Achieve Comprehensive Viral Detection Using NGS Target Enrichment
App Note / Case Study
Published: January 7, 2025

Credit: iStock
Next-generation sequencing (NGS) has revolutionized the detection and characterization of viral pathogens, enhancing global health efforts against outbreaks like SARS-CoV-2.
However, current reverse transcription polymerase chain reaction (RT-PCR) methods often fall short when detecting multiple viruses simultaneously, limiting their effectiveness. The latest viral detection technology enables the simultaneous detection and characterization of 29 common respiratory viruses with high sensitivity and uniform genome coverage.
This application note explores how an innovative targeted sequencing panel provides a comprehensive tool for infectious disease research and public health monitoring.
Download this application note to learn:
- How to detect and characterize multiple viral genomes in a single assay
- The benefits of NGS hybrid capture over conventional RT-PCR for viral surveillance
- Key findings on the panel's sensitivity, enrichment efficiency and performance
INTRODUCTION
Accurately detecting and identifying viral pathogens is a critical
global health concern exacerbated by the 2019 novel coronavirus
(SARS-CoV-2) pandemic. A wide range of viral pathogens
cause similar patient symptoms, making it difficult to identify
the underlying infectious agent. Currently, RT-PCR assays are
routinely used to detect viral pathogens. These common assays
are rapid, but are often used to test for just one pathogen at a time.
Multiplexed RT-PCR saves time, allowing for the simultaneous
detection and identification of multiple viruses, but it suffers from
other shortcomings, including lack of viral sequence information.
Next generation sequencing (NGS) hybrid capture combines
high-throughput capabilities with high sensitivity, making it
possible to quickly identify specific, whole viral genomes from
complex samples.
In the Twist SARS-CoV-2 Research Panel, we highlighted how
NGS-based target enrichment was successfully applied for the
detection and characterization of the SARS-CoV-2 viral genome.
We showed that target enrichment resulted in a nearly millionfold enrichment of viral fraction post-capture for low viral titer
samples and 99.9% genome coverage using only 25,000 reads
for samples with a viral titer of 1 thousand copies. Moreover, we
demonstrated the tolerance of the capture-based method to
virus mutations and successfully identified various mutations in
the SARS-CoV-2 virus. The ability to simultaneously detect and
characterize viral genomes makes hybrid capture a powerful
alternative to RT-PCR, that can monitor viral evolution and
conduct population scale surveillance.
We’ve built upon Twist’s expertise in utilizing target capture for
infectious diseases by creating a panel that is capable of detecting
multiple viral pathogens associated with common symptoms of
respiratory illnesses. This panel, called the Twist Respiratory Virus
Research Panel, is designed to enrich for 29 common respiratory
viruses. To validate this panel, we synthesized 15 viral controls,
both ssRNA and ssDNA, that were used in addition to the Twist
SARS-CoV-2 Synthetic RNA control. These controls were spiked
into human reference RNA to generate TruSeq-compatible DNA
libraries for target enrichment. Here, we demonstrate the:
1 Capture of 16 different synthetic viral strains at 1,000,000 copies.
2 Capture of 3 viral strains across viral titers of 100–1,000,000
copies.
3 Simultaneous capture of two viral genomes at 10,000 copies
each in simulations of co-infections.
4 Efficient multiplexed capture of diverse viral genomes at viral
titers ranging from 100–1,000,000 copies.
RESULTS
The Twist Respiratory Virus Research Panel is targeted against the
reference sequences for 29 common human respiratory viruses,
including coronavirus (CoV), influenza virus, adenovirus, bocavirus
(hBoV), enterovirus, metapneumovirus, parainfluenza (hPIV),
human rhinovirus (HRV), measles morbillivirus (MeV), mumps
virus (MuV), rubella virus, and respiratory syncytial virus (RSV)
(Table 4). Additional probes were also designed to target diverse
genomes representing major influenza A and B outbreaks since
the year 2000, and to incorporate diversity from 77 additional
rhinovirus strains.
To validate the performance of the Twist Respiratory Virus
Research Panel, a variety of synthetic viruses were designed
and synthesized as ssRNA or ssDNA, depending on their
native genome structure. The synthetic viruses were sequencevalidated, then diluted to a stock of ~1 million copies per ul for
downstream experiments.
Each viral synthetic control was spiked into 50 ng of human
reference RNA, which was then used to generate an Illumina
TruSeq-compatible library (1,000,000 copies for each sample).
The Twist Respiratory Virus Research Panel was then used to
capture viral sequences, following the Twist Target Enrichment
Protocol with a 16-hour hybridization time. In most cases, we
found that over 70 percent of reads came from viral genomes in
these libraries, representing at least a 2500-fold enrichment over
the spiked-in content (Figure 1).
