Sanger Sequencing

Sanger sequencing is the process of selective incorporation of chain-terminating

dideoxynucleotides by DNA polymerase during in vitro DNA replication;

From: Genomics, Circuits, and Pathways in Clinical Neuropsychiatry, 2016

Related terms:

Single-Nucleotide Polymorphism, Exon, Pyrosequencing, Plasmids, Polymerase

Chain Reaction, Phenotype, Proteome, DNA, RNA

View all Topics

Somatic Mosaicism and Neurological

Diseases
Saumya S. Jamuar, ... Christopher A. Walsh, in Genomics, Circuits, and Pathways in
Clinical Neuropsychiatry, 2016

Sanger Sequencing
Sanger sequencing is the process of selective incorporation of chain-terminating
dideoxynucleotides by DNA polymerase during in vitro DNA replication; it is the
most widely used method for the detection of SNVs. Because both alleles of an
autosomal locus are sequenced concurrently and are displayed as an analogue
electropherograms, Sanger sequencing is unable to detect mosaic alleles below a
threshold of 15–20% (Rohlin et al., 2009) and can miss a significant proportion of
low-level mosaic mutations (Jamuar et al., 2014). In addition, mosaic mutations at
higher allele fractions are miscalled “germ line,” which highlights the limitations of
Sanger sequencing in detecting mosaicism on both ends of the spectrum (Jamuar
et al., 2014).

> Read full chapter

Overview of Technical Aspects and
Chemistries of Next-Generation Se-
quencing
Ian S. Hagemann, in Clinical Genomics, 2015

Applications in Clinical Genomics

Sanger sequencing is a “first-generation” DNA sequencing method. Despite the ad-
vantages of next-generation sequencing techniques, where throughput is orders of
magnitude higher, Sanger sequencing retains an essential place in clinical genomics
for at least two specific purposes.

First, Sanger sequencing serves as an orthogonal method for confirming sequence

variants identified by NGS. When validating clinical NGS tests, reference materials
sequenced by Sanger approaches provide ground truth against which the NGS
assay can be benchmarked. These materials may include well-characterized publicly
available reagents, such as cell lines studied in the HapMap project, or archival
clinical samples previously tested by Sanger methods.

As an orthogonal method, Sanger sequencing provides a means to confirm variants

identified by NGS. It would be impractical to Sanger-confirm every variant, given
the large number of primers, reactions, and interpretations that would be required.
However, there may be instances where the veracity of a specific variant is in doubt;
e.g., called variants that are biologically implausible or otherwise suspected of being
spurious. Sanger sequencing is the easiest method to resolve these uncertainties
and is therefore an invaluable protocol in any clinical genomics laboratory.

Second, Sanger sequencing provides a means to “patch” the coverage of regions that
are poorly covered by NGS. In targeted NGS testing, there may be regions that are
resistant to sequencing, due to poor capture, amplification, or other idiosyncrasies.
These regions are often rich in GC content. One approach to restoring coverage of
these areas is to increase the quantity of input DNA, but the quantity available may
be limited. It may be possible to redesign the amplification step or capture reagents,
or otherwise troubleshoot the NGS technology. However, a very practical approach,
when the area to be backfilled is small, is to use Sanger sequencing to span the
regions poorly covered by NGS.

When Sanger sequencing is used for backfilling NGS data, the NGS and Sanger data
must be integrated together for purposes of analysis and reporting, which represents
a challenge since these data are obtained by different methods and do not have a
one-to-one correspondence to one another. Analyses that are natural for NGS data
may be difficult to map onto data obtained by Sanger. For example, measures
of sequence quality that are meaningful for NGS are not applicable to Sanger; the
concept of depth of coverage can only be indirectly applied to Sanger data; allele
frequencies are indirectly and imprecisely ascertained in Sanger sequence from peak
heights rather than read counts; and Sanger data do not have paired ends. While
NGS may potentially be validated to allow meaningful variant calling from a single
nonreference read, the sensitivity of Sanger sequencing has a floor of approximately
20%: variants with a lower allele frequency may be indistinguishable from noise or
sequencing errors (discussed below). Thus the performance of an NGS assay may be
altered in areas of Sanger patching, and these deviations in performance must be
documented and/or disclaimed.

