Thermo Fisher Scientific AmpliSeq Reference Manual
Ion AmpliSeq™ SARS‑CoV‑2 Research Panel
Instructions for use on an Ion GeneStudio™ S5 Series System
Pub. No. MAN0019277 Rev. B.0
80 samples per sequencing run), fast turnaround time, and
WARNING! Read the Safety Data Sheets (SDSs)
and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves. Safety Data
Sheets (SDSs) are available from thermofisher.com/
support.
minimal hands‑on time in SARS‑CoV‑2 research studies.
Ordering instructions
To order the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel, follow
these steps.
QUICK REFERENCE
This quick reference provides guidelines and instructions for using
the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel to prepare Ion
AmpliSeq™ libraries from SARS-CoV-2 samples, sequence the
libraries on an Ion GeneStudio™ S5 System, Ion GeneStudio™ S5
Plus System, or Ion GeneStudio™ S5 Prime System, then analyze
the sequencing results with Torrent Suite™ Software.
Product description .............................. 1
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Ordering instructions ............................. 1
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Isolate and quantify viral RNA ...................... 1
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Prepare libraries with the Ion AmpliSeq™ SARS‑CoV‑2
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Research Panel .................................. 3
Install plugins and import panel files ................. 5
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Create a Planned Run for the Ion AmpliSeq™ SARS‑CoV‑2
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Research Panel .................................. 6
Prepare template and load chips on the Ion Chef
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Instrument ..................................... 7
Start a sequencing run ............................ 7
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Analyze sequencing data from SARS‑CoV‑2 samples .... 7
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(Optional) Re-run the SARS‑CoV‑2 plugins after the
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sequencing run is complete ........................ 8
Guidelines for sample quality, viral copy number, and variant
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calling ......................................... 9
Limited product warranty .......................... 9
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1. Go to AmpliSeq.com and sign in, or register for a new
account.
2. In the navigation bar, go to the Fixed Panels dropdown
menu, then select Community Panels.
3. In the Research Area navigation pane on the left side of the
screen, select the Infectious Disease checkbox to filter the
list. Find the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel in
the filtered list, then click Preview Order.
Note: Alternatively, enter SARS-CoV-2 in the search field at
the top of the screen to find the panel page.
4. In the Order options window, select GeneStudio in the
Choose instrument section, then click Next.
5. In the Format option window, select Manual or Ion Chef,
then click Next.
6. In the Order summary window, review the order, then
select Proceed to cart. As an option, select the List
recommended consumables checkbox, then click Preview
to see a list of additional products that you may need. Select
the items, then click Add all to cart.
7. Click Proceed to checkout to complete the order at
thermofisher.com.
Unless otherwise indicated, all other materials listed in this
quick reference are available at thermofisher.com.
Product description
The Ion AmpliSeq™ SARS‑CoV‑2 Research Panel consists of
two 5X primer pools that target 237 amplicons specific to the
SARS‑CoV‑2 (the virus that causes COVID-19) and 5 human
expression controls. With an amplicon length range of 125–
275 bp, the panel provides >99% coverage of the SARS‑CoV‑2
genome (~30 kb), and covers all potential serotypes. The panel
is a community Ion AmpliSeq™ panel available for order through
AmpliSeq.com.
When used in conjunction with the Ion Chef™ System and an Ion
GeneStudio™ S5 Series System, the Ion AmpliSeq™ SARS‑CoV‑2
Research Panel oers high sensitivity, high throughput (up to
Isolate and quantify viral RNA
Guidelines for RNA isolation and sample normalization
For Research Use Only. Not for use in diagnostic procedures.
• A sample containing as little as 20 copies of viral RNA
(10 copies per target amplification reaction) can be used
to prepare an Ion AmpliSeq™ SARS‑CoV‑2 Research Panel
library. For optimal results, we recommend a viral copy
number in the 200 to 200,000 range, or an amount of
total RNA between 1–10 ng. For more information, see
“Guidelines for sample quality, viral copy number, and variant
calling” on page 9.
• The amount of viral RNA among samples should be
approximately equivalent so that the target amplification
conditions you select are optimal for all samples.
