scholarly journals Beyond Homopolymer Errors: a Systematic Investigation of Nanopore-based DNA Sequencing Characteristics Using HLA-DQA2

2017 ◽  
Vol 61 (3) ◽  
pp. 231 ◽  
Author(s):  
Péter Sárközy ◽  
Viktor Molnár ◽  
Dóra Fogl ◽  
Csaba Szalai ◽  
Péter Antal
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Gene Sequencing ◽  
Systematic Investigation ◽  
Sequencing Technology ◽  
Sequencing Platform ◽  
Lower Accuracy ◽  
Pcr Products ◽  
Long Range Pcr ◽  
Generation Sequencing

Electronic, nanopore based single molecule real-time DNA sequencing technology offers very long, albeit lower accuracy reads in sharp contrast to existing next-generation sequencing methods, which offer short, high-accuracy reads in abundance. We provide a systematic review of the error characteristics of this new sequencing platform, and demonstrate the most challenging aspects in the field of whole gene sequencing through the human HLA-DQA2 gene using long-range PCR products on multiplexed samples. We consider the limitations of these errors for the applications of this technology, and also indicate prospective improvements and expected thresholds with respect to these errors.

scholarly journals Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets

2020 ◽  
Vol 48 (22) ◽  
pp. e132-e132
Author(s):  
Tim J Puchtler ◽  
Kerr Johnson ◽  
Rebecca N Palmer ◽  
Emma L Talbot ◽  
Lindsey A Ibbotson ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Direct Detection ◽  
Gc Content ◽  
Cost Effective ◽  
Epigenetic Modifications ◽  
Fluorescence Signal ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Lower Accuracy

Abstract Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.

scholarly journals Next-Generation Sequencing: From Basic Research to Diagnostics

2009 ◽  
Vol 55 (4) ◽  
pp. 641-658 ◽  
Author(s):  
Karl V Voelkerding ◽  
Shale A Dames ◽  
Jacob D Durtschi
Keyword(s):  
Next Generation Sequencing ◽  
Dna Sequencing ◽  
Single Molecule ◽  
Basic Research ◽  
Clinical Diagnostics ◽  
Massively Parallel ◽  
Next Generation ◽  
New Paradigm ◽  
The Impact ◽  
Generation Sequencing

Abstract Background: For the past 30 years, the Sanger method has been the dominant approach and gold standard for DNA sequencing. The commercial launch of the first massively parallel pyrosequencing platform in 2005 ushered in the new era of high-throughput genomic analysis now referred to as next-generation sequencing (NGS). Content: This review describes fundamental principles of commercially available NGS platforms. Although the platforms differ in their engineering configurations and sequencing chemistries, they share a technical paradigm in that sequencing of spatially separated, clonally amplified DNA templates or single DNA molecules is performed in a flow cell in a massively parallel manner. Through iterative cycles of polymerase-mediated nucleotide extensions or, in one approach, through successive oligonucleotide ligations, sequence outputs in the range of hundreds of megabases to gigabases are now obtained routinely. Highlighted in this review are the impact of NGS on basic research, bioinformatics considerations, and translation of this technology into clinical diagnostics. Also presented is a view into future technologies, including real-time single-molecule DNA sequencing and nanopore-based sequencing. Summary: In the relatively short time frame since 2005, NGS has fundamentally altered genomics research and allowed investigators to conduct experiments that were previously not technically feasible or affordable. The various technologies that constitute this new paradigm continue to evolve, and further improvements in technology robustness and process streamlining will pave the path for translation into clinical diagnostics.

scholarly journals Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the ACKR1 Gene Encoding the Duffy Antigens

2019 ◽  
Vol 47 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Yann Fichou ◽  
Isabelle Berlivet ◽  
Gaëlle Richard ◽  
Christophe Tournamille ◽  
Lilian Castilho ◽  
...  
Keyword(s):  
Blood Group ◽  
Single Molecule ◽  
Pcr Amplification ◽  
Null Alleles ◽  
Sequencing Technology ◽  
Gene Encoding ◽  
Next Generation Sequencing Technology ◽  
Sequencing Technologies ◽  
Long Read ◽  
Long Range Pcr

