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Single-Molecule Analysis by Biological Nanopores
Published in Shuo Huang, Single-Molecule Tools for Bioanalysis, 2022
Biological nanopores are a type of proteins which form pores and were developed for in vitro single-molecule sensing. In view of their ease of use, consistency and precision of preparation, biological nanopores can be further engineered or modified for highly specialized sensing applications. Using single-channel recording, the identity of an analyte is reported from its interaction with the pore restriction during its translocation. Being geometrically compatible with single-stranded DNA or single-stranded RNA, biological nanopores, such as α-hemolysin (α-HL) or Mycobacterium smegmatis porin A (MspA), have long been considered the most promising candidates for third-generation sequencing. After research of ~3 decades, the prototype of a nanopore sequencer was first demonstrated in 2012 and is now widely used in a variety of genomics research programs. Sustained research of nanopore sequencing has also stimulated its other applications, such as sensing of single ions, small molecules, macromolecules, biomacromolecules, or their combinations. In this chapter, we introduce the mechanism and the methodology of the biological nanopore techniques along with a tutorial protocol. We hope the reader will benefit from reading this chapter by successfully carrying out a highly simplified nanopore measurement or becoming inspired for their own research.
Bioinformatics and Applications in Biotechnology
Published in Ram Chandra, R.C. Sobti, Microbes for Sustainable Development and Bioremediation, 2019
The conventional Sanger sequencing of genomes is slow and has many rate-limiting steps, which have been overcome using the techniques of parallel or deep sequencing. Using next-generation sequencing (NGS), an entire human genome can be sequenced in almost a day, which otherwise took many years of hard labor using the Sanger sequencing (Behjati & Tarpey, 2013). In NGS, the DNA is fragmented to small readouts from 30–40 bp to about 500bp, denatured, and attached to beads with each bead in a nanowell; the DNA fragment is amplified usually by emulsion PCR and the fragments are sequenced. Extensive bioinformatics tools are then used to reassemble the genome. The first massively parallel signature sequencing incorporating parallel, adaptor/ligation mediated bead based sequencing technologies were made available by Lynx Therapeutics in 2000 (Brenner et al., 2000). Three technologies have revolutionized NGS:ion torrent personal genome machine (PGM), single-molecule real-time sequencing (SMRT) by PacBio, sequencing by synthesis by Illumina, among others. In the ion torrent PGM, the protons released as nucleotides are incorporated to make a double strand of single-stranded DNA fragment amplified in nanowells are captured by proton-sensing silicon wafers in the nanowells. As nucleotides are added to wells, signal emanates from only those wells in which the nucleotide is incorporated. The PacBio technique involves binding of DNA polymerase to the single-stranded amplified DNA strands, and when a γ-phosphate-labeled nucleotide is added to form the double strand, a distinct pulse of incorporated fluorophore is detected in real time. In illumina technique, the clonally amplified DNA immobilized on acrylamide coating on the surface of a glass flow cell sequencing by synthesis approach is used using fluorescently labeled nucleotides (Quail et al., 2012). Of late DNA, nanopore sequencing pioneered by Oxford Nanopore technologies offers real-time, scalable, and direct DNA sequencing. Single DNA/RNA molecules can be analyzed by passing them through a nano-orifice in a protein such asalpha hemolysin using electrophoresis. The system also bypasses the need for PCR amplification and is set to become the benchmark in NGS with substantially reduced operating costs.
Growth and genetic analysis of Pseudomonas BT1 in a high-thiourea environment reveals the mechanisms by which it restores the ability to remove ammonia nitrogen from wastewater
Published in Environmental Technology, 2022
Jingxuan Deng, Zhenxing Huang, Wenquan Ruan
The toxicity of thiourea to the nitrification system has been reported widely in previous studies. Even at low concentrations, thiourea can have significant impacts on sewage treatment systems, which leads to numerous economic losses. As a wild heterotrophic nitrifying strain, Pseudomonas BT1 shows high ammonia removal efficiency. To analyze the possible molecular mechanisms, we assembled its genomes and performed a transcriptome analysis in this study. Compared with next-generation sequencing, Nanopore sequencing technology has advantages in conducting long sequencing reads, which is helpful for bacterial assembly and was adopted in our study [28]. The complete circular assembly of Pseudomonas BT1 provides accurate gene annotation and a valuable resource to analyze the biological processes and molecular mechanisms of thiourea metabolism in Pseudomonas.