Sanger sequencing

Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on
the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in
vitro DNA replication. After first being developed by Frederick Sanger and colleagues in 1977, it
became the most widely used sequencing method for approximately 40 years. An automated
instrument using slab gel electrophoresis and fluorescent labels was first commercialized by
Applied Biosystems in March 1987.[1] Later, automated slab gels were replaced with automated
capillary array electrophoresis.[2]

Recently, higher volume Sanger sequencing has been replaced by next generation sequencing
methods, especially for large-scale, automated genome analyses. However, the Sanger method
remains in wide use for smaller-scale projects and for validation of deep sequencing results. It still
has the advantage over short-read sequencing technologies (like Illumina) in that it can produce
DNA sequence reads of > 500 nucleotides and maintains a very low error rate with accuracies
around 99.99%.[3] Sanger sequencing is still actively being used in efforts for public health initiatives
such as sequencing the spike protein from SARS-CoV-2[4] as well as for the surveillance of
norovirus outbreaks through the United States Center for Disease Control and Prevention (CDC)'s
CaliciNet surveillance network.[5]

The Sanger (chain-termination) method for DNA sequencing

Method

Fluorescent ddNTP molecules

The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a
DNA polymerase, normal deoxynucleotide triphosphates (dNTPs), and modified di-
deoxynucleotide triphosphates (ddNTPs), the latter of which terminate DNA strand elongation.
These chain-terminating nucleotides lack a 3'-OH group required for the formation of a
phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of
DNA when a modified ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently
labelled for detection in automated sequencing machines.

The DNA sample is divided into four separate sequencing reactions, containing all four of the
standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each
reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP), while
the other added nucleotides are ordinary ones. The deoxynucleotide concentration should be
approximately 100-fold higher than that of the corresponding dideoxynucleotide (e.g. 0.5mM
dTTP : 0.005mM ddTTP) to allow enough fragments to be produced while still transcribing the
complete sequence (but the concentration of ddNTP also depends on the desired length of
sequence).[6] Putting it in a more sensible order, four separate reactions are needed in this process
to test all four ddNTPs. Following rounds of template DNA extension from the bound primer, the
resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. In the
original publication of 1977,[6] the formation of base-paired loops of ssDNA was a cause of serious
difficulty in resolving bands at some locations. This is frequently performed using a denaturing
polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T,
G, C). The DNA bands may then be visualized by autoradiography or UV light, and the DNA
sequence can be directly read off the X-ray film or gel image.
 Part of a radioactively
labelled sequencing gel

In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to
DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result
of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP).
The relative positions of the different bands among the four lanes, from bottom to top, are then
used to read the DNA sequence.

DNA fragments are labelled with a

radioactive or fluorescent tag on the
primer (1), in the new DNA strand with a
labeled dNTP, or with a labeled ddNTP.

Technical variations of chain-termination sequencing include tagging with nucleotides containing

radioactive phosphorus for radiolabelling, or using a primer labeled at the 5' end with a fluorescent
dye. Dye-primer sequencing facilitates reading in an optical system for faster and more
economical analysis and automation. The later development by Leroy Hood and coworkers[7][8] of
fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA
sequencing.

Sequence ladder by radioactive

sequencing compared to fluorescent
peaks

Chain-termination methods have greatly simplified DNA sequencing. For example, chain-
termination-based kits are commercially available that contain the reagents needed for sequencing,
pre-aliquoted and ready to use. Limitations include non-specific binding of the primer to the DNA,
affecting accurate read-out of the DNA sequence, and DNA secondary structures affecting the
fidelity of the sequence.

Dye-terminator sequencing

Capillary electrophoresis

Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits
sequencing in a single reaction rather than four reactions as in the labelled-primer method. In dye-
terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with
fluorescent dyes, each of which emits light at different wavelengths.

Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in
automated sequencing. Its limitations include dye effects due to differences in the incorporation of
the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and
shapes in the electronic DNA sequence trace electropherogram (a type of chromatogram) after
capillary electrophoresis (see figure to the left).

This problem has been addressed with the use of modified DNA polymerase enzyme systems and
dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-
terminator sequencing method, along with automated high-throughput DNA sequence analyzers,
was used for the vast majority of sequencing projects until the introduction of next generation
sequencing.

