What Limits How Far the Sequence Can Read Sanger Sequencing

Somatic Mosaicism and Neurological Diseases

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

Sanger Sequencing

Sanger sequencing is the process of selective incorporation of concatenation-terminating dideoxynucleotides past Deoxyribonucleic acid polymerase during in vitro Deoxyribonucleic acid replication; information technology is the most widely used method for the detection of SNVs. Considering both alleles of an autosomal locus are sequenced concurrently and are displayed as an analogue electropherograms, Sanger sequencing is unable to find mosaic alleles below a threshold of 15–20% ( Rohlin et al., 2009) and can miss a pregnant 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).

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Overview of Technical Aspects and Chemistries of Next-Generation Sequencing

Ian South. Hagemann , in Clinical Genomics, 2015

Applications in Clinical Genomics

Sanger sequencing is a "get-go-generation" DNA sequencing method. Despite the advantages of next-generation sequencing techniques, where throughput is orders of magnitude higher, Sanger sequencing retains an essential identify in clinical genomics for at least two specific purposes.

First, Sanger sequencing serves equally an orthogonal method for confirming sequence variants identified past NGS. When validating clinical NGS tests, reference materials sequenced by Sanger approaches provide ground truth confronting which the NGS assay can exist benchmarked. These materials may include well-characterized publicly available reagents, such as cell lines studied in the HapMap projection, or archival clinical samples previously tested by Sanger methods.

As an orthogonal method, Sanger sequencing provides a means to confirm variants identified past 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 exist instances where the veracity of a specific variant is in incertitude; east.one thousand., called variants that are biologically implausible or otherwise suspected of beingness 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, in that location 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 increment the quantity of input DNA, but the quantity bachelor may be express. It may exist possible to redesign the amplification step or capture reagents, or otherwise troubleshoot the NGS engineering. However, a very practical approach, when the area to be backfilled is small, is to use Sanger sequencing to bridge the regions poorly covered past NGS.

When Sanger sequencing is used for backfilling NGS data, the NGS and Sanger data must exist integrated together for purposes of analysis and reporting, which represents a challenge since these information are obtained past different methods and do not take a one-to-i correspondence to one another. Analyses that are natural for NGS data may be difficult to map onto data obtained past Sanger. For example, measures of sequence quality that are meaningful for NGS are non 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 accept 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 duplicate 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.

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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 denaturation of the double-stranded DNA. The single-stranded DNA is then annealed to oligonucleotide primers and elongated using a mixture of deoxynucleotide triphosphates (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 keep to extend with dNTPs until a ddNTP attaches. As the dNTPs and ddNTPs have an equal chance of attaching to the sequence, each sequence volition cease 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 past dark-green fluorescence, T past red, One thousand by black, and C by blue. A light amplification by stimulated emission of radiation inside 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 past two fluorescent dyes of equal intensity. When a homozygous variant is nowadays, the expected fluorescent color is replaced completely by the new base pair'south color (Fig. 5.7).

Types of variants detected

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Silent

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Missense

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Nonsense

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Truncating

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Deletion

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Insertion

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Splicing

Benefits

Sanger sequencing is a robust testing strategy able to determine whether a indicate 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 ramble variant in diagnostic testing. Primers can exist created to comprehend several regions (amplicons) to encompass any size region of involvement.

Limitations

Although one could employ individual Sanger sequencing reactions to cover any desired region, this testing approach can be plush when compared with other multiplex testing systems. Therefore, most currently available Sanger sequencing tests are cistron-specific or analyze a modest subset of genes. Sanger sequencing is able to identify mosaic mutations including as low equally 20% of the cells, but Sanger sequencing is not precisely quantifiable. For example, one cannot conclude if a mutation is nowadays in 25% versus forty% of cells based on peak sizes; boosted testing strategies must be used for quantification.

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Genomics of Infectious Diseases and Private Industry

Thousand. Vernet , in Genetics and Evolution of Infectious Diseases (Second Edition), 2017

ii.1 Sanger Sequencing

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

Sanger sequencing, although besides laborious and expensive for WGS, remains routinely 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 past culture and unmarried 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 washed on ABI sequencers (Thermo Fisher Scientific) instruments, a make 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 chemical science.

