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 High accuracy DNA sequencing on a small, scalable platform via electrical detection of single base incorporations

2019 ◽  
Author(s):  
Hesaam Esfandyarpour ◽  
Kosar B. Parizi ◽  
Meysam R. Barmi ◽  
Hamid Rategh ◽  
Lisen Wang ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Capital Investment ◽  
Sequence Data ◽  
Gc Content ◽  
Low Frequency ◽  
Single Base ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Electronic Detection ◽  
Data Output

AbstractHigh throughput DNA sequencing technologies have undergone tremendous development over the past decade. Although optical detection-based sequencing has constituted the majority of data output, it requires a large capital investment and aggregation of samples to achieve optimal cost per sample. We have developed a novel electronic detection-based platform capable of accurately detecting single base incorporations. The GenapSys technology with its electronic detection modality allows the system to be compact, accessible, and affordable. We demonstrate the performance of the system by sequencing several different microbial genomes with varying GC content. The platform is capable of generating up to 2 Gb of high-quality nucleic acid sequence in a single run. We routinely generate sequence data that exceeds 99% raw accuracy with read lengths of up to 175 bp. Average quality scores remain above Q30 (99.9% raw sequencing accuracy) beyond 150 bp, with more than 85% of total bases at or above Q30. The utility of the platform is highlighted by targeted sequencing of the human genome. We show high concordance of SNP detection on the human NA12878 HapMap cell line with data generated on the Illumina sequencing platform. In addition, we sequenced a targeted panel of cancer-associated genes in a well characterized reference standard. With multiple library preparation approaches on this sample, we were able to identify low frequency mutations at expected allele frequencies.

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 Deciphering Neurodegenerative Diseases Using Long-Read Sequencing

Neurology ◽  
2021 ◽  
pp. 10.1212/WNL.0000000000012466
Author(s):  
Yun Su ◽  
Liyuan Fan ◽  
Changhe Shi ◽  
Tai Wang ◽  
Huimin Zheng ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Single Molecule ◽  
Direct Detection ◽  
Gc Content ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Repeat Expansions ◽  
Genomic Regions

Neurodegenerative diseases exhibit chronic progressive lesions in the central and peripheral nervous systems with unclear causes. The search for pathogenic mutations in human neurodegenerative diseases has benefited from massively parallel short-read sequencers. However, genomic regions, including repetitive elements, especially with high/low GC content, are far beyond the capability of conventional approaches. Recently, long-read single-molecule DNA sequencing technologies have emerged and enabled researchers to study genomes, transcriptomes, and metagenomes at unprecedented resolutions. The identification of novel mutations in unresolved neurodegenerative disorders, the characterization of causative repeat expansions, and the direct detection of epigenetic modifications on naive DNA by virtue of long-read sequencers will further expand our understanding of neurodegenerative diseases. In this paper, we review and compare two prevailing long-read sequencing technologies, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), and discuss their applications in neurodegenerative diseases.

scholarly journals A Complete Genome Sequence of the Wood Stem Endophyte Bacillus velezensis BY6 Strain Possessing Plant Growth-Promoting and Antifungal Activities

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Ping Zhang ◽  
Jian Diao ◽  
Guangqiang Xie ◽  
Ling Ma ◽  
Lihai Wang
Keyword(s):  
Secondary Metabolite ◽  
Single Molecule ◽  
Complete Genome ◽  
Pathogenic Fungus ◽  
Gc Content ◽  
Endophytic Bacterium ◽  
Gene Clusters ◽  
Bacillus Velezensis ◽  
Sequencing Platform ◽  
Plant Growth Promoting

An endophytic bacterium Bacillus velezensis BY6 was isolated from the wood stems of healthy Populus davidiana × P. alba var. pyramidalis (PdPap). The BY6 strain can inhibit pathogenic fungus Alternaria alternate in PdPap and promote growth of PdPap seedlings. In the present study, we used the Pacific Biosciences long-read sequencing platform, a single-molecule real-time (SMRT) technology for strain BY6, to perform complete genome sequencing. The genome size was 3,898,273 bp, the number of genes was 4,045, and the average GC content was 47.33%. A complete genome of strain BY6 contained 110 secondary metabolite gene clusters. Nine of the secondary metabolite gene clusters exhibited antifungal activity and promoted growth functions primarily involved in the synthesis of surfactin, bacteriocins, accumulated iron ions, and related antibiotics. Gene clusters provide genetic resources for biotechnology and genetic engineering, and enhance understanding of the relationship between microorganisms and plants.

scholarly journals Genetic Biomonitoring and Biodiversity Assessment Using Portable Sequencing Technologies: Current Uses and Future Directions

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 858 ◽  
Author(s):  
Krehenwinkel ◽  
Pomerantz ◽  
Prost
Keyword(s):  
Dna Sequencing ◽  
Biodiversity Loss ◽  
Taxonomic Composition ◽  
Great Promise ◽  
Sequencing Platform ◽  
Biological Communities ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Sequencing Studies ◽  
High Throughput Dna Sequencing