Automation and sample preparation

View of the start of an example dye-

terminator read

Automated DNA-sequencing instruments (DNA sequencers) can sequence up to 384 DNA

samples in a single batch. Batch runs may occur up to 24 times a day. DNA sequencers separate
strands by size (or length) using capillary electrophoresis, they detect and record dye
fluorescence, and output data as fluorescent peak trace chromatograms. Sequencing reactions
(thermocycling and labelling), cleanup and re-suspension of samples in a buffer solution are
performed separately, before loading samples onto the sequencer. A number of commercial and
non-commercial software packages can trim low-quality DNA traces automatically. These
programs score the quality of each peak and remove low-quality base peaks (which are generally
located at the ends of the sequence).[9] The accuracy of such algorithms is inferior to visual
examination by a human operator, but is adequate for automated processing of large sequence
data sets.

Applications of dye-terminating sequencing

The field of public health plays many roles to support patient diagnostics as well as environmental
surveillance of potential toxic substances and circulating biological pathogens. Public health
laboratories (PHL) and other laboratories around the world have played a pivotal role in providing
rapid sequencing data for the surveillance of the virus SARS-CoV-2, causative agent for COVID-19,
during the pandemic that was declared a public health emergency on January 30, 2020.[10]
Laboratories were tasked with the rapid implementation of sequencing methods and asked to
provide accurate data to assist in the decision-making models for the development of policies to
mitigate spread of the virus. Many laboratories resorted to next generation sequencing
methodologies while others supported efforts with Sanger sequencing. The sequencing efforts of
SARS-CoV-2 are many, while most laboratories implemented whole genome sequencing of the
virus, others have opted to sequence very specific genes of the virus such as the S-gene,
encoding the information needed to produce the spike protein. The high mutation rate of SARS-
CoV-2 leads to genetic differences within the S-gene and these differences have played a role in
the infectivity of the virus.[11] Sanger sequencing of the S-gene provides a quick, accurate, and
more affordable method to retrieving the genetic code. Laboratories in lower income countries
may not have the capabilities to implement expensive applications such as next generation
sequencing, so Sanger methods may prevail in supporting the generation of sequencing data for
surveillance of variants.

Sanger sequencing is also the "gold standard" for norovirus surveillance methods for the Center for
Disease Control and Prevention's (CDC) CaliciNet network. CalciNet is an outbreak surveillance
network that was established in March 2009. The goal of the network is to collect sequencing data
of circulating noroviruses in the United States and activate downstream action to determine the
source of infection to mitigate the spread of the virus. The CalciNet network has identified many
infections as foodborne illnesses.[5] This data can then be published and used to develop
recommendations for future action to prevent tainting food. The methods employed for detection
of norovirus involve targeted amplification of specific areas of the genome. The amplicons are
then sequenced using dye-terminating Sanger sequencing and the chromatograms and sequences
generated are analyzed with a software package developed in BioNumerics. Sequences are
tracked and strain relatedness is studied to infer epidemiological relevance.

Challenges

Common challenges of DNA sequencing with the Sanger method include poor quality in the first
15–40 bases of the sequence due to primer binding and deteriorating quality of sequencing traces
after 700–900 bases. Base calling software such as Phred typically provides an estimate of quality
to aid in trimming of low-quality regions of sequences.[12][13]

In cases where DNA fragments are cloned before sequencing, the resulting sequence may contain
parts of the cloning vector. In contrast, PCR-based cloning and next-generation sequencing
technologies based on pyrosequencing often avoid using cloning vectors. Recently, one-step
Sanger sequencing (combined amplification and sequencing) methods such as Ampliseq and
SeqSharp have been developed that allow rapid sequencing of target genes without cloning or
prior amplification.[14][15]

Current methods can directly sequence only relatively short (300–1000 nucleotides long) DNA
fragments in a single reaction. The main obstacle to sequencing DNA fragments above this size
limit is insufficient power of separation for resolving large DNA fragments that differ in length by
only one nucleotide.