The one notable exception was for human bocavirus (hBoV). The
human bocavirus genome is the smallest among those tested.
Shorter templates will contribute less content to the library
at equal titers. Indeed, while the total percent of viral reads is
generally low for shorter templates, the fold-enrichment (which
is normalized to template length) is generally higher. (Figure 1).
Additionally, bocavirus has an ssDNA genome, while every other
tested virus has an ssRNA genome. Since similar length ssRNA
viruses show much higher enrichment, it’s likely that lower yield
for ssDNA during library preparation was what caused lower
capture efficiency.
Despite differences in capture between bocavirus and the other
ssRNA viruses, all synthetic controls were sequenced to high
depth in great uniformity with Fold 80 Base Penalty in the range
of 1.2 to 1.5. At 1 million sequenced reads, we find all templates to
be covered at a median depth of 1500x, with at least 99% of bases
covered to at least 30x depth (Figure 1), sufficient for confident
variant calling and de novo assembly.
NGS Target Enrichment of Viral Pathogens
using Twist Respiratory Virus Research Panel
APPLICATION NOTE
TWIST BIOSCIENCE | APPLICATION NOTE
VIRAL
STRAIN
VIRAL
TITER
(Copies)
READS EXPECTED
WITHOUT
ENRICHMENT
(Out of 1M total)
NUMBER OF
ON-TARGET
VIRAL READS
(Out of 1M total)
FOLD
ENRICHMENT
H1N1
100 <1 402 28704
10,000 1 53964 38531
1,000,000 140 715054 5106
H3N2
100 <1 349 24025
10,000 1 40081 27631
1,000,000 145 818155 5640
HRV
100 <1 1453 190849
10,000 1 26185 34393
1,000,000 76 682086 8959
Next, we assayed the sensitivity of the Twist Respiratory Virus
Research Panel by capturing three synthetic ssRNA viral genomes
(H3N2, H1N1, and HRV) at various titer loads (100, 10,000 and
1,000,000 copies per library), using a 16-hour hybridization
time. We found each virus to be enriched by at least 5000-fold
at every tested titer, with greater than 20,000-fold enrichment
for the viral template at low titers (Table 1). In summary, the Twist
Respiratory Virus Research Panel can efficiently enrich for viral
sequences present at titers spanning several orders of magnitude,
demonstrating a limit of detection as low as 100 copies.
The Twist Respiratory Virus Research Panel is designed to
target several viruses in a single capture, for the detection
or characterization of several viruses in one reaction. Human
respiratory pathogen co-infections often occur among patients
suffering from respiratory distress and discomfort. Some
common co-infections include respiratory syncytial virus (RSV)
and coronaviruses (Martin et al 2012) or human bocavirus and
parainfluenza viruses (Zhang et al 2012).
Co-infections were simulated by spiking in multiple synthetic
viral controls into a single sample during library preparation. The
following controls were spiked into 50ng human reference RNA at
10,000 copies of each virus per library:
• Human rhinovirus 89 (ssRNA) with Human bocavirus 1 (ssDNA)
• SARS-CoV-2 (ssRNA) with Human coronavirus 229E (ssRNA)
• SARS-CoV-2 (ssRNA) with Influenza H3N2 (ssRNA)
• SARS-CoV-2 (ssRNA) with Human rhinovirus 89 (ssRNA)
The libraries then underwent capture using the Twist Respiratory
Virus Research Panel, using a 16-hour hybridization time.
We assessed capture by measuring total viral content, foldenrichment, and template coverage at different depths (Table 2).
Both templates were completely covered at 1x in each co-infection
experiment, with generally good uniformity across the template
(Table 2 and Figure 2). This was true for viruses in the same
family (such as the coronavirus 229E and SARS-CoV-2) as well
as viruses in different families (such as human rhinovirus and
human bocavirus). All the viruses in the experiment had >99%
of the bases covered at 30x or above except human bocavirus.
The relatively lower coverage at 30x and 100x depth for human
bocavirus was likely due to lower library preparation efficiency
from the ssDNA.