Background: In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. Materials and Methods: After long-range PCR amplification spanning the whole ACKR1 gene locus (∼2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. Results: High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. Discussion: Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a “longitudinal” overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.

scholarly journals Systematic Comparison of Three Methods for Fragmentation of Long-Range PCR Products for Next Generation Sequencing

PLoS ONE ◽  
2011 ◽  
Vol 6 (11) ◽  
pp. e28240 ◽  
Author(s):  
Ellen Knierim ◽  
Barbara Lucke ◽  
Jana Marie Schwarz ◽  
Markus Schuelke ◽  
Dominik Seelow
Keyword(s):  
Next Generation Sequencing ◽  
Long Range ◽  
Next Generation ◽  
Systematic Comparison ◽  
Pcr Products ◽  
Long Range Pcr ◽  
Generation Sequencing

scholarly journals Assessing the quality of Astragalus fermented using Lactobacillus plantarum and Enterococcus faecium by its physicochemical properties and monitoring the bacterial composition with single molecule, real-time sequencing technology

2018 ◽  
Author(s):  
Hongxing Qiao ◽  
Xiaojing Zhang ◽  
Hongtao Shi ◽  
Yuzhen Song ◽  
Chuanzhou Bian
Keyword(s):  
Real Time ◽  
Lactobacillus Plantarum ◽  
Single Molecule ◽  
Enterococcus Faecium ◽  
Sequencing Technology ◽  
Production Rates ◽  
Microbial Composition ◽  
Sequencing Platform ◽  
Positive Effects

Background. Astragalus was a well-known traditional herbal medicine, widely used in human s , livestock and poultry in China and E ast Asia. Fermentation could improve health-promoting biological substance by probiotics. Methods. We investigated Astragalus that was fermented using probiotics including Enterococcus faecium , Lactobacillus plantarum and E nterococcus faecium + L actobacillus plantarum and applied the PacBio single molecule, real-time sequencing technology (SMRT) to evaluat e the quality of Astragalus fermentation production. Results. We found the production rates of acetic acid, methylacetic acid , ethylacetic acid and lactic acid using E. faecium + L. plantarum fermentation were 1866.24 mg/kg on day 15 , 203.80 mg/kg on day 30 , 996.04 mg/kg on day 15 and 3081.99 mg/kg on day 20 , respectively. Other production rates were: polysaccharide s, 9.43%, 8.51% and 7 . 59% on day 10; saponins , 19.6912 mg/g, 21.6630 mg/g and 20.2084 mg/g on day 15; and flavonoid s, 1.9032 mg/g, 2.0835 mg/g and 1.7086 mg/g on day 20 using E . faecium , L . plantarum and E. faecium + L. plantarum , respectively. According to SMRT analysis of the microbial composition s of nine Astragalus samples, we found after fermentation on day 3 , E. faecium and L. plantarum became the most prevalent species. Moreover, E. faecium + L. plantarum gave more positive effects than single strains in the Astragalus solid state fermentation process. Inclusion. Our data have demonstrate d that the SMRT sequencing platform is applicable to assessing the quality of Astragalus fermentation.

scholarly journals Assessment of the Application and Clinical Value of Next-Generation Sequencing for the Diagnosis of Inherited Thrombocytopenia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4993-4993
Author(s):  
Miao Jiang ◽  
Qi Wang ◽  
Yiming Zhao ◽  
Ziqiang Yu ◽  
Suning Chen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Conflicts Of Interest ◽  
Gene Sequencing ◽  
Hereditary Diseases ◽  
Next Generation ◽  
Clinical Value ◽  
Sequencing Platform ◽  
Pathogenic Variants ◽  
Inherited Thrombocytopenia ◽  
Generation Sequencing