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A Clinical Guide to Monogenic Diabetes

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

Deletion Assay

Sanger sequencing can readily identify small indels (insertions or deletions). While partial or whole gene deletions brand upward a minority of monogenic diabetes cases, these may exist missed using Sanger sequencing alone. ⁷⁸ Medium-sized deletion longer than the PCR amplicons also are not detected because they cannot be amplified and may non always exist identified through multiplex ligation-dependent probe distension, the most commonly used test to screen for deletions. ⁷⁹ Dedicated studies to identify deletions are particularly of import when assessing families with an HNF1B-MODY (MODY5) phenotype as deletions are more mutual 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 tin exist maintained. ⁷⁷

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Integrating Molecular Diagnostics With Surgical Neuropathology

David A. Solomon Physician, PhD , in Applied Surgical Neuropathology: A Diagnostic Approach (Second Edition), 2018

Sanger Sequencing for Detection of Single Nucleotide Mutations

Sanger sequencing is a method developed by Frederick Sanger and colleagues in the 1970s that is based on selective incorporation of chain-terminating dideoxynucleotides by Deoxyribonucleic acid polymerase during in vitro DNA replication. ³¹ Modern Sanger sequencing typically uses fluorescently labeled dideoxynucleotides that are detected past a light amplification by stimulated emission of radiation 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 mutations or pocket-sized insertions/deletions in oncogenes and tumor suppressor genes in surgically resected pathology specimens. First, genomic Deoxyribonucleic acid is extracted from snap-frozen or formalin-fixed, alkane-embedded tumor tissue. And then, polymerase chain reaction (PCR) is performed using oligonucleotide primers and genomic Deoxyribonucleic acid isolated from the tumor tissue as a template to amplify the genetic region of interest (e.thou., exon 4 of IDH1 containing codon p.R132). Sanger sequencing reactions are and so 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:

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IDH1 exon four containing the p.R132 hotspot and IDH2 exon 4 containing the p.R172 hotspot, which are oftentimes mutated in WHO class 2 and Three oligodendrogliomas, form Two and III diffuse/anaplastic astrocytomas, and IDH-mutant glioblastomas (Fig. v.5)

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H3F3A and HIST1H3B containing the p.K27 hotspot often mutated in diffuse midline gliomas and rarely other midline tumor entities, including ganglioglioma and pilocytic astrocytoma

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BRAF exon 15 containing the p.V600 hotspot frequently mutated in pleomorphic xanthoastrocytoma, ganglioglioma, extracerebellar pilocytic astrocytoma, epithelioid glioblastoma, diffuse gliomas in children, and other tumor entities

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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 observe some of the recurrent mutations institute in oncogenes in brain tumors (e.chiliad., IDH1 p.R132H, histone H3 p.K27M, and BRAF p.V600E), mutant-specific antibodies for other important unmarried 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-generation sequencing is required for their assessment. Occasionally, mutant-specific antibodies may yield equivocal staining, therefore requiring confirmation past Sanger sequencing or other methodology. Additionally, while approximately eighty% to 90% of lengthened lower grade gliomas harbor the IDH1 p.R132H mutation that is detectable using the mutant-specific antibiotic, a subset instead harbor one of the less common IDH1 mutations (due east.thousand., p.R132C or p.R132S) or mutation of the equivalent p.R172 codon in IDH2. Determining that a lengthened glioma is "IDH-wildtype" ultimately requires Sanger sequencing or other sequencing methodology post-obit a negative immunohistochemical stain using the IDH1 R132H mutant-specific antibody (Fig. five.5). While the vast bulk of activating mutations in BRAF are the p.V600E mutation detectable by mutant-specific antibiotic, occasional p.V600K or p.V600M mutations accept been identified, likewise as pocket-sized in-frame insertions such as p.T599_V600insT that can all exist detected by Sanger sequencing but not by immunohistochemistry.

Equally with all molecular testing, limitations and interpretational pitfalls of Sanger sequencing should exist recognized. The major limitations are related to the quantity of tissue required and the sensitivity of detection. Dissimilar immunohistochemistry using mutant-specific antibodies, which requires only a single unstained department, Sanger sequencing and other sequencing methodologies typically require at least a few unstained slides in order to obtain sufficient genomic Deoxyribonucleic acid. Thus, in some cases, small biopsies taken from critical structures such as the brainstem or spinal string may be insufficient. The sensitivity of detection for Sanger sequencing is more often than not recognized as being approximately xv% to xx% mutant allele frequency, meaning that xv% to 20% of the DNA molecules existence sequenced demand to comprise the mutation in order to exist reliably detected. Since man 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.due east., present in only one of ii alleles) and fully clonal (i.e., present in all of the tumor cells). Extracting DNA from tissue that contains an acceptable quantity of tumor cells relative to admixed non-neoplastic cells can be challenging for some infiltrative gliomas or tumors with arable inflammatory infiltrate. In such cases, Sanger sequencing may be falsely negative for the mutation beingness tested. Lastly, Sanger and other DNA sequencing methodologies rely on extraction of intact genomic DNA. Tissue stock-still in formalin for an extended period of time (greater than a few days) may comprise such all-encompassing crosslinking of Dna that PCR amplification becomes challenging. In well-nigh cases, decalcification of tissue prior to processing and embedding is non uniform with molecular testing, including Sanger sequencing.

