EasySeq™

SARS-CoV-2 (novel coronavirus)

Whole Genome Sequencing
NGS library prep by Reverse Complement PCR

Version: RC-COVID096-v1.9
Revision date: March 2, 2021

Protocol

-1-
Version: RC-COVID096-v1.9
Product and Company Information

Product name: EasySeq™ SARS-CoV-2 Whole Genome NGS Sequencing kit

Product Info: www.nimagen.com/covid19
Product use: Research Use Only
Company: NimaGen BV
Lagelandseweg 56
6545 CG Nijmegen, The Netherlands
Telephone: +31 (0)24 820 02 41
Email: [email protected]

-2-
Version: RC-COVID096-v1.9
Product Use
Multiplex Amplicon based NGS Library preparation for sequencing the genome of SARS-CoV-2 virus. To be used
to detect mutations, monitoring viral populations for epidemiology and outbreak events. This Reverse-
Complement PCR based library prep contains all reagents to generate Illumina® compatible libraries in a
simple, sensitive and robust method for fast and cost-effective WGS of the viral genome.

Kit Content.
Note: One complete kit consists of three part numbers, to be ordered separately:

NimaGen Part# RC-COV096 Box # Content

SARS CoV2 WGS Panel A (RC-PCR probe mix, black cap) 1 (Frozen) Tube 24 µL
SARS CoV2 WGS Panel B (RC-PCR probe mix, red cap) 1 (Frozen) Tube 24 µL
RC-PCR probe dilution mix (blue cap) 1 (Frozen) Tube 500 µL
AmpliClean Bead solution* 2 (Cooled) Tube 1.7 mL
RC-PCR Low-TE Buffer (yellow cap) 2 (Cooled) Tubes 2 x 1100 µL

NimaGen Part# MMHS-25 Content

PCR 2x Hotstart HiFi Mastermix, compatible with RC-PCR
1 (Frozen) Tubes 2 x 1100 µL
(white cap). p/n MMHS-25

Choose one of the index plates of choice

NimaGen Part# IDX96-U0xD Content

2 x 96-well plates
2 x IDX Index Primer Plates*. Choose one of the 5 available
(pre-spotted with
Index Plate (U01D, U02D, U03D, U05D, U06D) with 96 1 (Frozen)
4 µL Index Primers
Unique Dual, 10 bp Indexes for Illumina®. p/n IDX96-U0xD
per well)

*IDX plates are semi-skirted, ABI stye PCR Plates, dividable per 24 rxns, containing 4 µL of Unique Dual Index
primer pairs in each well, ready to use. Index Sequences can be downloaded from the product page,
section “downloads”

-3-
Version: RC-COVID096-v1.9
Needed, but not included
Description Vendor

Adjustable Pipette Set (P10, P20, P200, P1000) Multiple Vendors

LunaScript (p/n E3010L) cDNA synthesis kit (download protocol) New England Biolabs
Agarose Gel system, TapeStation, Bioanalyzer Instrument, or
Agilent® or other
equivalent, incl. consumables
Ethanol absolute, mol. biol. grade Multiple Vendors
General plasticware, DNAse free (1.5mL tubes, pipette tips
Multiple Vendors
w/filter)
Ice or tabletop cooling block Multiple Vendors
Illumina® NGS Sequencing instrument (iSeq, MiSeq, MiniSeq,
NextSeq) Illumina®
Illumina® Sequencing Reagent kit (min. 2x150 bp run) Illumina®

Mini Spinner for 1.5 mL tubes Multiple Vendors

Plate Spinner for 96-well plates Multiple Vendors

Magnetic Stand for 1.5 mL Eppendorf tubes Multiple Vendors

PCR Grade Water Multiple Vendors

Qubit™ Fluorometer including dsDNA High Sensitivity kit Thermofisher®

Thermocycler with heated lid, (0.2 mL standard PCR tubes),
compatible with semi-skirted ABI style PCR plates and option for Multiple Vendors
ramp rate programming.
Example: Applied Biosystems™ Veriti™, SimpliAmp or MiniAmp Thermal Cycler
The next three items are only necessary when sequencing in-house:
(not needed in case of sending samples to a core facility)

