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PubMed1998

The Sanger Centre.

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Academies and Institutes↗
PubMed2006

454 sequencing put to the test using the complex genome of barley.

BACKGROUND: During the past decade, Sanger sequencing has been used to completely sequence hundreds of microbial and a few higher eukaryote genomes. In recent years, a number of alternative technologies became available, among them adaptations of the pyrosequencing procedure (i.e. "454 sequencing"), promising an approximately 100-fold increase in throughput over Sanger technology--an advancement which is needed to make large and complex genomes more amenable to full genome sequencing at affordable costs. Although several studies have demonstrated its potential usefulness for sequencing small and compact microbial genomes, it was unclear how the new technology would perform in large and highly repetitive genomes such as those of wheat or barley. RESULTS: To study its performance in complex genomes, we used 454 technology to sequence four barley Bacterial Artificial Chromosome (BAC) clones and compared the results to those from ABI-Sanger sequencing. All gene containing regions were covered efficiently and at high quality with 454 sequencing whereas repetitive sequences were more problematic with 454 sequencing than with ABI-Sanger sequencing. 454 sequencing provided a much more even coverage of the BAC clones than ABI-Sanger sequencing, resulting in almost complete assembly of all genic sequences even at only 9 to 10-fold coverage. To obtain highly advanced working draft sequences for the BACs, we developed a strategy to assemble large parts of the BAC sequences by combining comparative genomics, detailed repeat analysis and use of low-quality reads from 454 sequencing. Additionally, we describe an approach of including small numbers of ABI-Sanger sequences to produce hybrid assemblies to partly compensate the short read length of 454 sequences. CONCLUSION: Our data indicate that 454 pyrosequencing allows rapid and cost-effective sequencing of the gene-containing portions of large and complex genomes and that its combination with ABI-Sanger sequencing and targeted sequence analysis can result in large regions of high-quality finished genomic sequences.

Base Pairing↗
PubMed2026

Simultaneous detection of glyphosate and glufosinate target-site resistance in Eleusine indica via multiplex TaqMan qPCR.

BACKGROUND: Continuous use of glyphosate followed by glufosinate-ammonium has selected for multiple resistance to both herbicides in Eleusine indica worldwide. Managing such resistant weeds requires fast, accurate molecular detection assay. To address this critical need, we developed a robust multiplex TaqMan quantitative (q)PCR assay that simultaneously detects five well-characterized target-site resistance markers in E. indica: EPSPS copy number variation; T102I in EPSPS; P106A and P106S in EPSPS; and S59G in GS1-1. RESULTS: The multiplex qPCR assay showed analytical specificity when tested on genomic DNA from nine reference accessions: three susceptible, three glyphosate-resistant (with EPSPS CNV) and three multiple-resistant. Subsequent analysis of 56 field-collected samples demonstrated 98.2% concordance (55 of 56) with Sanger sequencing across all five resistance-associated markers: EPSPS CNV, T102I, P106A, P106S and GS1-1 S59G, confirming the reliability and practical value of the multiplex qPCR assay. Only samples 7-8 showed discordance at EPSPS position 102, where Sanger chromatograms showed overlapping peaks at this position, which is likely to be a result of heterozygous mutation distribution among amplified EPSPS gene copies. This case further underscores the advantages of the multiplex qPCR assay over Sanger sequencing in detection sensitivity and accuracy. Moreover, a strong correlation (R2 = 0.8935) in gene copy number estimation between the two methods across all samples further supports the reliability of the qPCR assay. CONCLUSIONS: In summary, this study delivers a simple, robust and high-throughput diagnostic tool for the rapid, simultaneous identification of dual herbicide target-site resistance in goosegrass, offering superior sensitivity, quantitative resolution and throughput compared with Sanger sequencing. © 2026 Society of Chemical Industry.

Herbicides↗
PubMed1996

Analyzing sequencing reactions from bacteriophage M13 by matrix-assisted laser desorption/ionization mass spectrometry.

The current demand for improved DNA sequencing methodologies posed by the Human Genome Project has spurred the investigation of alternatives to gel electrophoresis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has great potential for the rapid analysis of DNA fragments. Mock Sanger sequencing mixtures have been successfully analyzed by MALDI by pooling synthesized oligonucleotides corresponding to the M13 bacteriophage sequence. More recently, analyses of Sanger sequencing fragments enzymatically generated from synthetic templates of 45 or 50 bases were reported. In the present study, these feasibility demonstrations are extended to show MALDI sequencing from the M13 bacteriophage DNA template commonly used in actual Sanger sequencing. The results show sequence determination for extension products up to 35 bases in length. Different desalting and purification procedures were investigated and it was found that salt could be efficiently reduced by removal of the template in a post-reaction step. Work in progress to stabilize DNA by chemical modification, employed in conjunction with the methods described here, should enable significant extension of the length of readable sequence.

