scholarly journals Extraction of Microbial and Host DNA, RNA, and Proteins from Oak Bark Tissue

2019 ◽  
Vol 2 (1) ◽  
pp. 15 ◽  
Author(s):  
Martin Broberg ◽  
James E. McDonald
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Protein Extraction ◽  
Quercus Petraea ◽  
Protein Sequencing ◽  
Tree Bark ◽  
Protein Mass ◽  
Dna And Rna ◽  
Bark Tissue

The application of high-throughput nucleic acid and protein sequencing technologies is transforming our understanding of plant microbiomes and their interactions with their hosts in health and disease. However, progress in studying host-microbiome interactions in above-ground compartments of the tree (the phyllosphere) has been hampered due to high concentrations of phenolic compounds, lignin, and other compounds in tree bark that severely limit the success of DNA, RNA, and protein extraction. Here we present modified sample-preparation and kit-based protocols for the extraction of host and microbiome DNA and RNA from oak (Quercus robus and Quercus petraea) bark tissue for subsequent high-throughput sequencing. In addition, reducing the quantity of bark tissue used for an established protein extraction protocol yielded high quality protein for parallel analysis of the oak-microbiota metaproteome. These procedures demonstrate the successful extraction of nucleic acids and proteins from oak tissue using as little as 50 mg of sample input, producing sufficient quantities for nucleic acid sequencing and protein mass spectrometry of tree stem tissues and their associated microbiota.

scholarly journals A Systematic Evaluation of High-Throughput Sequencing Approaches to Identify Low-Frequency Single Nucleotide Variants in Viral Populations

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1187
Author(s):  
David J. King ◽  
Graham Freimanis ◽  
Lidia Lasecka-Dykes ◽  
Amin Asfor ◽  
Paolo Ribeca ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Low Frequency ◽  
Illumina Miseq ◽  
Read Length ◽  
Systematic Evaluation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Dna And Rna

High-throughput sequencing such as those provided by Illumina are an efficient way to understand sequence variation within viral populations. However, challenges exist in distinguishing process-introduced error from biological variance, which significantly impacts our ability to identify sub-consensus single-nucleotide variants (SNVs). Here we have taken a systematic approach to evaluate laboratory and bioinformatic pipelines to accurately identify low-frequency SNVs in viral populations. Artificial DNA and RNA “populations” were created by introducing known SNVs at predetermined frequencies into template nucleic acid before being sequenced on an Illumina MiSeq platform. These were used to assess the effects of abundance and starting input material type, technical replicates, read length and quality, short-read aligner, and percentage frequency thresholds on the ability to accurately call variants. Analyses revealed that the abundance and type of input nucleic acid had the greatest impact on the accuracy of SNV calling as measured by a micro-averaged Matthews correlation coefficient score, with DNA and high RNA inputs (107 copies) allowing for variants to be called at a 0.2% frequency. Reduced input RNA (105 copies) required more technical replicates to maintain accuracy, while low RNA inputs (103 copies) suffered from consensus-level errors. Base errors identified at specific motifs identified in all technical replicates were also identified which can be excluded to further increase SNV calling accuracy. These findings indicate that samples with low RNA inputs should be excluded for SNV calling and reinforce the importance of optimising the technical and bioinformatics steps in pipelines that are used to accurately identify sequence variants.

scholarly journals Implementation of High-Throughput Sequencing (HTS) in Aptamer Selection Technology

2020 ◽  
Vol 21 (22) ◽  
pp. 8774
Author(s):  
Natalia Komarova ◽  
Daria Barkova ◽  
Alexander Kuznetsov
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Combinatorial Libraries ◽  
Structure And Function ◽  
Huge Number ◽  
Systematic Evolution ◽  
And Function ◽  
Exponential Enrichment

Aptamers are nucleic acid ligands that bind specifically to a target of interest. Aptamers have gained in popularity due to their high potential for different applications in analysis, diagnostics, and therapeutics. The procedure called systematic evolution of ligands by exponential enrichment (SELEX) is used for aptamer isolation from large nucleic acid combinatorial libraries. The huge number of unique sequences implemented in the in vitro evolution in the SELEX process imposes the necessity of performing extensive sequencing of the selected nucleic acid pools. High-throughput sequencing (HTS) meets this demand of SELEX. Analysis of the data obtained from sequencing of the libraries produced during and after aptamer isolation provides an informative basis for precise aptamer identification and for examining the structure and function of nucleic acid ligands. This review discusses the technical aspects and the potential of the integration of HTS with SELEX.