> Read full chapter

Genetic Testing Techniques

Alicia Gomes MS, Bruce Korf MD, PhD, in Pediatric Cancer Genetics, 2018

Sanger Sequencing

Methodology
Sanger sequencing is a targeted sequencing technique that uses oligonucleotide
primers to seek out specific DNA regions. Sanger sequencing begins with denatu-
ration of the double-stranded DNA. The single-stranded DNA is then annealed to
oligonucleotide primers and elongated using a mixture of deoxynucleotide triphos-
phates (dNTPs), which provide the needed arginine (A), cytosine (C), tyrosine (T), and
guanine (G) nucleotides to build the new double-stranded structure. In addition, a
small quantity of chain-terminating dideoxynucleotide triphosphates (ddNTPs) for
each nucleotide is included. The sequence will continue to extend with dNTPs until
a ddNTP attaches. As the dNTPs and ddNTPs have an equal chance of attaching to
the sequence, each sequence will terminate at varying lengths.

Each ddNTP (ddATP, ddGTP, ddCTP, ddTTP) also includes a fluorescent marker.
When a ddNTP is attached to the elongating sequence, the base will fluoresce based
on the associated nucleotide. By convention, A is indicated by green fluorescence,
T by red, G by black, and C by blue. A laser within the automated machine used to
read the sequence detects a fluorescent intensity that is translated into a “peak.”
When a heterozygous variant occurs within a sequence, loci will be captured by
two fluorescent dyes of equal intensity. When a homozygous variant is present, the
expected fluorescent color is replaced completely by the new base pair’s color (Fig.
5.7).
Types of variants detected
• Silent

• Missense

• Nonsense

• Truncating

• Deletion

• Insertion

• Splicing

Benefits
Sanger sequencing is a robust testing strategy able to determine whether a point
mutation or small deletion/duplication is present. It has been widely used for several
decades in many settings, including defining the mutational spectrum of a tumor
as well as identifying a constitutional variant in diagnostic testing. Primers can be
created to cover several regions (amplicons) to cover any size region of interest.

Limitations
Although one could use individual Sanger sequencing reactions to cover any desired
region, this testing approach can be costly when compared with other multiplex
testing systems. Therefore, most currently available Sanger sequencing tests are
gene-specific or analyze a small subset of genes. Sanger sequencing is able to iden-
tify mosaic mutations including as low as 20% of the cells, but Sanger sequencing is
not precisely quantifiable. For example, one cannot conclude if a mutation is present
in 25% versus 40% of cells based on peak sizes; additional testing strategies must
be used for quantification.

> Read full chapter

Genomics of Infectious Diseases and

Private Industry
G. Vernet, in Genetics and Evolution of Infectious Diseases (Second Edition), 2017

2.1 Sanger Sequencing

Sanger sequencing uses the SBS approach in which a DNA polymerase generates
DNA reads from a template that is the DNA molecule to be analyzed. The nature of
the nucleotide at a given position is now determined using specific dyes.

Sanger sequencing, although too laborious and expensive for WGS, remains rou-
tinely used when sequencing of specific genes or fragment of genes is needed, for
example, for viral or bacterial genotyping or for resistance testing when SNPs are
associated with specific genome regions. For bacterial WGS, biological amplification
by culture and single colony picking is needed whereas PCR amplification of specific
genes is done for both viruses and bacteria before amplicons are sequenced. Since
1987 and during the last four decades, Sanger sequencing has been mostly done on
ABI sequencers (Thermo Fisher Scientific) instruments, a brand that now proposes
a series of capillary electrophoresis sequencers ranging from 1 to 96 capillaries and
covering the needs of different laboratories in terms of throughput. All current ABI
DNA sequencing kits use cycle sequencing protocols with two different chemistries:
dye primer chemistry or dye terminator chemistry.

