-2 0 5,000 10,000 15,000 20,000 25,000 30,000
and the given species. COORD IN AT E AL ON G V IRAL GE N OM E

6,000
50
COPIES PER µL

4,000
COPIE S PE R µ L

25

2,000 0
9E

8
N1

N2

oV

S
eV
uV

63
43
RS

er
D6

IV

IV

ER
HR

IB

at
NL
OC
22

M
hB

M
H1

H3

SA
hP

hP

W
M

VIRUS

0
9E

8
N1

N2

oV

S
eV

uV

63

43

RS

V2

er
D6

IV

IV

ER
HR

IB

at
NL

OC
22

M
hB

SC
M
H1

H3

SA
hP

hP

W
M

VIRUS

Figure 5: Boxplot showing qPCR data using the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel for each of the Twist Respiratory Virus Controls. The Twist
SARS-CoV-2 RNA control 2 was added at 5,000 copies as a positive control. All of the viruses show similar behavior to the no-template water control, with fewer than
1 copy per µl of SARS-CoV-2. Full names for abbreviated viruses are present in Table 1. The zoomed-in boxplot shows qPCR data from the above RNA controls between 0
and 50 copies per µl.

SARS and SARS-CoV-2 genomes are close relatives and approximately 80% identical at the sequence level (Figure 4A). For these
genomes, we observed a very low-level (less than 0.05% of reads) of reciprocal cross-alignment that occurs in regions of high
homology between the two viruses (Figure 4B). Although the vast majority of reads were assigned to the correct genome between
these two controls, care should still be taken in interpreting alignments to the SARS genome when using SARS-CoV-2 controls, or
alignments to the SARS-CoV-2 genome when using SARS controls.
We also put these Twist Respiratory Viral Controls through a FDA-EUA approved qPCR diagnostic assay for SARS-CoV-2 to determine
if the Twist Respiratory Viral controls could potentially serve as negative controls in diagnostic assays. Using the CDC 2019-nCoV
Real-Time RT-PCR Diagnostic Panel with TaqPath 1-Step RT-qPCR Master Mix, we performed a series of assays with each of the Twist
Respiratory Viral Controls at approximately 106 copies per µl as templates. In addition, we used the Twist SARS-CoV-2 RNA Control 2 at
5,000 copies as a positive control against the ATCC Quantitative Synthetic SARS-CoV-2 RNA as a quantitative reference standard. As
expected, we found that SARS-CoV-2 was practically undetectable in 6 technical replicates for each virus, with SARS-CoV-2 genome
copies at fewer than 1 copy per µl. The Twist Respiratory Viral control essentially performs as well as the no-template water control. The
results below indicate these viral controls serve as valuable negative controls in routine SARS-CoV-2 detection assays.
TWIST BIOSCIENCE | TECH NOTE

SUMMARY

In this document we highlight the utility of 15 Twist Respiratory Viral Controls as positive controls for Multiplexed Pathogen Detection
via target enrichment and negative controls for qPCR assays detecting SARS-CoV-2. The 15 Twist Respiratory Viral controls resulted
in 99.9% sequence coverage with a minimum of 2500 fold enrichment after capture with the Twist Respiratory Panel. When used in a
FDA-EUA approved qPCR SARS-CoV-2 detection assay, we found the Twist Respiratory controls gave similar background levels as the
no-template water controls, indicating these controls serve as useful negative controls in SARS-CoV-2 assays.

NUCLEIC
PART NUMBER NAME ABBREVIATED NAME STORAGE
ACID SPECIES

102024 Twist Synthetic SARS-CoV-2 RNA Control 2 (MN908947.3) SCV-2 ssRNA –90 to –70°C
103016 Twist Synthetic Influenza H1N1 (2009) RNA control H1N1 ssRNA –90 to –70°C
103017 Twist Synthetic Influenza H3N2 RNA control H3N2 ssRNA –90 to –70°C
103018 Twist Synthetic Influenza B RNA control IBV ssRNA –90 to –70°C
103019 Twist Synthetic Human bocavirus 1 DNA control hBoV ssDNA –90 to –70°C
103020 Twist Synthetic Human enterovirus 68 RNA control D68 ssRNA –90 to –70°C
103021 Twist Synthetic Human rhinovirus 89 RNA control HRV ssRNA –90 to –70°C
103022 Twist Synthetic Mumps virus RNA control MuV ssRNA –90 to –70°C
103023 Twist Synthetic Human parainfluenza virus 1 RNA control hPIV1 ssRNA –90 to –70°C
103024 Twist Synthetic Measles virus RNA control MeV ssRNA –90 to –70°C
103025 Twist Synthetic Human parainfluenza virus 4 RNA control hPIV4 ssRNA –90 to –70°C
103026 Twist Synthetic Human coronavirus 229E RNA control 229E ssRNA –90 to –70°C
103027 Twist Synthetic Human coronavirus NL63 RNA control NL63 ssRNA –90 to –70°C
103028 Twist Synthetic Human coronavirus OC43 RNA control OC43 ssRNA –90 to –70°C
103029 Twist Synthetic SARS coronavirus Tor2 RNA control SARS ssRNA –90 to –70°C
103030 Twist Synthetic MERS coronavirus 2c EMC/2012 RNA control MERS ssRNA –90 to –70°C

Table 1: Twist Respiratory Synthetic Controls.

The Twist products referenced herein are for research use only, and subject to additional use restrictions as set forth in Twist’s Supply Terms and Conditions:
www.twistbioscience.com/supply-terms-and-conditions.

DOC-001174 REV 1.0