Bacteriophage M13↗
PubMed2006

Genomic rearrangements in MSH2, MLH1 or MSH6 are rare in HNPCC patients carrying point mutations.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease with high penetrance, caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, PMS2 and MLH3. Most reported pathogenic mutations are point mutations, comprising single base substitutions, small insertions and deletions. In addition, genomic rearrangements, such as large deletions and duplications not detectable by PCR and Sanger sequencing, have been identified in a significant proportion of HNPCC families, which do not carry a pathogenic MMR gene point mutation. To clarify whether genomic rearrangements in MLH1, MSH2 or MSH6 also occur in patients carrying a point mutation, we subjected normal tissue DNA of 137 colorectal cancer (CRC) patients to multiplex ligation-dependent probe amplification (MLPA) analysis. Patients fulfilled the following pre-requisites: all patients met at least one criterion of the Bethesda guidelines and their tumors exhibited high microsatellite instability (MSI-H) and/or showed loss of expression of MLH1, MSH2 or MSH6 proteins. PCR amplification and Sanger sequencing of all exons of at least one MMR gene, whose protein expression had been lost in the tumor tissue, identified 52 index patients without a point mutation (Group 1), 71 index patients with a pathogenic point mutation in MLH1 (n=38) or MSH2 (n=22) or MSH6 (n=11) (Group 2) and 14 patients with an unclassified variant in MLH1 (n=9) or MSH2 (n=3) or MSH6 (n=2) (Group 3). In 13 of 52 patients of group 1 deletions of at least one exon were identified. In addition, in group 3 one EX1_15del in MLH1 was found. No genomic rearrangement was identified in group 2 patients. Genomic rearrangements represent a significant proportion of pathogenic mutations of MMR genes in HNPCC patients. However, genomic rearrangements are rare in patients carrying point mutations in MMR genes. These findings suggest the use of genomic rearrangement tests in addition to Sanger sequencing in HNPCC patients.

Adaptor Proteins, Signal Transducing↗
PubMed2006

A Sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes.

Since its introduction a decade ago, whole-genome shotgun sequencing (WGS) has been the main approach for producing cost-effective and high-quality genome sequence data. Until now, the Sanger sequencing technology that has served as a platform for WGS has not been truly challenged by emerging technologies. The recent introduction of the pyrosequencing-based 454 sequencing platform (454 Life Sciences, Branford, CT) offers a very promising sequencing technology alternative for incorporation in WGS. In this study, we evaluated the utility and cost-effectiveness of a hybrid sequencing approach using 3730xl Sanger data and 454 data to generate higher-quality lower-cost assemblies of microbial genomes compared to current Sanger sequencing strategies alone.

Biotechnology↗
PubMed2025

Genetic analysis of three patients from two unrelated Chinese families with autosomal recessive spastic ataxia of Charlevoix-Saguenay.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare early-onset neurodegenerative disorder characterized by progressive cerebellar ataxia, spasticity, and sensorimotor peripheral neuropathy. This disorder is caused by homozygous or compound heterozygous variants in the sacsin (SACS) gene on chromosome 13q12.12. Three patients with ARSACS from two unrelated Chinese families were recruited for this study. Patient #1 was an 18-year-old male who had been walking unstably for 12 years. Patient #2, the younger sister of Patient #1, was a 5-year-old girl who had been walking unstably for 2 years. Patient #3 was a 19-year-old female who had been walking unstably and a tendency to fall for 17 years. For Patient #1, whole-exome sequencing (WES) identified a hemizygous variant c.8310_8313delAGAT (p.Asp2771fs4*) in SACS (NM_014363.6), with the father being heterozygous, the mother wild-type, and Patient #2 hemizygous, as verified by Sanger sequencing. Additional copy number variant analysis of the WES data indicated that Patient #1 had a heterozygous gross deletion of chr13q12.12 (chr13:23,808,732 - 24,890,322). Low-coverage whole-genome sequencing results revealed that Patient #2 carried a chr13q12.12 deletion (chr13:23,520,000-24,940,000). Together with Sanger sequencing results, this gross deletion was speculated to have been inherited from the mother, further explaining the hemizygous state of c.8310_8313delAGAT (p.Asp2771fs4*) in Patients #1 and #2. Through WES, Patient #3 was identified as having suspected compound heterozygous variants of c.2881 C > T (p.Arg961*) and c.6409 C > T (p.Gln2137*), inherited from the father and mother, respectively, as confirmed by Sanger sequencing. This study identified three variants in SACS. The c.8310_8313delAGAT (p.Asp2771fs4*) is novel, whereas c.2881 C > T (p.Arg961*) and c.6409 C > T (p.Gln2137*) have been reported previously. Moreover, this study highlights the growing trend that ARSACS has become increasingly prevalent worldwide rather than being localized to a specific region or race. As an increasing number of patients with ARSACS are diagnosed, the genetic spectrum of ARSACS will gradually broaden, providing an accurate genetic basis for prenatal diagnosis of mothers in the years ahead, if possible.