> Read full chapter

A Clinical Guide to Monogenic Dia-

betes
David Carmody, ... Louis H. Philipson, in Genetic Diagnosis of Endocrine Disorders
(Second Edition), 2016

Deletion Analysis
Sanger sequencing can readily identify small indels (insertions or deletions). While
partial or whole gene deletions make up a minority of monogenic diabetes cases,
these may be missed using Sanger sequencing alone.78 Medium-sized deletion
longer than the PCR amplicons also are not detected because they cannot be ampli-
fied and may not always be identified through multiplex ligation-dependent probe
amplification, the most commonly used test to screen for deletions.79 Dedicated
studies to identify deletions are particularly important when assessing families with
an HNF1B-MODY (MODY5) phenotype as deletions are more common in this
gene.80 As NGS technologies improve they may be used to detect large deletions
or duplications if deep; even coverage of the target genes can be maintained.77

> Read full chapter

Integrating Molecular Diagnostics
With Surgical Neuropathology
David A. Solomon MD, PhD, in Practical Surgical Neuropathology: A Diagnostic
Approach (Second Edition), 2018

Sanger Sequencing for Detection of Single Nucleotide Muta-

tions
Sanger sequencing is a method developed by Frederick Sanger and colleagues in
the 1970s that is based on selective incorporation of chain-terminating dideoxynu-
cleotides by DNA polymerase during in vitro DNA replication.31 Modern Sanger
sequencing typically uses fluorescently labeled dideoxynucleotides that are detected
by a laser after capillary electrophoresis to generate a sequence chromatogram with
fluorescent peaks corresponding to incorporation of the four different fluorescent
dyes coupled to ddATP, ddCTP, ddGTP, and ddTTP.32 Sanger sequencing has proven
useful for assessing the presence or absence of recurrent single nucleotide mu-
tations or small insertions/deletions in oncogenes and tumor suppressor genes
in surgically resected pathology specimens. First, genomic DNA is extracted from
snap-frozen or formalin-fixed, paraffin-embedded tumor tissue. Then, polymerase
chain reaction (PCR) is performed using oligonucleotide primers and genomic DNA
isolated from the tumor tissue as a template to amplify the genetic region of interest
(e.g., exon 4 of IDH1 containing codon p.R132). Sanger sequencing reactions are
then performed on these PCR amplicons to determine their nucleotide composition.
Examples of recurrent single nucleotide mutations with diagnostic, prognostic, or
therapeutic relevance that are now routinely assessed by Sanger sequencing in
surgical specimens include:

• IDH1 exon 4 containing the p.R132 hotspot and IDH2 exon 4 containing
the p.R172 hotspot, which are frequently mutated in WHO grade II and
III oligodendrogliomas, grade II and III diffuse/anaplastic astrocytomas, and
IDH-mutant glioblastomas (Fig. 5.5)
• H3F3A and HIST1H3B containing the p.K27 hotspot frequently mutated in
diffuse midline gliomas and rarely other midline tumor entities, including
ganglioglioma and pilocytic astrocytoma
• BRAF exon 15 containing the p.V600 hotspot frequently mutated in pleomor-
phic xanthoastrocytoma, ganglioglioma, extracerebellar pilocytic astrocytoma,
epithelioid glioblastoma, diffuse gliomas in children, and other tumor entities
• TERT promoter region containing the c.-124C and c.-146C hotspots upstream
of the ATG translational start site that are frequently mutated in IDH-wildtype
glioblastoma in adults, IDH-mutant and 1p/19-codeleted oligodendroglioma,
anaplastic (malignant) meningioma, and other tumor entities.

While mutant-specific antibodies have been generated to detect some of the recur-
rent mutations found in oncogenes in brain tumors (e.g., IDH1 p.R132H, histone
H3 p.K27M, and BRAF p.V600E), mutant-specific antibodies for other important
single nucleotide mutations are not available (e.g., TERT promoter c.-124C>T and
c.-146C>T mutations) and genetic analysis via Sanger sequencing or next-gener-
ation sequencing is required for their assessment. Occasionally, mutant-specific
antibodies may yield equivocal staining, therefore requiring confirmation by Sanger
sequencing or other methodology. Additionally, while approximately 80% to 90% of
diffuse lower grade gliomas harbor the IDH1 p.R132H mutation that is detectable
using the mutant-specific antibody, a subset instead harbor one of the less com-
mon IDH1 mutations (e.g., p.R132C or p.R132S) or mutation of the equivalent
p.R172 codon in IDH2. Determining that a diffuse glioma is “IDH-wildtype” ul-
timately requires Sanger sequencing or other sequencing methodology following
a negative immunohistochemical stain using the IDH1 R132H mutant-specific
antibody (Fig. 5.5). While the vast majority of activating mutations in BRAF are
the p.V600E mutation detectable by mutant-specific antibody, occasional p.V600K
or p.V600M mutations have been identified, as well as small in-frame insertions
such as p.T599_V600insT that can all be detected by Sanger sequencing but not by
immunohistochemistry.

