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Application of Molecular Tools and Biosensors for Monitoring Water Microbiota
Published in Maulin P. Shah, Wastewater Treatment, 2022
Pyrosequencing is a technique developed in 1996 by Mostafa Ronaghi and Pal Nyren at the Royal Institute of Technology in Stockholm. It is a DNA sequencing method that works on the principle of sequencing by synthesis. This technique is widely used for metagenomics detection of pathogens in various environmental and clinical samples. Pyrosequencing utilizes enzyme coupled reaction and bioluminescence in combination to monitor the release of pyrophosphate after the addition of a nucleotide in real time. This technique can be potentially applied to sequence a large number of reads in a single run. There are mainly four enzymes required for pyrosequencing: apyrase, luciferase, ATP sulfurylase, and Klenow fragment of DNA polymerase 1 (41). Apyrase is an enzyme incorporated into the process of pyrosequencing for degrading the free nucleotides and ATPs. The reaction mixture of pyrosequencing also demands luciferase, adenosine phosphosulfate, and the DNA template annealed to a primer as a starting material. The recognition and addition of a nucleotide to its complementary base in the single-stranded template leads to release of a pyrophosphate molecule (PPi) and the growth of the DNA strand. This released inorganic pyrophosphate is further converted to ATP in the presence of the enzyme ATP sulfurylase by utilizing the adenosine phosphosulfate molecule as a substrate. Finally, this ATP is utilized by the luciferase enzyme to generate a light signal. This generated light is identified and used as evidence for the incorporation of the nucleotide into the growing chain.
Associations between Genetic Polymorphisms and Heart Rate Variability
Published in Herbert F. Jelinek, David J. Cornforth, Ahsan H. Khandoker, ECG Time Series Variability Analysis, 2017
Anne Voigt, Jasha W. Trompf, Mikhail Tamayo, Ethan Ng, Yuling Zhou, Yaxin Lu, Slade Matthews, Brett D. Hambly, Herbert F. Jelinek
Methods for genetic analysis have relied on polymorphisms for more than two decades, but the current focus has shifted to single nucleotide polymorphisms (SNPs). This is the most common type of genetic variation in humans and is defined as a position in the sequence with at least two variants. The rarer variant has a frequency of at least 1%. SNPs are quite common and can be found as 1:1000 base pairs (bp) in the human genome. They are used in clinical tests, forensics, and, as in the case of HRV, to identify genes related primarily to CVD, but have also been shown to be important in renal disease and psychosis (Wang et al. 1998). SNP mapping has become cheaper and more efficient with the development of new methods. Mapping started with Sanger DNA sequencing and is now moving on to alternative methods, such as pyrosequencing. Unlike Sanger sequencing, which is time-consuming, labor-intensive, and requires labeling, pyrosequencing is efficient and the time necessary for the detection of SNPs has been significantly decreased. This makes pyrosequencing an ideal method for the comparison of variants in large-scale screening tests. It is based on the detection of fluorescence in proportion to the correct number of nucleotides incorporated into the sequence (Fodor et al. 1991; Southern at al, 1992; Ronaghi et al. 1998).
DNA Structure, Sequencing, Synthesis, and Modification: Making Biology Molecular
Published in Richard J. Sundberg, The Chemical Century, 2017
Methods for DNA sequencing have continued to improve beyond the pyrosequencing technique. As of 2010, rates of sequence acquisition have increased by 1012 and costs per genome has been reduced by a factor of at least 103.2
Key Microbes and Metabolic Potentials Contributing to Cyanide Biodegradation in Stirred-Tank Bioreactors Treating Gold Mining Effluent
Published in Mineral Processing and Extractive Metallurgy Review, 2020
Doyun Shin, Jeonghyun Park, Hyunsik Park, Jae-Chun Lee, Min-Seuk Kim, Jaeheon Lee
Pyrosequencing can be used to characterize microbial communities faster and at greater sequencing depth than was possible with cloning and Sanger sequencing (Huse and Welch 2011). Overall, 74,736 valid reads were obtained after invalid sequences were removed with denoising and chimera checks, and 5,377 OTUs were produced based on the normalized valid reads (1,299 reads per sample). All the samples, except those from the M3-50 reactor, showed high bacterial diversity (high Shannon index and low Simpson index) before feeding cyanide (SI Table S1). In the M3-50 reactor, the diversity was quite low in the initial stage, because it was inoculated with only three bacterial strains. The diversity of all samples inoculated with activated sludge decreased as feeding cyanide and increasing time. The number of OTUs showed similar trends to those of diversity. These results indicate that the initial sample hosted a range of bacterial strains, but after exposed to cyanide, cyanide-resistant or -degrading strains became dominant, resulting in a reduction in diversity.
Next-generation DNA sequencing of oral microbes at the Sir John Walsh Research Institute: technologies, tools and achievements
Published in Journal of the Royal Society of New Zealand, 2020
Nicholas C. K. Heng, Jo-Ann L. Stanton
In 2005, the first of several next-generation DNA sequencing technologies, ‘pyrosequencing’, was reported by Margulies et al. (2005). The chemical basis of pyrosequencing (also known as ‘454 sequencing’) was the detection of photons (light) upon the incorporation of a nucleotide into a growing DNA chain. Sequencing reactions were essentially conducted in nanolitre ‘PCR vessels’ that were microetched onto glass arrays. During a sequencing run, nucleotides were cycled one at a time into the polymerisation reaction and if that particular nucleotide was incorporated, a pyrophosphate molecule would be released with the concomitant production of a photon (in a secondary reaction involving luciferin and luciferase), which was then detected by a charge-coupled device. If multiple nucleotides were added, i.e. a homopolymer of thymines (TTTTT) or guanines (GGGGG), the photon intensity detected would be proportional to the number of nucleotides added. The prototype 454 sequencing system, the Roche GS20, allowed >200,000 individual sequencing reactions (of 100–150 bp length) to be analysed per run, generating 20 million basepairs (Mbp) of sequence data (Liu et al. 2012). At its peak, the most advanced 454 system was the Roche Genome Sequencer FLX (GS-FLX) Titanium which was capable of analysing over a million individual pyrosequencing reactions, yielding an unprecedented 700 Mbp of sequence data with an average read length of 400 bp (Liu et al. 2012). In our hands, the GS-FLX Titanium and GS Junior (a lower-cost GS-FLX Titanium configured as a smaller instrument) generated average read lengths of 450–480 bp, which allowed us to sequence through genes that contained many tracts of repeated sequences (e.g. cell surface adhesin genes). Roche eventually withdrew from the NGS market in 2016 due to the introduction of other more cost-effective competing technologies with much higher yields.