scholarly journals Comparison of the performance of ITS1 and ITS2 as barcodes in amplicon-based sequencing of bioaerosols

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8523 ◽  
Author(s):  
Hamza Mbareche ◽  
Marc Veillette ◽  
Guillaume Bilodeau ◽  
Caroline Duchaine
Keyword(s):  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Dairy Farms ◽  
Culture Methods ◽  
Specific Target ◽  
Taxonomic Profile ◽  
Differential Abundance Analysis ◽  
Metagenomic Approach ◽  
Its1 And Its2

This paper presents the performance of two eukaryotic genomic ribosomal regions, ITS1 and ITS2, in describing fungal diversity in aerosol samples using amplicon-based High-Throughput Sequencing (HTS). Composting sites, biomethanization facilities, and dairy farms, all affected by the presence of fungi, were visited to collect air samples. The amplicon-based HTS approach is a target enrichment method that relies on the amplification of a specific target using particular primers before sequencing. Thus, the results are highly dependent on the quality of amplification. For this reason, the authors of this paper used a shotgun metagenomic approach to compare its outcome with the amplicon-based method. Indeed, shotgun metagenomic does not rely on any amplification prior to sequencing, because all genes are sequenced without a specific target. In addition, culture methods were added to the analyses in biomethanization and dairy farms samples to validate their contribution to fungal diversity of aerosols. The results obtained are unequivocal towards ITS1 outperformance to ITS2 in terms of richness, and taxonomic coverage. The differential abundance analysis did demonstrate that some taxa were exclusively detected only by ITS2, and vice-versa for ITS1. However, the shotgun metagenomic approach showed a taxonomic profile more resembling to ITS1 than ITS2. Based on these results, neither of the barcodes evaluated is perfect in terms of distinguishing all species. Using both barcodes offers a broader view of the fungal aerosol population. However, with the actual knowledge, the authors strongly recommend using ITS1 as a universal fungal barcode for quick general analyses of diversity and when limited financial resources are available, primarily due its ability to capture taxonomic profiles similar to those obtained using the shotgun metagenomic. The culture comparison with amplicon-based sequencing showed the complementarity of both approaches in describing the most abundant taxa.

scholarly journals In Silico Study Suggesting the Bias of Primers Choice in the Molecular Identification of Fungal Aerosols

2021 ◽  
Vol 7 (2) ◽  
pp. 99
Author(s):  
Hamza Mbareche ◽  
Marc Veillette ◽  
Guillaume J. Bilodeau
Keyword(s):  
In Silico ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
In Silico Analysis ◽  
Its Region ◽  
Internal Transcribed Spacers ◽  
Sequence Length ◽  
Universal Method ◽  
Continuous Work ◽  
Its1 And Its2

This paper presents an in silico analysis to assess the current state of the fungal UNITE database in terms of the two eukaryote nuclear ribosomal regions, Internal Transcribed Spacers 1 and 2 (ITS1 and ITS2), used in describing fungal diversity. Microbial diversity is often evaluated with amplicon-based high-throughput sequencing approaches, which is a target enrichment method that relies on the amplification of a specific target using particular primers before sequencing. Thus, the results are highly dependent on the quality of the primers used for amplification. The goal of this study is to validate if the mismatches of the primers on the binding sites of the targeted taxa could explain the differences observed when using either ITS1 or ITS2 in describing airborne fungal diversity. Hence, the choice of the pairs of primers for each barcode concur with a study comparing the performance of ITS1 and ITS2 in three occupational environments. The sequence length varied between the amplicons retrieved from the UNITE database using the pair of primers targeting ITS1 and ITS2. However, the database contains an equal number of unidentified taxa from ITS1 and ITS2 regions in the six taxonomic levels employed (phylum, class, order, family, genus, species). The chosen ITS primers showed differences in their ability to amplify fungal sequences from the UNITE database. Eleven taxa consisting of Trichocomaceae, Dothioraceae, Botryosphaeriaceae, Mucorales, Saccharomycetes, Pucciniomycetes, Ophiocordyceps, Microsporidia, Archaeorhizomycetes, Mycenaceae, and Tulasnellaceae showed large variations between the two regions. Note that members of the latter taxa are not all typical fungi found in the air. As no universal method is currently available to cover all the fungal kingdom, continuous work in designing primers, and particularly combining multiple primers targeting the ITS region is the best way to compensate for the biases of each one to get a larger view of the fungal diversity.

