All cancers are diseases of the genome, since the cancer cell genome typically consists of 10,000s of passenger alterations, 5—10 biologically relevant alterations, and 1—2 “actionable” alterations. Therefore, somatic mutations in cancer cells can have diagnostic, prognostic, and predictive value. Traditional methods are widely used for testing, such as immunohistochemistry, Sanger sequencing, and allele-specific PCR. However, due to the low throughput, these methods are focused exclusively on testing the most common mutations in target genes. The modern next generation sequencing (NGS) is a technology that enables precision oncology in its current form. ESCAT and ESMO Guidelines defined NGS for routine use in patients with advanced cancers such as non-squamous non-small cell lung cancer, prostate cancer, ovarian cancer, and cholangiocarcinoma. The high sensitivity of the NGS method allows it to be used to search for specific mutations in circulating tumor DNA in blood plasma and other body fluids. NGS testing has evolved from hotspot panels, actionable gene panels, and disease-specific panels to more comprehensive panels. The exome and whole genome sequencing approaches are just beginning to emerge, that is why panel-based testing remains most optimal in oncology practice. NGS is also widely used to identify new and rare mutations in cancer genes and detect inherited cancer mutations.

Although next-generation sequencing (NGS) technologies have revolutionized our ability to sequence DNA with high-throughput, the chain termination-based Sanger sequencing method remains a widely used approach for DNA sequence analysis due to its simplicity, low cost and high accuracy. In particular, high accuracy makes Sanger sequencing the "gold standard" for sequence validation in basic research and clinical applications. During the early days of Sanger sequencing development, reverse transcriptase (RT)-based RNA sequencing was also explored and showed great promise, but the approach did not acquire popularity over time due to the limited processivity and low template unwinding capability of Avian Myeloblastosis Virus (AMV) RT, and other RT enzymes available at the time. RNA molecules have complex features, often containing repetitive sequences and stable secondary or tertiary structures. While these features are required for RNA biological function, they represent strong obstacles for retroviral RTs. Repetitive sequences and stable structures cause reverse transcription errors and premature primer extension stops, making chain termination-based methods unfeasible. MarathonRT is an ultra-processive RT encoded group II intron that can copy RNA molecules of any sequence and structure in a single cycle, making it an ideal RT enzyme for Sanger RNA sequencing. In this chapter, we upgrade the Sanger RNA sequencing method by replacing AMV RT with MarathonRT, providing a simple, ro

Accurate genotyping of small insertions and deletions (InDels; <5 bp) remains technically challenging in routine molecular breeding, largely due to the limited resolution of agarose gel electrophoresis and the labor-intensive nature of polyacrylamide-based assays. Here, we present the Tri-Primer Amplification Refractory Mutation System (TP-ARMS), a simple and cost-effective PCR-based strategy that enables high-resolution genotyping of small InDels using standard agarose gels. The TP-ARMS employs a universal reverse primer in combination with two allele-specific forward primers targeting insertion and deletion alleles, respectively. This design allows clear discrimination of homozygous and heterozygous genotypes using a two-tube PCR workflow. The method showed complete concordance with Sanger sequencing in detecting 1-5 bp InDels across multiple crop species, including rice (<i>Oryza sativa</i>) and quinoa (<i>Chenopodium quinoa</i>). In addition, using a TP-ARMS reduced experimental time by approximately 90% compared with PAGE-based approaches and avoided the high equipment and DNA quality requirements of fluorescence-based assays. The practical applicability of the TP-ARMS was demonstrated in breeding populations, including efficient genotyping of a 3-bp InDel in <i>OsNRAMP5</i> associated with cadmium accumulation and a 6-bp promoter InDel in <i>OsSPL10</i> underlying natural variation in rice trichome density across 370 accessions. Collectively, the TP-ARMS provides a robust, scalable, and low-cost solution for precise small InDel genotyping, with broad applicability in marker-assisted breeding and functional genetic studies.

Sometimes, leftover materials from select species are also passed on to the expert Natural History Museum curators. Frozen test tubes - species sample management - credit Wellcome Sanger Institute Dive into the laboratory Once the WWT specimens have finished their time at the Natural History Museum, these are sent to the Sanger Institute where the team are ready to prepare samples for DNA extraction, genome sequencing, and experts assemble these genomes for researchers around the world to use. Sanger Institute laboratory staff, Amy Denton, trained as a veterinarian but was always drawn to research in the laboratory. Amy extracts DNA from all sorts of taxa - such as bird, small mammal and frog samples. As genomes are all as different as the species they represent, the methods used are constantly being updated and improved. Amy remembers one WWT sample - the elegant tundra swan, was particularly tricky. High-quality DNA for each WWT sample is extracted by a well-established process, developed by the Sanger Institute. Once Amy has extracted DNA, the samples are checked and sent for DNA sequencing.