• See “Recommended materials for isolation and
quantification” on page 2 for recommended Thermo Fisher
Scientific kits and master mix.
Recommended materials for isolation and quantification
We recommend the following Thermo Fisher Scientific kits and
master mix for the isolation and quantification of SARS‑CoV‑2
RNA.
Item Cat. No.
Isolation
MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit A42352 or
A48310
Quantify by real-time PCR
To determine the optimal number of target amplification cycles
to use in library preparation, quantify viral RNA copy number
in your SARS‑CoV‑2 samples using the TaqMan™ 2019-nCoV
Assay Kit v1, the TaqMan™ 2019-nCoV Control Kit v1, and the
TaqPath™ 1-Step RT-qPCR Master Mix, CG. For more information
on reaction set up, see the TaqMan™ 2019-nCoV Assay Kit v1
Product Information Sheet (Pub. No. MAN0019096).
If you are unable to quantify viral RNA copy number in your
samples, start with 16 target amplification cycles for manual
library preparation, or 17 target amplification cycles for library
preparation on the Ion Chef™ Instrument, then optimize if
needed. For more information, see “Prepare libraries manually” on
page 4, or “Prepare libraries on the Ion Chef™ Instrument” on
page 5.
Quantification
TaqMan™ 2019-nCoV Assay Kit v1 A47532
TaqMan™ 2019-nCoV Control Kit v1 A47533
TaqPath™ 1-Step RT-qPCR Master Mix, CG A15299 or
A15300
Additional positive controls are available at the BEI Resources
Repository at https://www.beiresources.org, or through other
commercial providers.
Isolate viral RNA
The MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit can be
used in either a manual or a high-throughput automated mode
using the MagMAX™ Express Magnetic Particle Processor or
KingFisher™ Purification System. Follow these basic steps to
isolate SARS-CoV-2 RNA using the MagMAX™ Viral/Pathogen
Nucleic Acid Isolation Kit (manual extraction). For detailed
information on how to use the kit, and required materials not
supplied, see the following user guides, which are available for
download at thermofisher.com.
• MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (manual
extraction) User Guide (Pub. No. MAN0018072) or the
• MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit
(automated extraction) User Guide (Pub. No. MAN0018073)
1. For each 2019-nCoV assay (N Protein, S Protein, and
ORF1ab), combine the following components per reaction
to make a reaction mix for the total number of reactions, plus
10% overage.
Component
TaqPath™ 1-Step RT-qPCR Master Mix,
CG (4X)
2019-nCoV assay (20X; N Protein,
S Protein, or ORF1ab)
RNAse P assay (20X) 1.25 µL
RT-PCR Grade Water 11.25 µL
Total reaction mix volume 20.0 µL
Volume per
reaction
6.25 µL
1.25 µL
2. For each reaction, combine the following components in a
MicroAmp™ Optical 96-Well Reaction Plate (0.2‑mL) well.
Component
Reaction mix (from step 1) 20.0 µL
• Nucleic acid research sample or
• 1 µL 2019-nCoV Control v1 + 4 µL
RT-PCR Grade Water or
• NTC
Volume per well
5.0 µL
1. Digest 200–400 μL of each sample with Proteinase K in
a deep-well 96-well plate, then bind RNA to Nucleic Acid
Binding Beads.
2. Wash the Nucleic Acid Binding Beads.
3. Elute the RNA from the Nucleic Acid Binding Beads.
Use 1–10 ng total RNA in library target amplification reactions.
We recommend quantifying viral copy number by real-time PCR,
described in “Quantify by real-time PCR”.
2 Ion AmpliSeq
™
SARS‑CoV‑2 Research Panel on an Ion GeneStudio™ S5 Series System Quick Reference
Total reaction volume 25.0 µL
3. Set up and run the reactions on a real-time PCR instrument
using the following settings:
• Analysis method: Comparative C
t
Note: You must use Comparative Ct to analyze 2019-
nCoV assay data using QuantStudio™ Design and
Analysis Software v2 and ExpressionSuite™ Software.