scholarly journals High-throughput Sequencing for Species Authentication and Contamination Detection of 63 Cell Lines

2021 ◽  
Author(s):  
Oliver Lung ◽  
Rebecca Candlish ◽  
Michelle Nebroski ◽  
Peter Kruckiewicz ◽  
Cody Buchanan ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Classical Swine Fever ◽  
Virus Genome ◽  
Cox1 Gene ◽  
Species Identity ◽  
Cell Line Authentication ◽  
Dna And Rna

Abstract Cell lines are widely used in research and for diagnostic tests and are often shared between laboratories. Lack of cell line authentication can result in the use of contaminated or misidentified cell lines, potentially affecting the results from research and diagnostic activities. Cell line authentication and contamination detection based on metagenomic high-throughput sequencing (HTS) was tested on DNA and RNA from 63 cell lines available at the Canadian Food Inspection Agency’s National Centre for Foreign Animal Disease. Through sequence comparison of the cytochrome c oxidase subunit 1 (COX1) gene, the species identity of 53 cell lines was confirmed, and eight cell lines were found to show a greater pairwise nucleotide identity in the COX1 sequence of a different species within the same expected genus. Two cell lines, LFBK-αvβ6 and SCP-HS, were determined to be composed of cells from a different species and genus. Mycoplasma contamination was not detected in any cell lines. However, several expected and unexpected viral sequences were detected, including part of the classical swine fever virus genome in the IB-RS-2 Clone D10 cell line. Metagenomics-based HTS is a useful laboratory QA tool for cell line authentication and contamination detection that should be conducted regularly.

scholarly journals A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

2020 ◽  
Author(s):  
Justin P. Shaffer ◽  
Clarisse Marotz ◽  
Pedro Belda-Ferre ◽  
Cameron Martino ◽  
Stephen Wandro ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Sequence Data ◽  
Rna Virus ◽  
Microbial Community Composition ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Dna And Rna

AbstractOne goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols.Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX).Methods summaryTo allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

scholarly journals Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples

PLoS ONE ◽  
2018 ◽  
Vol 13 (5) ◽  
pp. e0197456 ◽  
Author(s):  
Stine H. Kresse ◽  
Heidi M. Namløs ◽  
Susanne Lorenz ◽  
Jeanne-Marie Berner ◽  
Ola Myklebost ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Dna And Rna ◽  
Ffpe Samples ◽  
Rna Extraction Methods

scholarly journals The effect of reverse transcription enzymes and conditions on high throughput amplicon sequencing of the 16S rRNA

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7608
Author(s):  
Adam Šťovíček ◽  
Smadar Cohen-Chalamish ◽  
Osnat Gillor
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
Ribosomal Rna ◽  
Reverse Transcription ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Amplicon Sequencing ◽  
Soil Microbiology ◽  
Active Population ◽  
Dna And Rna

It is assumed that the sequencing of ribosomes better reflects the active microbial community than the sequencing of the ribosomal RNA encoding genes. Yet, many studies exploring microbial communities in various environments, ranging from the human gut to deep oceans, questioned the validity of this paradigm due to the discrepancies between the DNA and RNA based communities. Here, we focus on an often neglected key step in the analysis, the reverse transcription (RT) reaction. Previous studies showed that RT may introduce biases when expressed genes and ribosmal rRNA are quantified, yet its effect on microbial diversity and community composition was never tested. High throughput sequencing of ribosomal RNA is a valuable tool to understand microbial communities as it better describes the active population than DNA analysis. However, the necessary step of RT may introduce biases that have so far been poorly described. In this manuscript, we compare three RT enzymes, commonly used in soil microbiology, in two temperature modes to determine a potential source of bias due to non-standardized RT conditions. In our comparisons, we have observed up to six fold differences in bacterial class abundance. A temperature induced bias can be partially explained by G-C content of the affected bacterial groups, thus pointing toward a need for higher reaction temperatures. However, another source of bias was due to enzyme processivity differences. This bias is potentially hard to overcome and thus mitigating it might require the use of one enzyme for the sake of cross-study comparison.

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