NaOH solution (2N) Multiple Vendors

Tris/HCl (200 mM), pH 7 Multiple Vendors

Low TE (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA) Multiple Vendors

-4-
Version: RC-COVID096-v1.9
General precautions

Use Pre-PCR environment for setting up the RC-PCR reaction. Pooling, cleaning and library
preparation should be performed in a Post-PCR environment

1. Thermocycling program

Temp: Duration: Ramping rate: (from previous step) Cycles

98°C 2 minutes N/A 1x

98°C 10 seconds Max

80°C 1 second Max
1x
58°C 10 minutes 0.1°C/sec (or 2% of max)
72°C 1 minute Max

98°C 10 seconds Max

80°C 1 second Max
2x
62°C 90 minutes 0.1°C/sec (or 2% of max)
72°C 1 minute Max

95°C 10 seconds Max

80°C 1 second Max
40 x
62°C 2 minutes 0.5°C/sec (or 10% of max)
72°C 1 minute Max
Heated lid at 105°C
Depending on the instrument, this protocol takes 6-7 hours to complete

NOTE: For Applied Biosystems 96-well (0.2 mL) Thermal Cyclers Veriti, SimpliAmp, MiniAmp, download the
method files from the product page (section “downloads”), copy to a USB stick and import directly into your
cycler.

-5-
Version: RC-COVID096-v1.9
2. Reverse Complement PCR

The target specific RC-probes will be transformed into the functional, tailed and indexed PCR primers, followed by
multiplex cDNA amplification of the target regions. Click here for example protocols to convert extracted RNA to cDNA.
2.1 Thaw on ice:
- SARS CoV2 WGS Panel A probe mix (Black Cap)
- SARS CoV2 WGS Panel B probe mix (Red cap)
- RC-PCR probe dilution buffer (Blue cap)
- PCR 2x Hotstart HiFi Mastermix (White cap)
Note: The 2x mastermix contains isostabilizers and may not freeze completely, even when stored at -15°C to -25°C. The
mastermix may contain precipitates when thawed at +2°C to +8°C. Always ensure that the mastermix is fully thawed and
thoroughly mixed before use.

2.2. Take the two IDX PCR plates and cut off the number of strips needed from both plates. Mark
the plates with “A” and “B”.
Note: Register the indexes used (IDX set / and well position for each sample). Download the index details for setting up
Illumina® samplesheets at the download section of https://www.nimagen.com/covid19

Note: For each sample, two PCR reactions are needed (pool A and pool B). Use always the same strip and well position for
the same sample, in order to have identical indexes for the sample in both pools. Example: For setting up 24 samples, cut
off strip 1-3 from both IDX plates and store the remaining of the plates immediately at -20°C.

2.3. Prepare in a fresh 1.5 mL Eppendorf tube the Probe-Polymerase premix A, by combining and
mixing:
- 0.2 µL panel A probe mix per reaction (black cap)
- 0.8 µL RC-PCR Probe dilution buffer per reaction (blue cap)
- 10 µL RC-PCR 2x mastermix per reaction (white cap)

Prepare in a fresh 1.5 mL Eppendorf tube the Probe-Polymerase premix B, by combining and
mixing:
- 0.2 µL panel B probe mix per reaction (red cap)
- 0.8 µL RC-PCR probe dilution buffer per reaction (blue cap)
- 10 µL RC-PCR 2x mastermix per reaction (white cap)

Example: 48 samples + 10% extra volume*

- Tube A:
▪ 10.56 µL Panel A probe mix (black cap)
▪ 42.24 µL RC-PCR probe dilution buffer (blue cap)
▪ 528 µL RC-PCR 2x mastermix (white cap)
- Tube B:
▪ 10.56 µL Panel A probe mix (red cap)
▪ 42.24 µL RC-PCR probe dilution buffer (blue cap)
▪ 528 µL RC-PCR 2x mastermix (white cap)

* It is recommended to allow for a 10% extra when preparing the mastermix to correct for pipetting loss.
The kit contains extra reagent for this.

