Utilize multiple overlapping amplicons in a single tube, using a rapid, 2-hour workflow to prepare ready-to-sequence libraries. The PCR1+PCR2 workflow generates robust libraries even from low input quantities of DNA that may be subsequently quantified and normalized with conventional methods such as Qubit® or Agilent Bioanalyzer, or optionally using the included Swift Normalase reagents. Provides coverage of >99% of the SARS-CoV-2 genome from limited viral titers

1. swiftbiosci.com
Swift SARS-CoV-2 Amplicon Panel: NGS in a SNAP
June 3, 2020

2. swiftbiosci.com
Today’s Speakers
Drew McUsic, PhD
Dir. Product Management
mcusic@swiftbio.com
Kayla Peck, PhD
Sr. R&D Scientist
peck@swiftbio.com
Jon Irish, PhD
Dir. of Data Sciences
irish@swiftbio.com

3. swiftbiosci.com
Swift Bioscience’s Amplicon Technology
Accel-Amplicon
PCR1 + Ligation

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SNAP = Swift Normalase Amplicon Panel
SNAP
PCR1 + PCR2 (+ Normalase)
Accel-Amplicon
PCR1 + Ligation

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SNAP = Swift Normalase Amplicon Panel
SNAP
PCR1 + PCR2 (+ Normalase)
SNAP features:
• More robust yields
• More flexible indexing options
• Libraries can be analyzed by
conventional means
• Can seamlessly incorporate
Normalase

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SARS-CoV-2 NGS
Kim et al., 2020

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• Variable sample type
• Variable sample quality
• Low viral titers in samples
• Variable RNA extraction methods
• Contamination by host gRNA
Challenges for SARS-CoV-2 NGS

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Workflow options for SARS-CoV-2 NGS
Ribodepletion
RNA-Seq
Library Prep
cDNA
synthesis
Amplicon
Panel
1 | Not Targeted 2 | Targeted

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1. Synthesize cDNA
2. Make libraries with SNAP
3. Produce an equimolar pool with
Normalase (optional)
4. Sequence (Illumina-compatible)
5. Data analysis
SNAP SARS-CoV-2 Workflow
1 hour
2 hours
1 hour
(variable)
minutes per sample

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Method Recommended Kit
Reverse Transcription to
generate 1st-strand cDNA
Superscript® IV First-Strand Synthesis System
(ThermoFisher Cat. No. 18091050/18091200)
1. Synthesize cDNA
Reverse Transcription
First-strand cDNA Second-strand cDNA
Swift’s protocol provides workflow integration for the output of this module into the SNAP library prep.
Compatible RT modules:
• Random primers
• Two-step RT-PCR
• Processivity >1 kb

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Swift technology allows for a
2-hour single-tube assay
Sophisticated primer design
targeting the full-length
SARS-CoV-2 genome
2. Make libraries with SNAP

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341 amplicons
116 – 255 bp (mean = 150 bp)
29,313 / 29,903 bases covered
2. Make libraries with SNAP
Wuhan isolate (NC_045512.2)

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Feature SNAP Kit Specification
Design Coverage 98% (29,313 of 29,903 total bases in NC_045512.2)
Panel Information 341 amplicons, sized 116-255 bp (average 150 bp)
Input Material
1st or 2nd strand cDNA
Minimum 10-100+ viral copies (qRT-PCR Ct value 30-40)
Time
2 hours cDNA-to-Library or
3 hours cDNA-to-Normalized-Library-Pool
Components Provided
Target-specific multiplex primer pool • PCR and library prep reagents
Swift Normalase • Combinatorial Dual Indexed Adapters
Note: kits do not include RT module or magnetic beads
Multiplexing Capability Up to 384 CDI • Inquire for custom indexing and UDIs
Recommended Depth for
high titer samples
<10K reads/library (+/- detection); <50K reads per library (variant calling)
Recommended Depth for
low titer samples
<50K reads/library (+/- detection); <1M reads per library (variant calling)
2. Make libraries with SNAP