We live in an era of unprecedented biodiversity loss, affecting the taxonomic composition of ecosystems worldwide. The immense task of quantifying human imprints on global ecosystems has been greatly simplified by developments in high-throughput DNA sequencing technology (HTS). Approaches like DNA metabarcoding enable the study of biological communities at unparalleled detail. However, current protocols for HTS-based biodiversity exploration have several drawbacks. They are usually based on short sequences, with limited taxonomic and phylogenetic information content. Access to expensive HTS technology is often restricted in developing countries. Ecosystems of particular conservation priority are often remote and hard to access, requiring extensive time from field collection to laboratory processing of specimens. The advent of inexpensive mobile laboratory and DNA sequencing technologies show great promise to facilitate monitoring projects in biodiversity hot-spots around the world. Recent attention has been given to portable DNA sequencing studies related to infectious organisms, such as bacteria and viruses, yet relatively few studies have focused on applying these tools to Eukaryotes, such as plants and animals. Here, we outline the current state of genetic biodiversity monitoring of higher Eukaryotes using Oxford Nanopore Technology’s MinION portable sequencing platform, as well as summarize areas of recent development.

scholarly journals A new full-length virus genome sequencing method reveals that antiviral RNAi changes geminivirus populations in field-grown cassava

2017 ◽  
Author(s):  
Devang Mehta ◽  
Matthias Hirsch-Hoffmann ◽  
Mariam Were ◽  
Andrea Patrignani ◽  
Hassan Were ◽  
...  
Keyword(s):  
Single Molecule ◽  
Deep Sequencing ◽  
Cost Effective ◽  
Virus Genome ◽  
Full Length ◽  
Dna Viruses ◽  
Circular Dna ◽  
Sequencing Technologies ◽  
Virus Genomes ◽  
And Control

ABSTRACTDeep-sequencing of virus isolates using short-read sequencing technologies is problematic since viruses are often present in complexes sharing a high-degree of sequence identity. The full-length genomes of such highly-similar viruses cannot be assembled accurately from short sequencing reads. We present a new method, CIDER-Seq (Circular DNA Enrichment Sequencing) which successfully generates accurate full-length virus genomes from individual sequencing reads with no sequence assembly required. CIDER-Seq operates by combining a PCR-free, circular DNA enrichment protocol with Single Molecule Real Time sequencing and a new sequence deconcatenation algorithm. We apply our technique to produce more than 1,200 full-length, highly accurate geminivirus genomes from RNAi-transgenic and control plants in a field trial in Kenya. Using CIDER-Seq we can demonstrate for the first time that the expression of antiviral doublestranded RNA (dsRNA) in transgenic plants causes a consistent shift in virus populations towards species sharing low homology to the transgene derived dsRNA. Our results show that CIDER-seq is a powerful, cost-effective tool for accurately sequencing circular DNA viruses, with future applications in deep-sequencing other forms of circular DNA such as transposons and plasmids.

scholarly journals Diagnostic optical sequencing

2019 ◽  
Author(s):  
Lee E. Korshoj ◽  
Prashant Nagpal
Keyword(s):  
Single Molecule ◽  
Point Of Care ◽  
Cost Effective ◽  
Diagnostic Methods ◽  
Sequencing Platform ◽  
Molecular Variants ◽  
Cost Effective Method ◽  
Wide Range ◽  
Lactamase Gene ◽  
Modified Nucleobases

AbstractAdvances in precision medicine require high-throughput, inexpensive, point-of-care diagnostic methods with multi-omics capability for detecting a wide range of biomolecules and their molecular variants. Optical techniques have offered many promising advances towards such diagnostics. However, the inability to squeeze light with several hundred-nanometer wavelengths into angstrom-scale volume for single nucleotide measurements has hindered further progress. Recently, a block optical sequencing (BOS) method has been shown for determining relative nucleobase content in DNA k-mer blocks with Raman spectroscopy, and a block optical content scoring (BOCS) algorithm was developed for robust content-based genetic biomarker database searching. Here, we performed BOS measurements on positively-charged silver nanoparticles to achieve 93.3% accuracy for predicting nucleobase content in DNA k-mer blocks (where k=10), as well as measurements on RNA and chemically-modified nucleobases for extensions to transcriptomic and epigenetic studies. Our high-accuracy BOS measurements were then used with BOCS to correctly identify a β-lactamase gene from the MEGARes antibiotic resistance database and confirm the Pseudomonas aeruginosa pathogen of origin from <12 content measurements (<15% coverage) of the gene. These results prove the integration of BOS/BOCS as a diagnostic optical sequencing platform. With the versatile range of available plasmonic substrates offering simple data acquisition, varying resolution (single-molecule to ensemble), and multiplexing, this optical sequencing platform has potential as the rapid, cost-effective method needed for broad-spectrum biomarker detection.

Abstract 1154: Direct detection of DNA methylation and mutagenic damage through single-molecule, real-time (SMRTTM) DNA sequencing

2010 ◽  
Author(s):  
Benjamin Flusberg ◽  
Dale Webster ◽  
Kevin Travers ◽  
Eric Olivares ◽  
Jonas Korlach ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Sequencing ◽  
Real Time ◽  
Single Molecule ◽  
Direct Detection
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