scholarly journals PCR-Sequencing Approaches to Assess Informative Mutations in SARS-Cov-2 Spike (S) and ORF7, ORF8 and N Genes Characterizing Variants of Concern and Variants of Interest

2021 ◽  
Author(s):  
Glauco Akelinghton Freire Vitiello ◽  
Isabela Medeiros Oliveira ◽  
Fernanda Ivanski ◽  
Bárbara Luisa Fermino ◽  
Emanuele Cristina Gustani-Buss ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Regions Of Interest ◽  
Chain Reaction ◽  
Viral Genomes ◽  
The World ◽  
Prolonged Duration ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
High Infectivity

Abstract Background: The high infectivity rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the prolonged duration of coronavirus disease 2019 (COVID-19) pandemics have contributed to the emergence of viral variants endowed with evolutionary advantages, leading to enhanced infectivity. The tracking of these lineages is urgent. However, the need to sequence whole-viral genomes through next-generation sequencing (NGS) represents a barrier hampering the massive identification of these variants. Therefore, in the current study, we developed Sanger-sequencing approaches targeting regions of interest containing vast lineage-defining mutations in the SARS-CoV-2 S gene and ORF8 region, allowing for unambiguous identification of all SARS-CoV-2 variants of concern (VOCs) and of interest (VOIs).Methods and results: Primers were designed for polymerase chain reaction (PCR) and nested-PCR to amplify and sequence samples with a low-viral burden. The primers’ annealing sites conservancy were checked in a large group of sequences. Amplification protocols were standardized, and sequencing reactions were performed in a cohort of samples for validation. The primers were highly efficient and sequencing of the targeted regions matched those generated by NGS in the same samples. The sequencing results allowed for the unambiguous identification of B.1.1.7, P.1 and P.2 samples, and would also allow the identification of B.1.617.2, B.1.351 and B.1.427/B.1.429 lineages, which were absent in our cohort.Conclusion: Implementing Sanger-sequencing-based approaches to identify SARS-CoV-2 lineages may represent an alternative to tracking these variants by more laboratories around the world and providing valuable molecular and epidemiologic information to inform health systems.

scholarly journals METODE EKSTRAKSI DNA TANAMAN TANPA PRESIPITASI ETANOL UNTUK KEGIATAN POLYMERASE CHAIN REACTION (PCR)

2019 ◽  
Vol 6 (1) ◽  
pp. 29
Author(s):  
Kristianto Nugroho ◽  
Rerenstradika Tizar Terryana ◽  
. Reflinur ◽  
Puji Lestari
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Simple Sequence Repeat ◽  
Sequence Repeat ◽  
Chain Reaction ◽  
Ethanol Precipitation ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Simple Sequence

A Simplified Plant DNA Extraction Protocol without Ethanol Precipitation for Polymerase Chain Reaction (PCR) Activities ABSTRACTMolecular-based research in agriculture includes DNA extraction stage involving DNA precipitation using ethanol or isopropanol which tends to take a long time. The purpose of this study was to obtain a plant DNA extraction method for Polymerase Chain Reaction (PCR) activities without going through the ethanol precipitation stage. Five important agricultural commodity crops, namely rice, corn, soybeans, chilies, and shallots were extracted by DNA using the modified Doyle and Doyle method. After the extraction phase using chloroform and isoamil alcohol solvents, the supernatant obtained was not precipitated using ethanol but was directly diluted and used as a template in PCR activities using two pairs of Simple Sequence Repeat (SSR) markers. The results showed that all samples could be well amplified, and amplicon tape visualized in both 1% agarose gel and 6% polyacrylamide gel were clearly visible. This method could save time and material, and reduce the dependence on liquid nitrogen. But this method is still limited to PCR requirements only, and cannot be used for activities that require high quality and quantity of DNA such as Next Generation Sequencing (NGS), digestion, and hybridization.Keywords: DNA extraction, ethanol precipitation, liquid nitrogen, PCR, SSR,  ABSTRAKPenelitian berbasis molekuler pada bidang pertanian mencakup tahapan ekstraksi DNA yang melibatkan presipitasi DNA menggunakan etanol atau isopropanol yang cenderung memakan waktu lama. Tujuan penelitian ini adalah untuk memperoleh metode ekstraksi DNA tanaman untuk kegiatan Polymerase Chain Reaction (PCR) tanpa melalui tahapan presipitasi etanol. Lima tanaman komoditas pertanian penting yaitu padi, jagung, kedelai, cabai, dan bawang merah diekstraksi DNA-nya menggunakan metode Doyle and Doyle yang dimodifikasi. Setelah tahap ekstraksi menggunakan pelarut kloroform dan isoamil alkohol, supernatan yang terbentuk tidak dipresipistasi menggunakan etanol melainkan langsung diencerkan dan digunakan sebagai template dalam kegiatan PCR menggunakan dua pasang marka Simple Sequence Repeat (SSR). Hasil menunjukkan bahwa seluruh sampel dapat teramplifikasi dengan baik serta pita hasil amplikon yang tervisualisasi baik pada gel agarosa 1% maupun gel poliakrilamid 6% terlihat jelas. Metode ini dapat menghemat waktu dan bahan serta mengurangi ketergantungan pemakaian nitrogen cair. Tetapi metode ini masih terbatas hanya untuk kebutuhan PCR saja dan tidak dapat digunakan untuk kegiatan yang membutuhkan DNA dengan kualitas serta kuantitas tinggi seperti Next Generation Sequencing (NGS), digesti, maupun hibridisasi.Kata Kunci: ekstraksi DNA, nitrogen cair, PCR, presipitasi etanol, SSR