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3. Produce an equimolar pool with
Normalase (optional)

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3. Produce an equimolar pool with
Normalase (optional)
Library amplification and
conditioning to > 12 nM
Enzymatic equal molar
normalization
Enzymatic selection
of 4 nM fraction
Equal
Molar Pool
SNAP
Indexing
PCR

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4. Sequence (Illumina compatible)
Libraries Per Sequencing Run
MiSeq® MiniSeq®
Application v2 Nano v2 Micro v2 v3
Mid-
Output
High-
Output
+/- Detection 40 160 600 1000 320 1000
Variant
Calling
2 8 30 50 16 50
* Note: custom indexing primers are required to multiplex >384 per run. Please inquire
for compatibility with your primers, or to order custom Normalase indexing primers.
• 2 x 150 PE
• No PhiX required (for most instruments)
• 50,000+ reads per library are recommended for +/- detection
• <1M reads per library are recommended for variant calling
• Examples of number of libraries to achieve this depth per sequencing run
for low viral titer samples:

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5. Data Analysis
Pipeline features:
• Open-source tools
• Dockerized data analysis
workflow script available
• Demo data available
Variant calling (GATK Haplotype Caller)
On-target and coverage metrics calculation and reporting
Primer trimming (Primerclip)
Alignment (BWA MEM)
Adapter trimming and filtering out of reads < 30 bases
long (Trimmomatic)

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SNAP SARS-CoV-2 Data
SARS-CoV-2 Control RNA
6 x 5 kb fragments
Twist 102024
10 – 106 viral genome copies
+
50 ng Universal Human Reference RNA
Wuhan-Hu-1

19. swiftbiosci.com
SNAP SARS-CoV-2 Data
SARS-CoV-2 Control RNA
6 x 5 kb fragments
Twist 102024
10 – 106 viral genome copies
+
50 ng Universal Human Reference RNA
SARS-CoV-2 Cell Lysate
Gamma-irradiated full-length RNA
BEI NR-52287
1 – 1:103 dilution series
𝛾⚡️
USA-WA1/2020Wuhan-Hu-1

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SNAP SARS-CoV-2 Data
Sequenced on a MiniSeq with 2 x 150 PE and downsampled to 50K or 1M reads
Detection down to
10 viral copies
10X coverage
at 102 viral copies
(1M reads) or 103
copies (50K reads)
SARS-CoV-2 Control RNA

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SNAP SARS-CoV-2 Data
Sequenced on a MiniSeq with 2 x 150 PE and downsampled to 50K or 1M reads
10X coverage
down to ~25
viral copies
(as estimated
by qPCR)
SARS-CoV-2 Cell Lysate
𝛾⚡️

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SNAP SARS-CoV-2 Data
SARS-CoV-2 Cell Lysate
Confident detection of known mutations
𝛾⚡️
Sequenced on a MiSeq with 2 x 150 PE and downsampled to 1M reads
USA-WA1/2020 (MT985325.1)
C8782T C18060T T28144C

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Collaborators at NYU Langone
• Nasopharyngeal swabs
• Ct values ranging from 16 to 42
(via qPCR targeting the Orf1ab/E)
• RT module = Superscript IV
• Low-pass sequencing (50K reads)
SARS-CoV-2 SNAP Performance on
Clinical Specimen

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A nasopharyngeal swab specimen processed with the Swift SNAP SARS-CoV-2 Kit and sequenced with
Illumina® MiSeq® to 50,000 reads demonstrates coverage across the 29.9 kb genome. The
A23403G/D614G mutation was detected in this sample, which affects the viral spike protein and is a key
variant of interest for its hypothesized role in transmissibility of COVID-19.
Complete Coverage from Clinical Specimen
A23403GCt value = 36.7