% ONTARGET VIRAL READS
FOLD ENRICHMENT
MEDIAN COVERAGE
% BASES AT >=30X
TEMPLATE SIZE (NT)
VIRAL
STRAIN
hBoV HRV D68 H1N1 H3N2 IBV MuV hPIV1
MeV hPIV4 229E NL63 SARS SCV-2 MERS OC43
0%
50%
100%
0
5,000
10,000
0
2,500
5,000
7,500
90.0%
97.5%
95.0%
92.5%
100.0%
0
10,000
20,000
30,000
Figure 1: Detection of viral standards from different viral families. Each Twist synthetic
viral control was spiked into 50 ng of human carrier RNA at 1,000,000 copies prior to
cDNA synthesis. For each standard, we show percent of on-target viral reads, foldenrichment above input, median coverage across the genome, the percent of bases
with at least 30x coverage, and the template length. Viral genomes are ordered by
template length (smallest to longest) among all genome segments. Full names for each
virus abbreviation used are given in Table 3.
Table 1: Viral capture at different titer points using the Twist Respiratory Virus
Research Panel. Synthetic viral standards for Influenza H1N1, Influenza H3N2, or HRV
were spiked into a background of human RNA at 100, 10,000 and 1,000,000 copies.
Expected and observed read counts from the viral template are shown, as well as
fold-enrichment from capture. Numbers represent the mean of two replicates.The full
names for each virus abbreviation are given in Table 3.
TWIST BIOSCIENCE | APPLICATION NOTE
Finally, we tested the Twist Respiratory Virus Research Panel
in a multiplex capture system. This system allows investigators
to decrease assay costs and time while maintaining equally
efficient viral capture as compared to a single-plex capture. We
demonstrate this by pooling 8 samples (with unique library indices)
at various viral titers into a single hybridization tube, creating an
8-plex capture reaction.
In Figure 3, the first graph shows comparisons using a multiplex
and single-plex hybridization reaction using 1,000,000 copies
per library, while the second graph shows a similar comparison
using 100, 10,000, and 1,000,000 copies per library. Each viral
control was captured using the Twist Standard Target Enrichment
workflow and the Twist Respiratory Virus Research Panel.
This data demonstrates that 8-plex capture gives comparable
efficient enrichment as a single plex capture when using the Twist
Respiratory Virus Research Panel.
Due to the high capture efficiency of the Twist Target Enrichment
Protocol and Twist Respiratory Virus Research Panel, extremely low
levels of contamination between samples can sometimes be seen
in the negative control data results. As a general note, to minimize
risk of cross contamination between samples and/or controls,
viral control and negative control libraries should be generated
at different times, or in physically separated lab workspaces. If
physical separation is not possible, cross contamination can be
reduced by leaving an empty well between each sample during
the library preparation process.
Table 2: Detection of simulated co-infections. Two synthetic viral standards were spiked at 10,000 copies per virus into a background of 50ng of human RNA. Percent on-target
reads, enrichment, and coverage at 1x, 30x and 100x depth are shown for both strains in each experiment. Numbers represent the mean of two replicates. Full names for each
virus abbreviation are given in Table 3.
Figure 2: Genome browser views of read density from co-infection capture experiments using synthetic SARS-CoV-2 and human rhinovirus (HRV) standards. Full names for each
virus abbreviation are given in Table 3.
GROUP VIRAL STRAIN PERCENT ON-TARGET
VIRAL READS FOLD ENRICHMENT 1X COVERAGE 30X COVERAGE 100X COVERAGE
1
HRV 2.1% 27263 100.0% 99.4% 91.4%
hBoV 0.2% 4218 100.0% 63.6% 0.0%
2
SCV-2 23.4% 73403 99.9% 99.9% 99.7%
229E 6.8% 23380 99.9% 99.7% 90.9%
3
SCV-2 21.9% 68657 99.9% 99.9% 99.7%
H3N2 3.3% 22678 100.0% 99.3% 86.6%
4
SCV-2 30.2% 94734 99.9% 99.9% 99.8%
HRV 2.2% 29070 100.0% 99.8% 93.6%
HRV/SCV-2
capture replicate 1
SARSCOV2 GENOME HRV GENOME
HRV/SCV-2
capture replicate 2
29 kb
10 kb 20 kb 30 kb
6,090 bp
1,000 bp 2,000 bp 3,000 bp 4,000 bp 5,000 bp 6,000 bp
PERCENT ONTARGET VIRAL READS PERCENT ONTARGET VIRAL READS
Single-plexed Multiplexed
H1N1
100
hBoV HRV D68 H1N1 H3N2 IBV MuV hPIV1 MeV hPIV4 229E NIL63 SARS SCV-2 MERS OC43
H1N1
10,000 H1N1
100,000 H3N2
100 H3N2
10,000 H3N2
100,000 HRV
100 HRV
10,000 HRV
100,000
0.1%
1.0%
10.0%
100.0%
0%
20%
40%
60%
80%
100%
Figure 3. A comparison of the percent on-target viral reads captured using the Twist
Synthetic Viral Controls at different viral titers, using a multiplex and single-plex
hybridization reaction. Full names for each virus abbreviation are given in Table 3.
