Thermo Fisher SARS-CoV-2 User Manual

PROTOCOL Sanger sequencing

Protocol for Sanger sequencing of the SARS-CoV-2

spike (S) gene

SARS-CoV-2 infections continue to be a challenge across
the globe. Part of the challenge, often seen with viruses, is
that the nucleic acid genome quickly mutates, producing
new strain lineages. These new lineages may spread more
quickly, cause either milder or more severe disease, may
have decreased susceptibility to therapeutic agents, and
may evade vaccine-induced immunity. Importantly, they
can also have the ability to evade detection by sequence­based diagnostic tests, complicating epidemiological
monitoring. While the SARS-CoV-2 mutation rate is thought
to be lower than those of other RNA viruses, the sheer
number of infections raises the chances that novel strain
lineages will appear in circulation [1]. Recently, two new
lineages that appear to have increased infectivity have
been identified [2,3]. Interestingly, both of these lineages
have many new mutations in the SARS-CoV-2 spike (S)
gene. Because mutations in the S gene have the potential
to aect interactions with the angiotensin-converting
enzyme 2 (ACE2) receptor [4], it is important to monitor
S gene sequences for new mutations.

We therefore developed a protocol for analyzing the entire
S gene by Sanger sequencing. The primer sequences
used here are based on those published by the Centers
for Disease Control and Prevention (CDC) [5]. Briefly,
cDNA synthesis is performed on a sample containing
viral RNA. Next, the cDNA is used in specific regions

of target amplification using tailed primers that cover
the S gene. For this, the Applied Biosystems™ BigDye™
Direct Cycle Sequencing Kit and M13 sequence–tagged
primer sets are used. The amplified sequences are then
subjected to cycle sequencing using either M13-forward or
M13-reverse primers provided in the BigDye Direct Cycle
Sequencing Kit. Unincorporated nucleotides and primers
are next removed using the Applied Biosystems™ BigDye
XTerminator™ Purification Kit, and the sequences are read
by standard capillary electrophoresis (CE). The sequences
obtained can be read by any sequencing program, such as
SeqA or Geneious™ software, and compared with known or
expected SARS-CoV-2 sequences (Figure 1).

Some of the sequences generated by this method will
produce CE traces that may be dicult to interpret.
To determine whether a sequencing trace was useful,
we employed quality control metrics generated by
Applied Biosystems™ Sequence Scanner Software v2.0.
These metrics include trace score (average of basecaller
quality values for bases in the clear range), contiguous
read length (CRL), and QV20+ (total number of bases in
the entire trace that have a basecaller quality value of ≥20).
Guidelines for using these metrics for QC and analysis
of results are given at the end of the protocol. However,
standard analysis of sequencing traces is often sucient to
determine whether a novel sequence is present.

RNA

purification

Figure 1. Workflow detection of new SARS-CoV-2 lineages using Sanger sequencing.

RNA is purified from samples using standard techniques. cDNA is synthesized from the RNA, and
specific M13 sequence–tagged amplicons are generated by PCR. The amplicons are sequenced
in the forward and reverse directions using universal M13 primers and the BigDye Direct Cycle
Sequencing Kit. The sequencing reactions are cleaned using the BigDye XTerminator kit and
subjected to CE. The resulting sequencing traces can be analyzed and compared to reference
SARS-CoV-2 sequences to determine if the lineages are present.

For Research Use Only. Not for use in diagnostic procedures.

Amplicon

generation

Cycle

sequencing

Capillary

electrophoresis

Analysis

IMPORTANT: This protocol is very sensitive; therefore, utmost care must be taken to prepare the stock solutions and
set up the amplification reactions in an amplicon-free environment.

1. Materials needed

1.1 Equipment

Product Supplier Cat. No.

Veriti 96-Well Fast Thermal Cycler, ProFlex 96-Well PCR System,
or similar thermal cycler

MicroMixer E-36 for 96-well plates Taitec 0027765-000

Single-channel and multichannel micropipettes of various sizes
capable of pipetting volumes from 1.00 µL to 1,000.0 µL

Cold block or ice MLS Any

Plate centrifuge MLS Any

Microcentrifuge or mini centrifuge MLS Any

Vortex mixer MLS Any

1.2 Reagents, kits, and consumables

Thermo Fisher Scientific 4375305 or 4484075

MLS Any

Product Supplier Cat. No.

SuperScript IV VILO Master Mix Thermo Fisher Scientific 1175 6 55 00

Nuclease-Free Water Thermo Fisher Scientific AM9937 or equivalent

BigDye Direct Cycle Sequencing Kit Thermo Fisher Scientific 4458688 or equivalent

BigDye XTerminator Purification Kit Thermo Fisher Scientific 4376486 or equivalent

MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL Thermo Fisher Scientific 4346906 or 4366932

MicroAmp Optical Adhesive Film Thermo Fisher Scientific

Nonstick, RNase-Free Microcentrifuge Tubes, 1.5 mL Thermo Fisher Scientific AM12450 or equivalent

5 mL tube, PCR clean MLS Any

Sterilized aerosol barrier (filter) pipette tips MLS Any

4311971, 4313663, or
4360954

1.3 Prime r s

• Primer sequences are given in Table 1.