25. swiftbiosci.com
Mutation Profiling: NYU Clinical Specimens
A23403G
“Swift has been a valued research partner,
and we look forward to working with them
to continually improve the ability of
amplicon-based methods to achieve
greater coverage in fewer reads, which
would enable us to achieve good genome
coverage for low viral load samples.”
– Adriana Heguy, PhD, Professor of
Pathology at NYU Langone Health,
NYU Grossman School of Medicine
A total of 29 nasopharyngeal swab specimens with qRT-PCR Ct values ranging from 16 to 42 were processed with the
Swift SNAP SARS-CoV-2 Kit by NYU Langone Health and sequenced with Illumina® MiSeq® to 50,000 reads. Variants
were called using GATK Haplotype Caller. Variants with allele fractions of 0.5 or greater are shown.

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nextstrain.org
A23403G (D614G)

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• Fast, single-tube workflow
• >98% genome coverage from >10 viral genome copies
• Confidently detect nucleotide variants
• Cost-effective SARS-CoV-2 NGS library prep
• Multiplexing capabilities offer scalability and cost-effective sequencing

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SNAP Ordering Information
PRODUCT INFO US LIST PRICE
Product Component Product Name Cat. No. Rxns Kit Per Rxn
Amplicon primers SARS-CoV-2 Panel (primers only) COVG1-96 96 $960.00 $10.00
SNAP Core
Swift Normalase Amplicon Protocol SNAP Core
(96 rxns, no indexing)
SN-5X296 96 $2,400.00 $25.00
Indexing Primers
SNAP Combinatorial Dual Index Primer Kit
(Set 1A, 96 rxns)
SN-5S1A96 96 $600.00 $6.25
SNAP Combinatorial Dual Index Primer Kit
(Set 1B, 96 rxns)
SN-5S1B96 96 $600.00 $6.25
SNAP Combinatorial Dual Index Primer Kit
(Set 2A, 96 rxns)
SN-5S2A96 96 $600.00 $6.25
SNAP Combinatorial Dual Index Primer Kit
(Set 2B, 96 rxns)
SN-5S2B96 96 $600.00 $6.25

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Thank You!
For further inquiries, contact us at techsupport@swiftbio.com

30. swiftbiosci.com
SARS-CoV-2 Workflow Selection Guide
Swift RNA Library Kit
+
Arbor Bio myBaits
Swift Amplicon
SARS-CoV-2 Research Panel
Swift Normalase Amplicon
SARS-CoV-2 Panel (SNAP)
Viral Copy Number 100-1000+ 1000+ 10+
Input Material
Total RNA
> 70-100 nt
cDNA
> 255 bp
cDNA
> 255 bp
Coverage of SARS-CoV-2
(NC_045512.2)
100% 98% 98%
Enrichment Method Hybridization Capture Single Tube Multiplex PCR Single Tube Multiplex PCR
Workflow RNA Library + Hyb Capture PCR + Adapter Ligation PCR1 + PCR2
Library Quantification Qubit, Bioanalyzer, or Normalase qPCR required Qubit, Bioanalyzer, or Normalase
Time
(RNA to NGS Library)
2 days 3 hours 4 hours
Components Included
RT module
Library prep, polymerase, up to 768 CDI
adapters or 96 UDI plate primers for ILMN,
flexible to your own 384+ UDI primers
Capture probes, adapter blockers, wash
Primer pool, polymerase, up to 384
CDI adapters for ILMN
Primer pool, polymerase, up to
384 CDI adapters for ILMN and
Normalase, flexible to your own
384+ UDI primers
Recommended Reads
per Library
50,000 (+/- detection)
1M (variant calling)
50,000 (+/- detection)
1M (variant calling)
50,000 (+/- detection)
1M (variant calling)

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How to make the most of your RNA sample?
Can data be improved if ribodepletion is performed prior to cDNA synthesis?
Lexogen Ribocop performed on 50 ng UHR + SARS-CoV-2 genome copies.
Sequenced on a MiniSeq with 2 x 150 PE and downsampled to 50K reads