Adolescent↗
PubMed2000

Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins.

When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the host's cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning regions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody staining of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit a number of focal adhesion proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli.

Actinin↗
PubMed2024

Variants in HCFC1 and MN1 genes causing intellectual disability in two Pakistani families.

BACKGROUND: Intellectual disability (ID) is a neurodevelopmental condition affecting around 2% of children and young adults worldwide, characterized by deficits in intellectual functioning and adaptive behavior. Genetic factors contribute to the development of ID phenotypes, including mutations and structural changes in chromosomes. Pathogenic variants in the HCFC1 gene cause X-linked mental retardation syndrome, also known as Siderius type X-linked mental retardation. The MN1 gene is necessary for palate development, and mutations in this gene result in a genetic condition called CEBALID syndrome. METHODS: Exome sequencing was used to identify the disease-causing variants in two affected families, A and B, from various regions of Pakistan. Affected individuals in these two families presented ID, developmental delay, and behavioral abnormalities. The validation and co-segregation analysis of the filtered variant was carried out using Sanger sequencing. RESULTS: In an X-linked family A, a novel hemizygous missense variant (c.5705G > A; p.Ser1902Asn) in the HCFC1 gene (NM_005334.3) was identified, while in family B exome sequencing revealed a heterozygous nonsense variant (c.3680 G > A; p. Trp1227Ter) in exon-1 of the MN1 gene (NM_032581.4). Sanger sequencing confirmed the segregation of these variants with ID in each family. CONCLUSIONS: The investigation of two Pakistani families revealed pathogenic genetic variants in the HCFC1 and MN1 genes, which cause ID and expand the mutational spectrum of these genes.

Humans↗
PubMed1996

Affinity methods for purification of DNA sequencing reaction products for mass spectrometric analysis.

Time-of-flight mass spectrometry has the potential to replace gel electrophoresis for rapid and accurate analysis of DNA sequencing mixtures. However, impurities in the Sanger sequencing reaction solutions can complicate and degrade the mass spectrometric performance. Therefore, a fast purification procedure is necessary for mass spectrometric analysis. Two affinity methods were tested for the capture of the target fragments: a probe strand, complementary to the primer used to initiate synthesis of the Sanger fragments, is immobilized to a solid support either before or after hybridization so that the impurities are readily separated by filtering and washing the support material. The approaches were tested using mock sequencing mixtures assembled from synthetic DNA strands, to which representative impurities could be added. The results showed that the latter method has better overall yield. The recovered fragments were analyzed by matrix-assisted laser desorption/ionization.

Bacterial Proteins↗
PubMed1997

Gel electrophoresis of DNA fragments in narrow-bore capillaries.

In this work, we studied the behavior of electrophoretic columns, having an inner diameter (ID) of 2-10 microm, filled with a cross-linked polyacrylamide gel matrix. The usefulness of these columns for DNA sequencing is discussed. Evaluation of column performance included tests of gel stability and migration time reproducibility. Confocal laser-induced fluorescence (LIF)-detection was employed utilizing a 488 nm argon ion laser for separations of C- and T-terminated DNA Sanger fragments. Reducing the inner diameter of the column from 50 microm to 10 microm resulted in an approximately eightfold increase in lifetime, under conditions in which the columns were subjected to a field strength of 1000 V/cm. The 10 microm ID columns were utilized for separation of Sanger fragments, and adequate detection sensitivity was obtained by stacking of the fragments from a deionized sample solution. A linear algorithm for retention data synchronization between individual electropherograms was employed to provide a route towards a reliable automated base calling protocol.

Acrylic Resins↗
PubMed2026

Analysis of Genetic Factors in a Family With Short Stature.

BACKGROUND: To elucidate the genetic underpinnings of short stature in a familial cohort of five individuals. METHODS: A family with a history of short stature from Zhongnan Hospital of Wuhan University was the subject of this study. Peripheral blood samples were collected from family members for whole exome sequencing and Sanger sequencing to identify genetic anomalies. RESULTS: The male proband, aged 3&#x2009;years and 10&#x2009;months, had significant growth retardation, with a height of 91&#x2009;cm (<&#x2009;3rd percentile) and a weight of 13&#x2009;kg (<&#x2009;3rd percentile). Whole exome sequencing identified a missense mutation in the COL1A2 gene (c.577G>A, p.Gly193Ser) with maternal inheritance. Sanger sequencing confirmed this mutation in the mother and half-sister. According to American College of Medical Genetics and Genomics (ACMG) guidelines, this variant was classified as likely pathogenic. Additionally, a heterozygous mutation in the GH1 gene (c.291+1G>A) was detected in the father and grandfather, contributing to the familial short stature phenotype. CONCLUSION: In this family, we identified that variants in the COL1A2 and the GH1 can each cause short stature. This reflects both the genetic consistency and complexity of short stature, which is highly dependent on comprehensive genetic testing.

Humans↗