Microfluidic Sanger sequencing

Microfluidic Sanger sequencing is a lab-on-a-chip application for DNA sequencing, in which the
Sanger sequencing steps (thermal cycling, sample purification, and capillary electrophoresis) are
integrated on a wafer-scale chip using nanoliter-scale sample volumes. This technology generates
long and accurate sequence reads, while obviating many of the significant shortcomings of the
conventional Sanger method (e.g. high consumption of expensive reagents, reliance on expensive
equipment, personnel-intensive manipulations, etc.) by integrating and automating the Sanger
sequencing steps.

In its modern inception, high-throughput genome sequencing involves fragmenting the genome
into small single-stranded pieces, followed by amplification of the fragments by polymerase chain
reaction (PCR). Adopting the Sanger method, each DNA fragment is irreversibly terminated with
the incorporation of a fluorescently labeled dideoxy chain-terminating nucleotide, thereby
producing a DNA “ladder” of fragments that each differ in length by one base and bear a base-
specific fluorescent label at the terminal base. Amplified base ladders are then separated by
capillary array electrophoresis (CAE) with automated, in situ “finish-line” detection of the
fluorescently labeled ssDNA fragments, which provides an ordered sequence of the fragments.
These sequence reads are then computer assembled into overlapping or contiguous sequences
(termed "contigs") which resemble the full genomic sequence once fully assembled.[16]

Sanger methods achieve maximum read lengths of approximately 800 bp (typically 500–600 bp
with non-enriched DNA). The longer read lengths in Sanger methods display significant advantages
over other sequencing methods especially in terms of sequencing repetitive regions of the
genome. A challenge of short-read sequence data is particularly an issue in sequencing new
genomes (de novo) and in sequencing highly rearranged genome segments, typically those seen
of cancer genomes or in regions of chromosomes that exhibit structural variation.[17]

Applications of microfluidic sequencing technologies

Other useful applications of DNA sequencing include single nucleotide polymorphism (SNP)
detection, single-strand conformation polymorphism (SSCP) heteroduplex analysis, and short
tandem repeat (STR) analysis. Resolving DNA fragments according to differences in size and/or
conformation is the most critical step in studying these features of the genome.[16]

Device design

The sequencing chip has a four-layer construction, consisting of three 100-mm-diameter glass
wafers (on which device elements are microfabricated) and a polydimethylsiloxane (PDMS)
membrane. Reaction chambers and capillary electrophoresis channels are etched between the top
two glass wafers, which are thermally bonded. Three-dimensional channel interconnections and
microvalves are formed by the PDMS and bottom manifold glass wafer.

The device consists of three functional units, each corresponding to the Sanger sequencing steps.
The thermal cycling (TC) unit is a 250-nanoliter reaction chamber with integrated resistive
temperature detector, microvalves, and a surface heater. Movement of reagent between the top
all-glass layer and the lower glass-PDMS layer occurs through 500-μm-diameter via-holes. After
thermal-cycling, the reaction mixture undergoes purification in the capture/purification chamber,
and then is injected into the capillary electrophoresis (CE) chamber. The CE unit consists of a 30-
cm capillary which is folded into a compact switchback pattern via 65-μm-wide turns.

Sequencing chemistry

Thermal cycling
In the TC reaction chamber, dye-terminator sequencing reagent, template DNA, and primers are
loaded into the TC chamber and thermal-cycled for 35 cycles ( at 95 °C for 12 seconds and at
60 °C for 55 seconds).

Purification
The charged reaction mixture (containing extension fragments, template DNA, and excess
sequencing reagent) is conducted through a capture/purification chamber at 30 °C via a 33-
Volts/cm electric field applied between capture outlet and inlet ports. The capture gel through
which the sample is driven, consists of 40 μM of oligonucleotide (complementary to the
primers) covalently bound to a polyacrylamide matrix. Extension fragments are immobilized by
the gel matrix, and excess primer, template, free nucleotides, and salts are eluted through the
capture waste port. The capture gel is heated to 67–75 °C to release extension fragments.