As with all molecular testing, limitations and interpretational pitfalls of Sanger se-
quencing should be recognized. The major limitations are related to the quantity of
tissue required and the sensitivity of detection. Unlike immunohistochemistry using
mutant-specific antibodies, which requires only a single unstained section, Sanger
sequencing and other sequencing methodologies typically require at least a few
unstained slides in order to obtain sufficient genomic DNA. Thus, in some cases,
small biopsies taken from critical structures such as the brainstem or spinal cord
may be insufficient. The sensitivity of detection for Sanger sequencing is generally
recognized as being approximately 15% to 20% mutant allele frequency, meaning
that 15% to 20% of the DNA molecules being sequenced need to contain the
mutation in order to be reliably detected. Since human cells are diploid, the tissue
used for DNA extraction therefore needs to contain at least 30% to 40% tumor nuclei
for detection of somatic mutations in oncogenes that are heterozygous (i.e., present
in only one of two alleles) and fully clonal (i.e., present in all of the tumor cells).
Extracting DNA from tissue that contains an adequate quantity of tumor cells relative
to admixed non-neoplastic cells can be challenging for some infiltrative gliomas or
tumors with abundant inflammatory infiltrate. In such cases, Sanger sequencing
may be falsely negative for the mutation being tested. Lastly, Sanger and other DNA
sequencing methodologies rely on extraction of intact genomic DNA. Tissue fixed in
formalin for an extended period of time (greater than a few days) may contain such
extensive crosslinking of DNA that PCR amplification becomes challenging. In most
cases, decalcification of tissue prior to processing and embedding is not compatible
with molecular testing, including Sanger sequencing.

> Read full chapter

Pulmonary Adenocarcinoma—Patholo-
gy and Molecular Testing
Prodipto Pal MD, PhD, ... Ming-Sound Tsao MD, FRCPC, in Pulmonary Adenocar-
cinoma: Approaches to Treatment, 2019

Nontargeted assays
Sanger sequencing was the standard for EGFR testing in the first clinical trials with
erlotinib and gefitinib. This method incorporates fluorescent-tagged dideoxy termi-
nators to amplifying DNA strands, which can be sorted by size and the nucleotide
sequence read sequentially. A major consideration is the relatively low analytical
sensitivity of the assay, which usually requires specimens with high tumor content.
As such, this assay is no longer a method of choice for detection of EGFR mutations.
Another method, called pyrosequencing, involves measuring the chemiluminescent
signal released by pyrophosphate as triphosphate nucleotides are being incorporated
into the synthesized DNA strand. Although the fragment length required for py-
rosequencing is much shorter than those used for Sanger sequencing, this method
offers higher sensitivity and can detect mutations in samples with up to 5% tumor
cellularity.

> Read full chapter

Pathology, Biomarkers, and Molecular

Diagnostics
Wilbur A. Franklin, ... Marileila Varella Garcia, in Abeloff 's Clinical Oncology (Fifth
Edition), 2014

Sanger Sequencing
Sanger sequencing has been the gold standard for many years and, like many other
assays, is based on the dideoxy-chemistry illustrated in Figure 17-11. The sequencing
reaction is read as a color code that distinguishes oligonucleotides of variable length
with four specific color labels. A ladder of nucleotides is created that can be identified
by their electrophoretic mobility and color of terminal fluorescent nucleotide. The
identification of abnormal (mutant) peaks in chromatograms of this ladder can
be facilitated by computer programs that not only create the chromatogram but
compare them with reference sequences (RefSeq) and identify abnormalities that
represent mutations and polymorphisms.

Sanger sequencing is readily accomplished in DNA extracted from FFPE tissue that
has been amplified by PCR provided that the amplified sequence is short (<300 base
pair). This method has the advantage that it provides unbiased sequence results
that will detect virtually any mutation in the targeted region. However, the analytic
sensitivity is limited, with tumor cell concentration of approximately 50% required
for accurate results. Because of the PCR amplification, the amount of DNA starting
material required is usually small, depending on the number of DNA templates
selected for amplification.