TWIST BIOSCIENCE | APPLICATION NOTE
CATALOG # NAME ABBREVIATED NAME NUCLEIC
ACID SPECIES STORAGE
102023 Twist Synthetic SARS-CoV-2 RNA Control 2 (MN908947.3) SCV-2 ssRNA –90 to –70°C
103001 Twist Synthetic Influenza H1N1 (2009) RNA control H1N1 ssRNA –90 to –70°C
103002 Twist Synthetic Influenza H3N2 RNA control H3N2 ssRNA –90 to –70°C
103003 Twist Synthetic Influenza B RNA control IBV ssRNA –90 to –70°C
103004 Twist Synthetic Human bocavirus 1 DNA control hBoV ssDNA –90 to –70°C
103005 Twist Synthetic Human enterovirus 68 RNA control D68 ssRNA –90 to –70°C
103006 Twist Synthetic Human rhinovirus 89 RNA control HRV ssRNA –90 to –70°C
103007 Twist Synthetic Mumps virus RNA control MuV ssRNA –90 to –70°C
103008 Twist Synthetic Human parainfluenza virus 1 RNA control hPIV1 ssRNA –90 to –70°C
103009 Twist Synthetic Measles virus RNA control MeV ssRNA –90 to –70°C
103010 Twist Synthetic Human parainfluenza virus 4 RNA control hPIV4 ssRNA –90 to –70°C
103011 Twist Synthetic Human coronavirus 229E RNA control 229E ssRNA –90 to –70°C
103012 Twist Synthetic Human coronavirus NL63 RNA control NL63 ssRNA –90 to –70°C
103013 Twist Synthetic Human coronavirus OC43 RNA control OC43 ssRNA –90 to –70°C
SUMMARY
Due to their common transmission patterns, respiratory pathogens are extremely contagious and outbreaks are difficult to control.
The ability to monitor viral genome evolution and to characterize novel strains of these pathogens is crucial for containment and
epidemiological studies that may inform public health decisions. The Twist Respiratory Virus Research Panel, along with the Twist
Target Enrichment solution and Next Generation Sequencing, gives investigators the opportunity to collect meaningful data for multiple
respiratory pathogens in a single assay. These workflows are compatible with a multiplexed system, decreasing cost and time without
impacting the quality of sequencing results. The Twist Respiratory Virus Research Panel leverages a straightforward target enrichment
workflow for detection and characterization of respiratory pathogens.
MATERIALS AND METHODS
Twist synthetic RNA and DNA viral controls were spiked into a background of 50 ng human reference RNA (Agilent) with viral copy
numbers ranging from 100 to 1,000,000 copies per sample (Table 3). Co-infections were simulated by spiking multiple synthetic viral
controls into one sample. A negative control consisting solely of human reference RNA was processed in parallel. The samples were then
converted to single-stranded cDNA through random priming using NEB’s Random Primer 6 (S1230S), ProtoScript II First Strand cDNA
Synthesis Kit (E6560S). Single-stranded cDNA was then converted to dsDNA using the NEBNext Ultra II Non-Directional RNA Second
Strand Synthesis kit (E6111S). The samples were converted to Illumina TruSeq-compatible libraries using Twist Library Preparation Kit
using Enzymatic Fragmentation (PN 101059 and 100401) and Unique Dual Indices (UDI) (PN 101307).
Enrichment was performed using the Twist Respiratory Virus Research Panel (PN 103066, 103067 & 103068) and 500 ng of library
in single-plex capture reactions using a 16-hour hybridization. For multiplexing experiments, 8 libraries were pooled (187.5 ng each)
for a total of 1500 ng. Enriched libraries were sequenced with 2x75 bp paired-end reads on the Illumina NextSeq platform, using a
NextSeq500/550 High Output kit. Alignment was performed with BWA against a custom genome index comprising the human genome
(build hg38) concatenated with reference sequences for each virus in the panel. All data were downsampled to 1M mapped reads per
sample, unless otherwise noted.
REFERENCES
Martin ET, Kuypers J, Wald A, and Englund JA. Multiple versus single virus respiratory infections: viral load and clinical disease severity
in hospitalized children. Influenza Other Respir Viruses. 2012; 6(1): 71–77.
Zhang G, Hu Y, Wang H, Zhang L, Bao Y, and Zhou X. High Incidence of Multiple Viral Infections Identified in Upper Respiratory Tract
Infected Children under Three Years of Age in Shanghai, China. PLoS One. 2012; 7(9): e44568.