Capillary electrophoresis
Extension fragments are injected into the CE chamber where they are electrophoresed through a
125-167-V/cm field.
Platforms

The Apollo 100 platform (Microchip Biotechnologies Inc., Dublin, California)[18] integrates the first
two Sanger sequencing steps (thermal cycling and purification) in a fully automated system. The
manufacturer claims that samples are ready for capillary electrophoresis within three hours of the
sample and reagents being loaded into the system. The Apollo 100 platform requires sub-microliter
volumes of reagents.
Comparisons to other sequencing techniques

Performance values for genome sequencing technologies including Sanger methods and next-generation
methods[17][19][20]

Throughput
Injection Average
Number Analysis (including Gel Lane
Technology volume read
of lanes time analysis; pouring tracking
(nL) length
Mb/h)

Slab gel 96 500–1000 6–8 hours 700 bp 0.0672 Yes Yes

Capillary array
96 1–5 1–3 hours 700 bp 0.166 No No
electrophoresis

6–30
Microchip 96 0.1–0.5 430 bp 0.660 No No
minutes

454/Roche FLX
< 0.001 4 hours 200–300 bp 20–30
(2008)

Illumina/Solexa
2–3 days 30–100 bp 20
(2008)

ABI/SOLiD
8 days 35 bp 5–15
(2008)

Illumina MiSeq 2x75–2x300

1–3 days 170–250
(2019) bp

Illumina 2x50–2x150
1–2 days 22,000–67,000
NovaSeq (2019) bp

Ion Torrent Ion

2.5–4 hours 200–600 bp 110–920
530 (2019)

BGI MGISEQ-T7
1 day 2x150 bp 250,000
(2019)

Pacific
12–30
Biosciences 15–25 kb [21] 15,000
hours
Revio (2023)

Oxford
Nanopore 3 days 13–20 kb [22] 700
MinIon (2019)

The ultimate goal of high-throughput sequencing is to develop systems that are low-cost, and
extremely efficient at obtaining extended (longer) read lengths. Longer read lengths of each single
electrophoretic separation, substantially reduces the cost associated with de novo DNA
sequencing and the number of templates needed to sequence DNA contigs at a given redundancy.
Microfluidics may allow for faster, cheaper and easier sequence assembly.[16]
See also

Maxam–Gilbert sequencing

Second-generation sequencing

Third-generation sequencing

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Medicine. 15 (1): 42. doi:10.1186/s13073-023-01194-3 (https://doi.org/10.1186%2Fs13073-0
23-01194-3) . PMC 10266321 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10266321) .
PMID 37316925 (https://pubmed.ncbi.nlm.nih.gov/37316925) .

22. Tyson JR, O'Neil NJ, Jain M, Olsen HE, Hieter P, Snutch TP (February 2018). "MinION-based
long-read sequencing and assembly extends the Caenorhabditis elegans reference genome"
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793790) . Genome Research. 28 (2): 266–
274. doi:10.1101/gr.221184.117 (https://doi.org/10.1101%2Fgr.221184.117) . PMC 5793790
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793790) . PMID 29273626 (https://pubme
d.ncbi.nlm.nih.gov/29273626) .

Further reading

Dewey FE, Pan S, Wheeler MT, Quake SR, Ashley EA (February 2012). "DNA sequencing: clinical
applications of new DNA sequencing technologies" (https://www.ncbi.nlm.nih.gov/pmc/articles/
PMC3364518) . Circulation. 125 (7): 931–944. doi:10.1161/CIRCULATIONAHA.110.972828 (htt
ps://doi.org/10.1161%2FCIRCULATIONAHA.110.972828) . PMC 3364518 (https://www.ncbi.nl
m.nih.gov/pmc/articles/PMC3364518) . PMID 22354974 (https://pubmed.ncbi.nlm.nih.gov/223
54974) .

Sanger F, Coulson AR, Barrell BG, Smith AJ, Roe BA (October 1980). "Cloning in single-stranded
bacteriophage as an aid to rapid DNA sequencing". Journal of Molecular Biology. 143 (2): 161–
178. doi:10.1016/0022-2836(80)90196-5 (https://doi.org/10.1016%2F0022-2836%2880%29901
96-5) . PMID 6260957 (https://pubmed.ncbi.nlm.nih.gov/6260957) .

External links

MBI Says New Tool That Automates Sanger Sample Prep Cuts Reagent and Labor Costs (http
s://www.genomeweb.com/sequencing/mbi-says-new-tool-automates-sanger-sample-prep-cuts-
reagent-and-labor-costs)