scholarly journals Ancient human DNA: A history of hype (then and now)

2021 ◽  
pp. 146960532199011
Author(s):  
Elizabeth D Jones ◽  
Elsbeth Bösl
Keyword(s):  
Current Debate ◽  
Chain Reaction ◽  
Hype Cycle ◽  
Human Dna ◽  
Celebrity Status ◽  
History Of ◽  
Polymerase Chain ◽  
And Migration ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

In this article on the history of ancient DNA research, we argue that the innovation of next-generation sequencing (NGS) of the early 2000s has ushered in a second hype cycle much like the first hype cycle the field experienced in the 1990s with the advent of the polymerase chain reaction (PCR). While the first hype cycle centered around the search for the oldest DNA, the field’s current optimism today promotes the rhetoric of revolution surrounding the study of ancient human gnomes. This is evidenced from written sources and personal interviews with researchers who feel the vast amount of data, the conclusions being made from this data, and the ever-increasing celebrity status of the field are perhaps moving too fast for their own good. Here, we use the concept of contamination, in both a literal and figurative understanding of the term, to explore the field’s continuities and disparities. We also argue that a number of additional, figurative interpretations of “contamination” are useful for navigating the current debate between geneticists and archaeologists regarding the origin, evolution, and migration of ancient humans across space and time. Our historical outlook on aDNA’s disciplinary development, we suggest, is necessary to accurately appreciate the state of the field, how it came to be, and where it might go in the future.

Diagnostik der Sepsis – Teil 2: Erregeridentifikation

2019 ◽  
Vol 54 (01) ◽  
pp. 38-48 ◽  
Author(s):  
Daniel Richter ◽  
Alexandra Heininger ◽  
Karsten Schmidt ◽  
Thomas Schmoch ◽  
Michael Bernhard ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Chain Reaction ◽  
Antimikrobielle Therapie ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Kritisch Kranker

ZusammenfassungIm Rahmen der Sepsis und des septischen Schocks spielen, trotz der zunehmenden Verbreitung von neuen molekularbiologischen Verfahren, der kulturelle Erregernachweis und die Resistenztestung weiterhin die entscheidende Rolle in der antimikrobiellen Therapie auf der Intensivstation. Hierbei kann der Erregernachweis für die antimikrobielle Therapie einerseits direkt aus dem Patientenblut, andererseits aber auch aus diversen anderen Probenmaterialien (respiratorische Sekrete, Punktat, intraoperative Abstriche etc.) geführt werden. Ein Nachteil konventioneller kultureller Verfahren im Kontext kritisch kranker Patienten ist die zeitliche Latenz bis zum Erregernachweis bzw. zum Ergebnis der Resistenztestung. Molekularbiologische Verfahren wie Techniken der Erregerdiagnostik und Resistenztestung, die auf Polymerase Chain Reaction (PCR) oder vor allem Next-Generation Sequencing (NGS) basieren, versprechen hier zwar kürzere Umlaufzeiten, sind aber aktuell noch kein klinischer Standard. Trotzdem besitzen diese Verfahren das Potenzial, einen Paradigmenwechsel in der Erregerdiagnostik herbeizuführen.

scholarly journals Discovery of Negative-Sense RNA Viruses in Trees Infected with Apple Rubbery Wood Disease by Next-Generation Sequencing