APPLICATION OF THE ATP-BIOLUMINESCENCE METHOD TO ASSESS THE QUALITY OF SANITATION OF MILKING EQUIPMENT

2020 ◽  
Vol 1 (4) ◽  
pp. 468-472
Author(s):  
P.A. Popov ◽  
◽  
D.V. Gruznov ◽  
S.V. Tokarev ◽  
◽  
...  
Keyword(s):  
Dairy Farms ◽  
Laboratory Studies ◽  
Before And After ◽  
Atp Bioluminescence ◽  
Microbial Number ◽  
Proportional Relationship ◽  
The Relationship ◽  
Number Of Bacteria ◽  
Bioluminescence Method

The article presents the results of determining the relationship between the total microbial number and microbial ATP on the surface of milking equipment before and after treatment with neutral anolyte ANK-SUPER. The possibility of using the ATP-bioluminescence method to control the quality of sanitation of milking equipment on dairy farms is shown. Laboratory studies revealed a proportional relationship between the level of bacterial ATP and the number of bacteria on the surface of milking equipment before and after sanitation.

scholarly journals Great differences in performance and outcome of high-throughput sequencing data analysis platforms for fungal metabarcoding

MycoKeys ◽  
2018 ◽  
Vol 39 ◽  
pp. 29-40 ◽  
Author(s):  
Sten Anslan ◽  
R. Henrik Nilsson ◽  
Christian Wurzbacher ◽  
Petr Baldrian ◽  
Leho Tedersoo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Computation Time ◽  
Potential Effect ◽  
Data Sets ◽  
Sequencing Data ◽  
Operational Taxonomic Units ◽  
High Throughput Sequencing Data ◽  
Recent Developments

Along with recent developments in high-throughput sequencing (HTS) technologies and thus fast accumulation of HTS data, there has been a growing need and interest for developing tools for HTS data processing and communication. In particular, a number of bioinformatics tools have been designed for analysing metabarcoding data, each with specific features, assumptions and outputs. To evaluate the potential effect of the application of different bioinformatics workflow on the results, we compared the performance of different analysis platforms on two contrasting high-throughput sequencing data sets. Our analysis revealed that the computation time, quality of error filtering and hence output of specific bioinformatics process largely depends on the platform used. Our results show that none of the bioinformatics workflows appears to perfectly filter out the accumulated errors and generate Operational Taxonomic Units, although PipeCraft, LotuS and PIPITS perform better than QIIME2 and Galaxy for the tested fungal amplicon dataset. We conclude that the output of each platform requires manual validation of the OTUs by examining the taxonomy assignment values.

scholarly journals Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Francesca De Filippis ◽  
Manolo Laiola ◽  
Giuseppe Blaiotta ◽  
Danilo Ercolini
Keyword(s):  
Microbial Ecology ◽  
High Throughput ◽  
Biological Samples ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Amplicon Sequencing ◽  
Fungal Species ◽  
Fungal Communities ◽  
Its2 Region ◽  
Mock Community

ABSTRACT Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

scholarly journals Human Cytomegalovirus Genomes Sequenced Directly from Clinical Material: Variation, Multiple-Strain Infection, Recombination and Mutation

2018 ◽  
Author(s):  
Nicolás M. Suárez ◽  
Gavin S. Wilkie ◽  
Elias Hage ◽  
Salvatore Camiolo ◽  
Marylouisa Holton ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
High Throughput Sequencing ◽  
Clinical Pathology ◽  
Congenital Infection ◽  
Population Based ◽  
Single Strain ◽  
Natural Mutation ◽  
Future Population

ABSTRACTThe genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination and natural mutation. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analysed, in the process facilitating the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating diversity, (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination, (iii) mutants with non-functional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae, and (iv) intrahost diversity in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology and pathogenesis of HCMV.

scholarly journals Bulked Segregant Analysis Coupled With Whole-Genome Sequencing (BSA-Seq) and Identification of a Novel Locus, qGL3.5, That Regulates Grain Length

2021 ◽  
Author(s):  
Haiyan Zhao ◽  
Yuebin Zheng ◽  
Feng Bai ◽  
Yang Liu ◽  
Sicheng Deng ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Bulked Segregant Analysis ◽  
Functional Characterization ◽  
Quality Data ◽  
Rice Grain ◽  
Grain Length ◽  
Starting Point ◽  
New Gene ◽  
Membrane Bound

Abstract Rice grain length (GL) directly affects the yield and quality of this species. Very few GL-related genes cloned are applied in production because their yield-increasing effect was not obvious. In this study, the two bulk-DNA pools (L-pool and S-pool) and their parents’ (KJ01 and Huaye 4) DNAs were subjected to high-throughput sequencing. After assessing the quality of the data, we obtained a total of 100.22 Gb of high-quality data; the average coverage depth was 55x, and the genome coverage was 96.51%. After combining the association results of the ED and SNP index methods, we mapped the GL genes to a 0.34 Mb “hotspot” region on chromosome 3, which contains 37 genes related to various traits. The 37 predicted genes were further analyzed by the use of the Gene GO, COG database and so on. Thirty-three genes were annotated by GO functions. According to the GO annotations, three genes whose molecular function involved in the plasma membrane and intracellular membrane-bound organelles were detected via CRISPR/ Cas9 editing technology. ORF33 was verified to regulate GL and was the target gene qGL3.5. These results provides a new gene resource for rice grain shape breeding and a starting point for functional characterization of the wild rice GL gene.