Abstract Inherited thrombocytopenia is a group of hereditary diseases with a reduction in platelet count as the main clinical manifestation. Clinically, there is an urgent need for a convenient and rapid diagnosis method. We introduced a high-throughput next-generation sequencing (NGS) platform into the routine diagnosis of patients with unexplained thrombocytopenia and analyzed the gene sequencing results to evaluate the value of NGS technology in the screening and diagnosis of inherited thrombocytopenia. From a cohort of 182 patients with thrombocytopenia, we screened 78 patients with hereditary features. For the blood samples of these 78 patients, a gene sequencing platform for hemorrhagic and thrombotic diseases comprising 89 genes was used to perform gene detection using NGS technology. When we combined the screening results with clinical features and other findings, 23 of 78 patients (29.5%) were diagnosed with inherited thrombocytopenia. In addition, 29 pathogenic variants, including 11 previously unreported variants, were identified in these patients. In summary, NGS could play more important role in the molecular pathology diagnosis of inherited thrombocytopenia. Through the use of this detection platform, we expect to establish a more effective diagnostic approach to such disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Trichoderma reesei Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences

2021 ◽  
Vol 118 (8) ◽  
pp. e2007192118
Author(s):  
Wan-Chen Li ◽  
Chia-Yi Lee ◽  
Wei-Hsuan Lan ◽  
Tai-Ting Woo ◽  
Hou-Cheng Liu ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Single Molecule ◽  
Amino Acid Residues ◽  
Sequencing Technology ◽  
Structural Variations ◽  
Genome Sequences ◽  
Third Generation Sequencing ◽  
L1 And L2 ◽  
Generation Sequencing ◽  
Better Than

Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity. We applied multidisciplinary approaches (next- and third-generation sequencing technology, genetics, cytology, bioinformatics, biochemistry, and single-molecule biophysics) to show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and, like ScDmc1, TrRad51 possesses better mismatch tolerance than ScRad51 during homologous recombination. Our results also indicate that the ancestral TrRad51 evolved to acquire ScDmc1-like properties by creating multiple structural variations, including via amino acid residues in the L1 and L2 DNA-binding loops.

scholarly journals Current and Future Methodology for Quantitation and Site-specific Mapping the Location of DNA Adducts

2021 ◽  
Author(s):  
Gunnar Boysen ◽  
Intawat Nookaew
Keyword(s):  
Dna Sequencing ◽  
Dna Adducts ◽  
Single Molecule ◽  
State Of The Art ◽  
Dna Adduct ◽  
Sequencing Technology ◽  
Specific Mass ◽  
Comprehensive Picture ◽  
Specific Mapping ◽  
Altered Gene

Abstract: Formation of DNA adducts is a key event for a genotoxic mode of action and its formation is often use as surrogate for mutation and cancer. Interest in DNA adducts are twofold, first, to demonstrate exposure, and second, to link DNA adduct location to subsequent mutations or altered gene regulation. High chemically specific mass spectrometry methods have been established for DNA adduct quantitation and elegant bio-analytic methods utilizing enzymes, various chemistries, and molecular biology methods to visualize the location of DNA adducts. Traditionally, these highly specific methods cannot be combined, and the results are incomparable. Initially developed for single-molecule DNA sequencing, nanopore-type technologies are expected to enable simultaneous quantitation and location of DNA adducts across the genome. We will briefly summarize the current methodologies for state-of-the-art quantitation of DNA adduct levels and mapping of DNA adducts and describe novel single-molecule DNA sequencing technology that is expected to achieve both measures simultaneously. Emerging technologies are expected to soon provide a comprehensive picture of the exposome and identify gene regions susceptible to DNA adduct formation.

scholarly journals Genetic Adaptation of Porcine Circovirus Type 1 to Cultured Porcine Kidney Cells Revealed by Single-Molecule Long-Read Sequencing Technology

2017 ◽  
Vol 5 (5) ◽  
Author(s):  
Dóra Tombácz ◽  
Norbert Moldován ◽  
Zsolt Balázs ◽  
Zsolt Csabai ◽  
Michael Snyder ◽  
...  
Keyword(s):  
Single Molecule ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
Genetic Adaptation ◽  
Kidney Cells ◽  
Porcine Kidney ◽  
Sequencing Technology ◽  
Sequencing Platform ◽  
Long Read

ABSTRACT Porcine circovirus type 1 (PCV1) is a nonpathogenic circovirus, and a contaminant of the porcine kidney (PK-15) cell line. We present the complete and annotated genome sequence of strain Szeged of PCV1, determined by Pacific Biosciences RSII long-read sequencing platform.

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