Table 3: Synthetic Controls used in validating the Twist Respiratory Virus Research Panel
TWIST BIOSCIENCE | APPLICATION NOTE
VIRUS NAME ACCESSION NUMBER
Human adenovirus 14 JN032132
Human adenovirus B1 NC_011203.1
Human adenovirus E NC_003266
Human adenovirus type 7 AC_000018
Human bocavirus 1 MG953830.1
Human coronavirus 229E NC_002645.1
Human coronavirus HKU1 NC_006577.2
Human coronavirus NL63 NC_005831.2
Human coronavirus OC43 NC_006213.1
Human enterovirus 68 NC_038308.1
Human metapneumovirus NC_039199.1
Human parainfluenza virus 1 NC_003461.1
Human parainfluenza virus 3 NC_001796.2
Human parainfluenza virus 4 NC_021928.1
Human rhinovirus 3 NC_038312.1
Human rhinovirus 89 NC_001617.1
Human rhinovirus C NC_009996.1
Human rubulavirus 2 (parainfluenzavirus 2) NC_003443.1
Influenza B NC_002211, NC_002204, NC_002210, NC_002209,
NC_002208, NC_002207, NC_002206, NC_002205
Influenza H1N1 (2009) NC_026432, NC_026431, NC_026434, NC_026436,
NC_026433, NC_026437, NC_026435, NC_026438
Influenza H3N2 NC_007369, NC_007373, NC_007372, NC_007371,
NC_007366, NC_007368, NC_007367, NC_007370
Measles NC_001498.1
MERS JX869059.2
Mumps NC_002200.1
Respiratory syncytial virus (A) NC_001803.1
Respiratory syncytial virus (B) NC_001781
Rubella NC_001545.2
SARS NC_004718.3
SARS-CoV-2 NC_045512.2
Table 4: Viruses Targeted in Twist Respiratory Virus Research Panel
TWIST BIOSCIENCE | APPLICATION NOTE
The following trademark representations are believed to be accurate, but are not guaranteed to be so: Agilent®, Illumina®, NEB®, NEBNext®, NextSeq®, ProtoScript®, and TruSeq®. Registered or
registration-pending names, trademarks, etc used herein, even when not specifically marked as such, are not to be considered unprotected by applicable law.
The Twist products are for research use only, and subject to additional use restrictions set forth in Twist’s Supply Terms and Conditions: www.twistbioscience.com/supply-terms-and-conditions.
CATALOG # NAME DESCRIPTION STORAGE
101059: 16 rxn
101058: 96 rxn
Twist Library Preparation EF Kit Reagents for library construction
Twist Library Preparation
EF Kit 1
5x Fragmentation Enzyme
10x Fragmentation Buffer
DNA Ligation Mix
DNA Ligation Buffer
Amplification Primers, ILMN
(Tubes 100220, 100583 are not required when used
with universal adapters)
–25 to –15°C
Twist Library Preparation Kit 2 DNA Purification Beads 2 to 8°C
100401: 16 rxn
100573: 96 rxn Twist Library Preparation Kit 2
DNA Purification Beads
(as a Standalone Product, Bead Purification
is also needed during cDNA Synthesis)
2 to 8°C
101307: 16 rxn
101308, 101309,
101310, 101311: 96 rxn
Twist Universal Adapter System -
TruSeq Compatible
Twist Universal Adapters and Twist UDI Primers,
provides unique dual-indexed combinations with 1
reaction per index pair
–25 to –15°C
103066: 2 rxn
103067: 12 rxn
103068: 96 rxn
Twist Respiratory Virus Research
Panel & One Codex Software
Custom DNA Panel for Respiratory Viral Detection
& One Codex software analysis credits –25 to –15°C
100856: 2 rxn
100578: 12 rxn
100767: 96 rxn
Twist Universal Blockers For the prevention of nonspecific capture:
Universal Blockers Blocker Solution –25 to –15°C
101262: 2 rxn
100983: 12 rxn
100984: 96 rxn
Twist Binding and Purification
Beads
For target enrichment and purification: Streptavidin
Binding Beads DNA Purification Beads 2 to 8°C
101279: 2 rxn
101025: 12 rxn
101026: 96 rxn
Twist Hybridization and
Wash Kit (2 Boxes) For target enrichment with standard hybridization:
Twist Hybridization Reagents
(Box 1 of 2)
Hybridization Mix Hybridization Enhancer
Amplification Primers –25 to –15°C
Twist Wash Buffers
(Box 2 of 2)
Binding Buffer
Wash Buffer 1
Wash Buffer 2
2 to 8°C
KEY COMPONENTS
DOC-001186 REV 2.0
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