scholarly journals Tall fescue use on dairy farms

1997 ◽  
pp. 163-167 ◽  
Author(s):  
G.D. Milne ◽  
R. Shaw ◽  
R. Powell ◽  
B. Pirie ◽  
J. Pirie
Keyword(s):  
Tall Fescue ◽  
Festuca Arundinacea ◽  
Animal Health ◽  
Dairy Farms ◽  
Moisture Stress ◽  
Dairy Farmers ◽  
Costelytra Zealandica ◽  
Listronotus Bonariensis ◽  
Summer Growth

Tall fescue (Festuca arundinacea Schreb.) is now well proven on New Zealand dairy farms as a pasture species capable of producing high levels of milk production. Compared with perennial ryegrass (Lolium perenne) it offers benefits of higher annual and summer growth, higher clover contents in pasture, higher green leaf to stem ratios in summer, reduced plant pulling, better animal health, grass grub (Costelytra zealandica) and Argentine stem weevil (Listronotus bonariensis) tolerance, tolerance of wet soils, and drought tolerance and growth. Tall fescue can be recommended for dairy farmers in areas where summer growth and quality of ryegrass is reduced by moisture stress or high temperatures, and for those willing to adjust their grazing and establishment practices to suit tall fescue. Keywords: dairy farms, Festuca arundinacea, tall fescue

scholarly journals DNA metabarcoding of fungal diversity in air and snow of Livingston Island, South Shetland Islands, Antarctica

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Luiz Henrique Rosa ◽  
Otávio Henrique Bezerra Pinto ◽  
Tina Šantl-Temkiv ◽  
Peter Convey ◽  
Micheline Carvalho-Silva ◽  
...  
Keyword(s):  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Rank Order ◽  
Mesh Size ◽  
Diversity Indices ◽  
South Shetland Islands ◽  
Livingston Island ◽  
Dna Metabarcoding ◽  
Air Sample ◽  
Total Dna

AbstractWe assessed fungal diversity present in air and freshly deposited snow samples obtained from Livingston Island, Antarctica, using DNA metabarcoding through high throughput sequencing (HTS). A total of 740 m3 of air were pumped through a 0.22 µm membrane. Snow obtained shortly after deposition was kept at room temperature and yielded 3.760 L of water, which was filtered using Sterivex membranes of 0.22 µm mesh size. The total DNA present was extracted and sequenced. We detected 171 fungal amplicon sequence variants (ASVs), 70 from the air and 142 from the snow. They were dominated by the phyla Ascomycota, Basidiomycota, Mortierellomycota and Mucoromycota. Pseudogymnoascus, Cladosporium, Mortierella and Penicillium sp. were the most dominant ASVs detected in the air in rank order. In snow, Cladosporium, Pseudogymnoascus, Penicillium, Meyerozyma, Lecidea, Malassezia, Hanseniaspora, Austroplaca, Mortierella, Rhodotorula, Penicillium, Thelebolus, Aspergillus, Poaceicola, Glarea and Lecanora were the dominant ASVs present. In general, the two fungal assemblages displayed high diversity, richness, and dominance indices, with the assemblage found in snow having the highest diversity indices. Of the total fungal ASVs detected, 29 were only present in the air sample and 101 in the snow sample, with only 41 present in both samples; however, when only the dominant taxa from both samples were compared none occurred only in the air and, among the rare portion, 26 taxa occurred in both air and snow. Application of HTS revealed the presence of a more diverse fungal community in the air and snow of Livingston Island in comparison with studies using traditional isolation methods. The assemblages were dominated by cold-adapted and cosmopolitan fungal taxa, including members of the genera Pseudogymnoascus, Malassezia and Rhodotorula, which include some taxa reported as opportunistic. Our results support the hypothesis that the presence of microbiota in the airspora indicates the possibility of dispersal around Antarctica in the air column. However, further aeromycology studies are required to understand the dynamics of fungal dispersal within and beyond Antarctica.

High-throughput sequencing analysis of endophytic fungal diversity in cynanchum sp.

2020 ◽  
Vol 134 ◽  
pp. 349-358
Author(s):  
W.-H. Chen ◽  
S.-J. Wu ◽  
X.-L. Sun ◽  
K.-M. Feng ◽  
K. Rahman ◽  
...  
Keyword(s):  
High Throughput ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Sequencing Analysis
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