Table 1. Sequences of M13-tagged primers for analyzing the S gene. A subset of primer pairs that focus on specific regions of the S gene can be
chosen according to researchers’ needs; the complete list is provided in here. The M13 sequence tags are highlighted in red.

Coordinates*

20990-21562

21421-21916

21775-22345

22203-22697

22563-23128

22986-23519

23379-23876

23737-24231

24095-24623

24493-25003

24858-25369

25214-25790

* Based on N C_0 45512.2 coordinates.

Forward
primer name Forward primer sequence

SC 2M 1-5 4_
LE F T_ M13

SC2M1-55_
LE F T_ M13

SC 2M 1-5 6_
LE F T_ M13

SC 2M1-5 7_
LE F T_ M13

SC 2M 1-5 8_
LE F T_ M13

SC2M1-59_
LE F T_ M13

SC2M1-60_
LE F T_ M13

SC 2M 1-6 1_
LE F T_ M13

SC 2M 1-6 2b _
LE F T_ M13

SC 2M 1-6 3_
LE F T_ M13

SC2M1-64_
LE F T_ M13

SC2M1-65_
LE F T_ M13

TGTAAA ACGACGGCCAGTTGATTGGTG ATTGTGCAACTGTACA

TGTAAA ACGACGGCCAGTAGGGGTACTGCTGTTATGTCTTTAAA

TGTAAA ACGACGGCCAGTTGGGACCAATGGTACTAAGAGGT

TGTAAA ACGACGGCCAGTGTGATCTCCCTCAGGGT TTT TCG

TGTAAA ACGACGGCCAGTACTTGTGCCCT TTTGGTGAAGT

TGTAAA ACGACGGCCAGTCCGGTAGCACACCTTGTAATGG

TGTAAA ACGACGGCCAGTACCAGGTTGCTGTTCTTTATCAGG

TGTAAA ACGACGGCCAGTAATTCTACCAGTGTCTATGACCAAGAC

TGTAAA ACGACGGCCAGTTGCAGATGCTGGCTTCATCA

TGTAAA ACGACGGCCAGTAAATGATATCCTTTCACGTCTTGACAAA

TGTAAA ACGACGGCCAGTGCACACACTGGT TTGTAACACAA

TGTAAA ACGACGGCCAGTTAGGTTTTATAGCTGGCTTGATTGC

Reverse
primer name Reverse primer sequence

SC 2M 1-5 4_
RI GHT_M 13

SC2M1-55_
RI GHT_M 13

SC 2M 1-5 6_
RI GHT_M 13

SC 2M1-5 7_
RI GHT_M 13

SC 2M 1-5 8_
RI GHT_M 13

SC2M1-59_
RI GHT_M 13

SC2M1-60_
RI GHT_M 13

SC 2M 1-6 1_
RI GHT_M 13

SC 2M 1-6 2b _
RI GHT_M 13

SC 2M 1-6 3_
RI GHT_M 13

SC2M1-64_
RI GHT_M 13

SC2M1-65_
RI GHT_M 13

• Primers can be ordered from our custom oligo ordering web page
(https://www.thermofisher.com/order/custom-standard-oligo).

CAGGAAACAGCTATGACCTGTTCGTTTAGTTGTTAACAAGAACATCA

CAGGAAACAGCTATGACCAAGTAGGGACTGGGTCTTCGAA

CAGGAAACAGCTATGACCACCAGCTGTCCAACCTGAAGAA

CAGGAAACAGCTATGACCACTTAAAAGTGGAAAATGATGCGGAA

CAGGAAACAGCTATGACCTGCTGGTGCATGTAGAAGTTCA

CAGGAAACAGCTATGACCCCCCTATTAAACAGCCTGCACG

CAGGAAACAGCTATGACCCAGCTATTCCAGTTAAAGCACGGT

CAGGAAACAGCTATGACCGCACCAAAGGTCCAACCAGAAG

CAGGAAACAGCTATGACCCACAC TCTGACATT TTAGTAGCAGC

CAGGAAACAGCTATGACCTGAGTCTAATTCAGGTTGCAA AGGA

CAGGAAACAGCTATGACCTTTGACTCCTTTG AGCACTGGC

CAGGAAACAGCTATGACCCATTTCCAGCAAAGCCAAAGCC

– 25 nmol of dried and desalted primers can be ordered, but order can be scaled up as needed.

• Resuspend dried oligos to final concentration of 100 µM with TE buer.

1.4. Amplification mixes of primers

• Prepare the target-specific amplification primer mixes:

– Label clean microcentrifuge tubes for each primer pair (e.g., SC2M1-54, SC2M1-55, etc.).

Add 492 µL of TE buer to each tube.

– Add 4 µL each of both the left and right oligos of a pair to the appropriate tube (i.e.,

SC2M1-54_LEFT_M13 and SC2M1-54_RIGHT_M13 in one tube, SC2M1-55_LEFT_M13
and SC2M1-55_RIGHT_M13 to the next, etc.).

– These will be the 10X sequencing amplification primer mixes, with each oligo at 0.8 µM, that will

be used in step 3.1–3.2.