DNA sequencing, which deciphers DNA nucleotide sequence, has had a transformative impact on biology and medicine. The quest began in 1953 with Watson and Crick’s discovery of the DNA double helix, based on Franklin and Wilkins’ pioneering work in X-ray crystallography. Arthur Kornberg’s 1956 discovery of DNA polymerase further advanced the field, laying the groundwork for future sequencing technologies. In 1977, Frederick Sanger’s chain-termination method (Sanger sequencing) and Maxam-Gilbert’s chemical sequencing emerged as the first viable techniques for reading DNA. Sanger sequencing, in particular, remains widely used for analyzing shorter DNA sequences today. The 1980s saw the introduction of automated sequencers, which dramatically boosted throughput, precision, and accessibility. Launched in 1990, The Human Genome Project (HGP) utilized Sanger sequencing to map the human genome, marking a historic achievement that is often compared to landing on the moon. The early 2000s ushered in next-generation sequencing (NGS) technologies, such as pyrosequencing, sequencing by synthesis (SBS), and SOLiD, offering faster and more cost-effective sequencing. Today, third-generation technologies like Oxford Nanopore’s “nanopore” sequencing and PacBio’s Single-Molecule Real-Time (SMRT) sequencing enable longer reads and real-time data. These advancements have revolutionized genomics, driving progress in evolutionary biology, precision medicine, and forensic science.

The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of heritable muscle disorders with often difficult to interpret muscle pathology, making them challenging to diagnose. Serial Sanger sequencing of suspected CMD genes, while the current molecular diagnostic method of choice, can be slow and expensive. A comprehensive panel test for simultaneous screening of mutations in all known CMD-associated genes would be a more effective diagnostic strategy. Thus, the CMDs are a model disorder group for development and validation of next-generation sequencing (NGS) strategies for di

Traditional Sanger sequencing as well as Next-Generation Sequencing have been used for the identification of disease causing mutations in human molecular research. The majority of currently available tools are developed for research and explorative purposes and often do not provide a complete, efficient, one-stop solution. As the focus of currently developed tools is mainly on NGS data analysis, no integrative solution for the analysis of Sanger data is provided and consequently a one-stop solution to analyze reads from both sequencing platforms is not available. We have therefore developed a

during in vitro DNA replication. After first being developed by Frederick Sanger and colleagues in 1977, it became the most widely used sequencing method 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 el 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. Later, automated slab gels were replaced with automated capillary array electrophoresis. 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%. Sanger sequencing is still actively being used in efforts for public health initiatives such as sequencing the spike protein from SARS-CoV-2 as well as for the surveillance of norovirus outbreaks through the United States Center for Disease Control and Prevention (CDC)'s CaliciNet surveillance network.

Nanopore-based sequencing has emerged as a revolutionary tool for animal pathogen genomics, offering capabilities unattainable with Sanger and next-generation sequencing (NGS). Despite rapid technical progress, routine veterinary deployment still faces uncertainty in study design, sample preparation, and interpretation thresholds across diverse hosts and sample matrices. Accordingly, this review consolidates recent evidence and provides workflow-oriented guidance for veterinary diagnostics and One Health surveillance. Its portability, ability to generate real-time long-read data, and minimal infrastructure requirements enable rapid, on-site sequencing for veterinary diagnostics and surveillance. This review examines the principles of nanopore sequencing and its advantages over conventional methods, surveying recent applications across viral, bacterial (including antimicrobial resistance, AMR), and parasitic pathogen detection in animals. In viral diagnostics, it facilitates rapid whole-genome sequencing and outbreak tracing in field settings. For bacterial pathogens, it enables near-complete genome assembly and identification of plasmid-borne AMR genes. Emerging studies also demonstrate its utility in parasitology, from high-resolution species identification to whole-genome assemblies. We compare these advancements with traditional diagnostics, highlighting strengths in speed and comprehensiveness while addressing current limitations in accuracy and host-DNA interference. As technology matures through improvements in chemistry and adaptive sampling, nanopore sequencing is poised to transform veterinary pathogen detection and bolster One Health surveillance of emerging zoonoses.