Plant Disease ◽  
2018 ◽  
Vol 102 (7) ◽  
pp. 1254-1263 ◽  
Author(s):  
Michael E. Rott ◽  
Prasad Kesanakurti ◽  
Constanze Berwarth ◽  
Heidi Rast ◽  
Ian Boyes ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
New Genus ◽  
Fruit Trees ◽  
Next Generation ◽  
Chain Reaction ◽  
The Family ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs ◽  
Negative Sense ◽  
Generation Sequencing

Apple rubbery wood is a disease of apple found around the world, often associated with Apple flat limb disease, and regulated in many countries. Despite its long history in apple cultivation, the disease’s causal agent has remained elusive. In this study, next-generation sequencing (NGS) was used to identify and characterize several related novel viral agents from apple rubbery wood-infected plants, which have been named Apple rubbery wood virus (ARWV) 1 and 2. Additional specimens with apple rubbery wood disease tested positive by polymerase chain reaction with primers designed to ARWV 1 and 2 genomic RNA segments. In an NGS-based screening of over 100 Malus and 100 Prunus specimens from a collection of virus-infected trees, only one Malus specimen was found to be infected with ARWV not known to be infected with the disease, which strongly suggests that ARWV is not commonly found in Malus spp. or other fruit trees. The two viruses are most closely related to members of the order Bunyavirales. Three RNA segments (large, medium, and small) were characterized and the viruses likely represent a new genus under the family Phenuiviridae, with a suggested name of Rubodvirus (Rubbery wood virus).

scholarly journals Transcriptomic analysis revealed increased expression of genes involved in keratinization in the tears of COVID-19 patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Leonardo Mastropasqua ◽  
Lisa Toto ◽  
Luigi Chiricosta ◽  
Francesca Diomede ◽  
Agnese Gugliandolo ◽  
...  
Keyword(s):  
Cell Signaling ◽  
B Cell ◽  
Functional Evaluation ◽  
Healthy Individuals ◽  
Chain Reaction ◽  
B Cell Signaling ◽  
Expression Of Genes ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

AbstractRecent studies have focused their attention on conjunctivitis as one of the symptoms of coronavirus disease 2019 (COVID-19). Therefore, tear samples were taken from COVID-19 patients and the presence of SARS-CoV-2 was evidenced using Real Time reverse transcription polymerase chain reaction. The main aim of this study was to analyze mRNA expression in the tears of patients with COVID-19 compared with healthy subjects using Next Generation Sequencing (NGS). The functional evaluation of the transcriptome highlighted 25 genes that differ statistically between healthy individuals and patients affected by COVID-19. In particular, the NGS analysis identified the presence of several genes involved in B cell signaling and keratinization. In particular, the genes involved in B cell signaling were downregulated in the tears of COVID-19 patients, while those involved in keratinization were upregulated. The results indicated that SARS-CoV-2 may induce a process of ocular keratinization and a defective B cell response.

scholarly journals Evaluation of Ion Torrent next-generation sequencing for thalassemia diagnosis

2020 ◽  
Vol 48 (12) ◽  
pp. 030006052096777
Author(s):  
Peisong Chen ◽  
Xuegao Yu ◽  
Hao Huang ◽  
Wentao Zeng ◽  
Xiaohong He ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Ion Torrent ◽  
Next Generation ◽  
Large Deletions ◽  
Different Types ◽  
Variant Detection ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs ◽  
Accuracy And Repeatability ◽  
Generation Sequencing

Introduction To evaluate a next-generation sequencing (NGS) workflow in the screening and diagnosis of thalassemia. Methods In this prospective study, blood samples were obtained from people undergoing genetic screening for thalassemia at our centre in Guangzhou, China. Genomic DNA was polymerase chain reaction (PCR)-amplified and sequenced using the Ion Torrent system and results compared with traditional genetic analyses. Results Of the 359 subjects, 148 (41%) were confirmed to have thalassemia. Variant detection identified 35 different types including the most common. Identification of the mutational sites by NGS were consistent with those identified by Sanger sequencing and Gap-PCR. The sensitivity and specificities of the Ion Torrent NGS were 100%. In a separate test of 16 samples, results were consistent when repeated ten times. Conclusion Our NGS workflow based on the Ion Torrent sequencer was successful in the detection of large deletions and non-deletional defects in thalassemia with high accuracy and repeatability.

scholarly journals Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material.

1996 ◽  
Vol 44 (10) ◽  
pp. 1205-1207 ◽  
Author(s):  
A Dakhama ◽  
V Macek ◽  
J C Hogg ◽  
R G Hegele
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Housekeeping Gene ◽  
Chain Reaction ◽  
Viral Genomes ◽  
Negative Results ◽  
Beta Actin ◽  
Target Nucleic Acid ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.

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