> Read full chapter

Locked Nucleic Acid Technology for

Highly Sensitive Detection of Somatic
Mutations in Cancer
Takayuki Ishige, ... Kazuyuki Matsushita, in Advances in Clinical Chemistry, 2018

3.3 Sanger Sequencing

Sanger sequencing, also known as the chain termination method, was developed
by Sanger et al. [27]. Many clinical laboratories perform direct sequencing of PCR
products using this method. In cycle sequencing, PCR products, sequencing primers
(either forward or reverse), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs,
typically labeled with a different fluorescent dye), and thermostable DNA polymerase
are included in the reaction mixture. First, the sequencing primer hybridizes the PCR
products and is elongated by the DNA polymerase during PCR. ddNTPs are random-
ly incorporated in the DNA strands during elongation, thereby terminating strand
elongation at each location along the sequence. Subsequent capillary electrophoresis
separates the DNA strands with respect to the size, and the terminating nucleotides
are identified using each fluorescent dye. It is considered the gold standard method
for mutational analysis and can determine the entire sequence and identify unknown
mutations. The major limitations of this method are high cost, labor intensiveness,
and low sensitivity. The sensitivity of this method is 10%–20% mutations in the
wild-type background [38,43]. Thus, low frequent mutations (< 10%) in tumor
samples cannot be determined using Sanger sequencing.

> Read full chapter

Other Post-PCR Detection Technolo-

gies
P. Zhang, ... H. Fernandes, in Pathobiology of Human Disease, 2014

Sanger (Conventional) Sequencing

Sanger sequencing using capillary sequencers has become very popular in molecular
diagnostic laboratories. Multiple sequencing reactions are loaded onto multiwell
plates, which are injected into capillaries for electrophoresis on the instrument. In
sequencing reactions, primers that anneal to a single-stranded DNA template are
elongated by DNA (Taq) polymerase. Fluorescent-labeled deoxynucleotides are in-
troduced one at a time and the primer is extended in a template-dependent manner.
In the same reaction, Taq polymerase adds to denatured DNA, fluorescent-labeled
modified (dideoxy) nucleotides that terminate the formation of a new DNA strand as
they encounter their complementary nucleotides in the target sequence. This results
in DNA strands of variable length, which are separated on a gel by electrophoresis
and reflect the sequence being analyzed. Each capillary is separately calibrated for
the dyes used in the sequencing reactions so that the software can perform the
multi-component analysis to identify each of the dye-labeled fragments (Figure 5).
These sequencing reactions are then analyzed using special software.
Figure 5. Principle of Sanger sequencing. Fluorescence-labeled dNTPs are added to
amplicons as it is synthesized. The information is translated to a sequence in an
electropharagram.

Sanger sequencing requires a DNA template, a sequencing primer, a thermostable

DNA polymerase, nucleotides (dNTPs), dideoxynucleotides (ddNTPs), and buffer.
Thermal cycling in the sequencing reactions amplifies extension products that are
terminated by one of the four ddNTPs. The ratio of deoxynucleotides to ddNTPs
is optimized to produce a balanced population of long and short extension prod-
ucts similar to the conventional Sanger sequencing method. Detection in cycle
sequencing can be accomplished using two different dye-labeling chemistries: the
dye terminators or the dye primer labeling. The four ddNTP terminators are tagged
with different fluorescent dyes. Only one reaction is performed with all the reagents
such as unlabeled sequencing primer, enzyme, nucleotides, and all dye-labeled
ddNTPs in a single tube. Fluorescent fragments are generated by incorporation
of dye-labeled ddNTPs. Each different ddNTP (ddATP, ddCTP, ddGTP, or ddTTP)
will carry a different-colored dye. All terminated fragments (those ending with a
ddNTP), therefore, contain a dye at their 3 end. The products from this reaction
are injected into one capillary and are distinguished as individual nucleotides with
unique fluorophores that are recognized by the laser. The information from the
 laser is captured by photomultiplier tubes to generate an electropharagram that
represents the unique sequence.

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