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Pulmonary Adenocarcinoma—Pathology and Molecular Testing

Prodipto Pal MD, PhD , ... Ming-Audio Tsao MD, FRCPC , in Pulmonary Adenocarcinoma: Approaches to Treatment, 2019

Nontargeted assays

Sanger sequencing was the standard for EGFR testing in the start clinical trials with erlotinib and gefitinib. This method incorporates fluorescent-tagged dideoxy terminators to amplifying DNA strands, which can be sorted past size and the nucleotide sequence read sequentially. A major consideration is the relatively low analytical sensitivity of the assay, which commonly requires specimens with high tumor content. As such, this assay is no longer a method of selection for detection of EGFR mutations. Another method, called pyrosequencing, involves measuring the chemiluminescent indicate released by pyrophosphate as triphosphate nucleotides are being incorporated into the synthesized Dna strand. Although the fragment length required for pyrosequencing 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.

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Pathology, Biomarkers, and Molecular Diagnostics

Wilbur A. Franklin , ... Marileila Varella Garcia , in Abeloff'due south Clinical Oncology (5th Edition), 2014

Sanger Sequencing

Sanger sequencing has been the aureate standard for many years and, similar many other assays, is based on the dideoxy-chemistry illustrated in Effigy 17-11. The sequencing reaction is read equally a color lawmaking that distinguishes oligonucleotides of variable length with 4 specific color labels. A ladder of nucleotides is created that can exist identified by their electrophoretic mobility and color of terminal fluorescent nucleotide. The identification of abnormal (mutant) peaks in chromatograms of this ladder can exist facilitated by reckoner 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 Deoxyribonucleic acid 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 volition notice about any mutation in the targeted region. However, the analytic sensitivity is express, with tumor jail cell concentration of approximately 50% required for authentic results. Considering of the PCR distension, the corporeality of DNA starting textile required is usually pocket-size, depending on the number of DNA templates selected for amplification.

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Locked Nucleic Acid Engineering science for Highly Sensitive Detection of Somatic Mutations in Cancer

Takayuki Ishige , ... Kazuyuki Matsushita , in Advances in Clinical Chemical science, 2018

3.3 Sanger Sequencing

Sanger sequencing, also known equally the concatenation termination method, was developed by Sanger et al. [27]. Many clinical laboratories perform direct sequencing of PCR products using this method. In bike sequencing, PCR products, sequencing primers (either frontwards or opposite), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs, typically labeled with a dissimilar fluorescent dye), and thermostable Deoxyribonucleic acid polymerase are included in the reaction mixture. Kickoff, the sequencing primer hybridizes the PCR products and is elongated by the DNA polymerase during PCR. ddNTPs are randomly incorporated in the DNA strands during elongation, thereby terminating strand elongation at each location forth the sequence. Subsequent capillary electrophoresis separates the Deoxyribonucleic acid 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 assay and tin determine the entire sequence and identify unknown mutations. The major limitations of this method are high cost, labor intensiveness, and depression sensitivity. The sensitivity of this method is 10%–20% mutations in the wild-type background [38,43]. Thus, low frequent mutations (<   x%) in tumor samples cannot be determined using Sanger sequencing.

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Other Mail service-PCR Detection Technologies

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

Sanger (Conventional) Sequencing

Sanger sequencing using capillary sequencers has go very popular in molecular diagnostic laboratories. Multiple sequencing reactions are loaded onto multiwell plates, which are injected into capillaries for electrophoresis on the musical instrument. In sequencing reactions, primers that anneal to a single-stranded DNA template are elongated by Dna (Taq) polymerase. Fluorescent-labeled deoxynucleotides are introduced one at a fourth dimension 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 reverberate the sequence existence analyzed. Each capillary is separately calibrated for the dyes used in the sequencing reactions and so that the software can perform the multi-component analysis to identify each of the dye-labeled fragments ( Figure 5 ). These sequencing reactions are so analyzed using special software.

Figure five. 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 past ane of the four ddNTPs. The ratio of deoxynucleotides to ddNTPs is optimized to produce a counterbalanced population of long and brusk extension products similar to the conventional Sanger sequencing method. Detection in cycle sequencing tin exist accomplished using two different dye-labeling chemistries: the dye terminators or the dye primer labeling. The iv ddNTP terminators are tagged with different fluorescent dyes. Merely 1 reaction is performed with all the reagents such every bit 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 comport a dissimilar-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 1 capillary and are distinguished equally individual nucleotides with unique fluorophores that are recognized by the laser. The information from the light amplification by stimulated emission of radiation is captured past photomultiplier tubes to generate an electropharagram that represents the unique sequence.

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