• Cycling mode: Standard
• Thermal cycling protocol:
Stage Step Temperature Time
Hold
Hold
Hold Activation
UNG
incubation
Reverse
transcription
[2]
[1]
25°C 2 minutes
50°C 15 minutes
95°C 2 minutes
Denaturation 95°C 3 seconds
Cycling
(40 cycles)
[1]
Heat-labile UNG in TaqPath™ 1-Step RT-qPCR Master Mix, CG is
completely inactivated during the first ramp to 95°C.
[2]
Required for RT inactivation, first denaturation, and activation of the DNA
polymerase.
Anneal/
Extension
60°C 30 seconds
Use the Ct result for each 2019-nCoV assay to estimate copy
number in your sample. See example data in the table on
page 3.
Copy number determination by qPCR
Note: If your qPCR data give a dierent relationship between
Ct and copy number, this is likely a result of dierences in the
baseline or threshold selected. Determine the copy number of a
sample according to the known copy number in control reactions.
Copy number determination of SARS-CoV-2 with TaqMan
2019-nCoV Assay Kit v1 and TaqMan™ 2019-nCoV Control Kit
v1
Ct of N Protein
Ct of S Protein Ct of ORF1ab
qPCR reaction
34 36 37 5
33 35 36 10
32 34 35 20
31 33 34 39
30 32 33 78
29 31 32 156
28 30 31 312
27 29 30 625
26 28 29 1,250
25 27 28 2,500
24 26 27 5,000
23 25 26 10,000
22 24 25 20,000
21 23 24 40,000
20 22 23 80,000
19 21 22 160,000
18 20 21 320,000
17 19 20 640,000
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Copies in
Prepare libraries with the Ion AmpliSeq
™
SARS‑CoV‑2 Research Panel
After reverse transcription, two options are available for preparing
libraries using the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel:
• manual preparation using the Ion AmpliSeq™ Library Kit Plus
(Cat. Nos. 4488990, A35907, or A38875)—follow steps listed
in “Prepare libraries manually” on page 4
• automated preparation on the Ion Chef™ Instrument using the
Ion AmpliSeq™ Kit for Chef DL8 (Cat. No. A29024)—follow
steps listed in “Prepare libraries on the Ion Chef™ Instrument”
on page 5
For detailed information on using these kits, see the Ion
AmpliSeq™ Library Kit Plus User Guide (Pub. No. MAN0017003),
or the Ion AmpliSeq™ Library Preparation on the Ion Chef™ System
User Guide (Pub. No. MAN0013432), available for download at
thermofisher.com.
Reverse transcribe RNA with the SuperScript™ VILO
cDNA Synthesis Kit
Use the SuperScript™ VILO™ cDNA Synthesis Kit (Cat. No.
11754050) to reverse transcribe SARS‑CoV‑2 RNA for both
manual library preparation and automated library preparation on
the Ion Chef™ Instrument.
1. Warm the 5X VILO™ Reaction Mix to room temperature for at
least 20 minutes, then vortex to mix.
Note: If there is visible precipitate in the 5X VILO™ Reaction
Mix, vortex further until the mix is completely dissolved.
2. Combine the following components per reaction to make
a master mix for the total number of reactions, plus
10% overage. Up to 8 sample libraries can be prepared in
one Ion Chef™ Instrument run.
Volume
Component
5X VILO™ Reaction Mix (blue
cap)
10X SuperScript III™ Enzyme
Blend (black cap)
Total volume per well 3 µL 4.5 µL
3. For each reaction, combine the following components in a
MicroAmp™ Optical 96-Well Reaction Plate (0.2‑mL) well for
manual library preparation, or wells A1 to H1 of an IonCode
96 Well PCR Plate from the Ion AmpliSeq™ Kit for Chef DL8
for library preparation on the Ion Chef™ Instrument.
Component
Master mix (from step 2) 3 µL 4.5 µL
RNA (1–10 ng) 7 µL 10.5 µL
Total volume per well 10 µL 15 µL
Manual
2 µL 3 µL
1 µL 1.5 µL
Volume
Manual
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Ion Chef
Instrument
Ion Chef
Instrument
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Ion AmpliSeq™ SARS‑CoV‑2 Research Panel on an Ion GeneStudio™ S5 Series System Quick Reference 3
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