-6-
Version: RC-COVID096-v1.9
 -7-
Version: RC-COVID096-v1.9
 2.4. Quick-spin the PCR plates (A and B), and remove seals carefully.
2.5. Add to each tube of plate A: 11 µL of the Probe-Polymerase premix A.
2.6. Add to each tube of plate B: 11 µL of the Probe-Polymerase premix B.
2.7. Add to the same well position of both plate/strips A and B: 5 µL cDNA.
Example: Add 5 µL of cDNA from patient 1 to both wells A1 of plate A and B → total cDNA per sample needed
is 10 µL.

2.8. Close the tube strips carefully with caps (included in the kit) and mix by flicking.
2.9. Short spin.
2.10. Start the RC-PCR program in the thermal cycler(s) and place samples in the cycler when the
block is between 60°C and 98°C, close the lid.
Note: When running >48 samples in a run, two PCR cyclers are needed.

Safe stopping point after RC-PCR. Store at 4°C for max. 48H

The samples have now been amplified and tagged with sample specific indexes and sequencing tails. From this point, PCR
products will be pooled together in two tubes, as their corresponding pool A and B, purified by a “2x” AmpliClean
purification to remove primers, primer dimers and salts. In order to decrease read depth variation between samples with
low and high viral loads, it is recommended to follow a pooling strategy based on the Ct values from the Real-Time PCR
assay the samples were detected Covid19 positive with (see table at 3.3).

3. Pool, Purify and Sequence

Note: Before pooling, optionally check 3 µL of the unpurified PCR products on agarose (2%).

3.1. Bring the AmpliCleanTM beads solution (Brown Cap) to Room Temperature.
3.2. Mark 2 x 1.5 mL Eppendorf tubes with resp. “A” and “B”.
3.3. Create, in the tubes A and B, two pools, by combining RC-PCR products from all the wells in
each of the two plates (except negative controls), according to the table below.

Ct value from qPCR RC-PCR volume

< 20 (very high viral load) 2 µL
20-24 (high viral load) 4 µL
25-28 (medium viral load) 8 µL
> 28 (low viral load) 16 µL
Note: Keep pool A and B separated during the complete cleanup procedure in two tubes.
Note: If Ct values are unknow, pool 5 µL of each PCR product.

3.4. Mix well and transfer 40 µL of both pools to two new 1.5 mL Eppendorf tubes
(marked “A2” / “B2”).

3.5. Add 60 µL RC-PCR Low TE buffer (yellow cap) to both tubes A2 and B2 (total volumes are
now 100 µL each).

-8-
Version: RC-COVID096-v1.9
 3.6. Double AmpliClean™ beads purification:
a. Vortex beads thoroughly to resuspend.
b. Add 85 µL beads solution to the two 100 µL pools A2 and B2 (from step 3.5) and mix
well immediately by pipetting up and down 5 times.
c. Incubate for 5 minutes, off magnet.
d. Place both tubes on magnet for 3 minutes or for the solutions to be fully cleared.
e. Remove and discard liquid carefully without disturbing the beads.
f. Add 200 µL (freshly prepared) 75% ethanol, without disturbing the beads.
g. Wait for 1 minute.
h. Repeat steps e., f. and g. for a second ethanol wash step
i. Carefully remove all liquid without leaving traces of ethanol. (Optionally a quick spin
can be performed, then place tube back on magnet and remove excess ethanol)
j. Dry with open cap for 2-3 minutes at Room Temperature. Do not over-dry.
k. On Magnet: Add 110 µL RC-PCR Low TE buffer (Yellow cap).
l. Off Magnet: Re-suspend the beads by pipetting up and down or flicking.
m. Incubate for 2 minutes, off magnet.
n. Put on magnet and wait for 3-5 minutes or for the solution to be fully cleared.
o. Carefully bring 100 µL of the clear solution to two new 1.5 ml Eppendorf tubes (marked
“A3” and “B3”) making sure not to transfer any of the beads.
p. Add 85 µL resuspended beads solution to the two 100 µL pools A3 and B3 and mix well
immediately by pipetting up and down 5 times
q. Incubate for 5 minutes, off magnet.
r. Place both tubes on magnet for 3 minutes or for the solutions to be fully cleared.
s. Remove and discard liquid carefully without disturbing the beads.
t. Add 200 µL (freshly prepared) 75% ethanol, without disturbing the beads.
u. Wait for 1 minute.
v. Repeat steps s., t. and u. for a second ethanol wash step
w. Carefully remove all liquid without leaving traces of ethanol. (Optionally a quick spin
can be performed, then place tube back on magnet and remove excess ethanol)
x. Dry with open cap for 2-3 minutes at Room Temperature. Do not over-dry.

3.7. Elution
a. On Magnet: Add 50 µL RC-PCR Low TE buffer to the two tubes A3 and B3
b. Off Magnet: Re-suspend the beads by flicking or short vortexing.
c. Incubate for 2 minutes, off magnet.
d. Put on magnet and wait for 3-5 minutes or for the solutions to be fully cleared.
e. Carefully bring 40 µL of the clear solution to two new 1.5 ml Eppendorf tubes (marked
A4 and B4) making sure not to transfer any of the beads.

4. Safe stopping point after 2nd clean-up. Store at -20°C if not continuing same day.

-9-
Version: RC-COVID096-v1.9
 Libraries are now ready for quantification and qualification.

3.6 Determine final concentration of both A4 and B4 by a duplo Qubit (HS) measurement
according to manufacturer’s manual. Use the dedicated SARS-CoV-2 WGS calculator at the
download section of www.nimagen.com/covid19

Step 3Step 3.6: Download the SARS-CoV-2 WGS

calculator and just fill in (at the
green fields, step 1.) the
concentration of both A4 and B4, in
ng/µL. The sheet automatically
calculates the pipetting schemes for
creating 2 nM libraries (step 2),
followed by the pipetting scheme
for the denaturation (step 3) and
creating the final library for the
different Illumina instruments / kits
(step 4).

3.7 Verify library on TapeStation or Bioanalyzer, according to the manufacturer’s protocol. If

needed, dilute pool. Example: For TapeStation High Sensitivity kit, dilute to ~2 ng/µL

Example of a clean library on TapeStation:

3.8 Perform Sequencing on an Illumina® NGS platform, according to the manufacturer’s manual.
Appendix 1 outlines the detailed Illumina® NGS protocols.

NOTE: Download Index Sequences from www.nimagen.com/covid19, section “downloads” to create a

sample sheet. For technical assistance contact our technical support at [email protected].

- 10 -
Version: RC-COVID096-v1.9
Illumina® Systems Reference Guides

- iSeq100 System Guide

- Miseq System Guide
- Miseq Denature and Dilute Libraries Guide
- MiniSeq System Guide
- MiniSeq Denature and Dilute Libraries Guide
- Nextseq 550 System Guide
- NextSeq System Denature and Dilute Libraries Guide
- Illumina® experiment manager

- 11 -
Version: RC-COVID096-v1.9
 Version change Date
V1.5 -> v1.6 Miseq v2 loading changed from 5.5 to 9pM 19jan2021, WvdV
MiSeq v3 loading changed from 10 to 15pM
EBT buffer → LowTE
V1.6 -> v1.7 D: iSeq100 kit 20jan2021, wvdv
Save stopping points added
V1.7 -> v1.8 Reverse Transcription kit recommendation, safe 19feb2021, jth
stopping points info, tube labelling
V1.8 -> v1.9 safe stopping points info, tube labelling 02mar2021, jth

Reference:

Wolters et al., 2020: “Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse
Complement PCR enables fast and accurate outbreak analysis”.
doi:xhttps://doi.org/10.1101/2020.10.29.360578

Legal Notice:

RC-PCR is patent protected (PCT/GB2016/050558, WO2016146968A1) and exclusively licensed to

NimaGen B.V. Nijmegen

Published by:
NimaGen B.V.
Lagelandseweg 56
NL-6545CG Nijmegen
The Netherlands
www.nimagen.com
© 2021 NimaGen
All rights reserved.

- 12 -
Version: RC-COVID096-v1.9