scholarly journals Novel ITS1 Fungal Primers for Characterization of the Mycobiome

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Mykhaylo Usyk ◽  
Christine P. Zolnik ◽  
Hitesh Patel ◽  
Michael H. Levi ◽  
Robert D. Burk
Keyword(s):  
Pcr Primers ◽  
Fungal Communities ◽  
Rrna Gene ◽  
Its1 Region ◽  
Sequencing Technologies ◽  
Identification Of Fungi ◽  
Fungal Organisms ◽  
Primer Sets ◽  
Yeasts And Molds

ABSTRACT The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states. Studies of the human microbiome frequently omit characterization of fungal communities (the mycobiome), which limits our ability to investigate how fungal communities influence human health. The internal transcribed spacer 1 (ITS1) region of the eukaryotic ribosomal cluster has features allowing for wide taxonomic coverage and has been recognized as a suitable barcode region for species-level identification of fungal organisms. We developed custom ITS1 primer sets using iterative alignment refinement. Primer performance was evaluated using in silico testing and experimental testing of fungal cultures and human samples. Using an expanded novel reference database, SIS (18S-ITS1-5.8S), the newly designed primers showed an average in silico taxonomic coverage of 79.9% ± 7.1% compared to a coverage of 44.6% ± 13.2% using previously published primers (P = 0.05). The newly described primer sets recovered an average of 21,830 ± 225 fungal reads from fungal isolate culture samples, whereas the previously published primers had an average of 3,305 ± 1,621 reads (P = 0.03). Of note was an increase in the taxonomic coverage of the Candida genus, which went from a mean coverage of 59.5% ± 13% to 100.0% ± 0.0% (P = 0.0015) comparing the previously described primers to the new primers, respectively. The newly developed ITS1 primer sets significantly improve general taxonomic coverage of fungal communities infecting humans and increased read depth by an order of magnitude over the best-performing published primer set tested. The overall best-performing primer pair in terms of taxonomic coverage and read recovery, ITS1-30F/ITS1-217R, will aid in advancing research in the area of the human mycobiome. IMPORTANCE The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states.

scholarly journals Molecular Characterization of Ciliate Diversity in Stream Biofilms

2008 ◽  
Vol 74 (6) ◽  
pp. 1740-1747 ◽  
Author(s):  
Andrew Dopheide ◽  
Gavin Lear ◽  
Rebecca Stott ◽  
Gillian Lewis
Keyword(s):  
18S Rrna ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Sequence Diversity ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Specific Pcr ◽  
Stream Biofilms

ABSTRACT Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

scholarly journals Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities

2013 ◽  
Vol 79 (8) ◽  
pp. 2519-2526 ◽  
Author(s):  
Nicholas A. Bokulich ◽  
David A. Mills
Keyword(s):  
High Throughput ◽  
Internal Transcribed Spacer ◽  
High Throughput Sequencing ◽  
Work Flow ◽  
Fungal Communities ◽  
Amplification Bias ◽  
Sequencing Technologies ◽  
Yeast Community ◽  
Primer Sets ◽  
Sequencing Platforms

ABSTRACTUltra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predictedin silicoand by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities.

scholarly journals The ITS region as a target for characterization of fungal communities using emerging sequencing technologies

2009 ◽  
Vol 296 (1) ◽  
pp. 97-101 ◽  
Author(s):  
Rolf Henrik Nilsson ◽  
Martin Ryberg ◽  
Kessy Abarenkov ◽  
Elisabet Sjökvist ◽  
Erik Kristiansson
Keyword(s):  
Its Region ◽  
Fungal Communities ◽  
Sequencing Technologies

scholarly journals Detection of Enterotoxic Bacillus cereus andBacillus thuringiensis Strains by PCR Analysis

2001 ◽  
Vol 67 (1) ◽  
pp. 185-189 ◽  
Author(s):  
Bjarne Munk Hansen ◽  
Niels Bohse Hendriksen
Keyword(s):  
Bacillus Cereus ◽  
Protein Complexes ◽  
Pcr Primers ◽  
Gastrointestinal Diseases ◽  
23S Rrna ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Internal Transcribed Spacer Region ◽  
Primer Sets ◽  
Different Strains

ABSTRACT Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereusand B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceTgene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of thebceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes inB. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.

scholarly journals Benchmark of 16S rRNA gene amplicon sequencing using Japanese gut microbiome data from the V1–V2 and V3–V4 primer sets

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Shoichiro Kameoka ◽  
Daisuke Motooka ◽  
Satoshi Watanabe ◽  
Ryuichi Kubo ◽  
Nicolas Jung ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Analysis Data ◽  
Rrna Gene ◽  
Bacterial Composition ◽  
Sequencing Technologies ◽  
Primer Sets ◽  
Microbiome Data

Abstract Background 16S rRNA gene amplicon sequencing (16S analysis) is widely used to analyze microbiota with next-generation sequencing technologies. Here, we compared fecal 16S analysis data from 192 Japanese volunteers using the modified V1–V2 (V12) and the standard V3–V4 primer (V34) sets to optimize the gut microbiota analysis protocol. Results QIIME1 and QIIME2 analysis revealed a higher number of unclassified representative sequences in the V34 data than in the V12 data. The comparison of bacterial composition demonstrated that at the phylum level, Actinobacteria and Verrucomicrobia were detected at higher levels with V34 than with V12. Among these phyla, we observed higher relative compositions of Bifidobacterium and Akkermansia with V34. To estimate the actual abundance, we performed quantitative real-time polymerase chain reaction (qPCR) assays for Akkermansia and Bifidobacterium. We found that the abundance of Akkermansia as detected by qPCR was close to that in V12 data, but was markedly lower than that in V34 data. The abundance of Bifidobacterium detected by qPCR was higher than that in V12 and V34 data. Conclusions These results indicate that the bacterial composition derived from the V34 region might differ from the actual abundance for specific gut bacteria. We conclude that the use of the modified V12 primer set is more desirable in the 16S analysis of the Japanese gut microbiota.

scholarly journals Characterization of the upper respiratory tract microbiome of turkeys

2020 ◽  
Author(s):  
Olimpia Kursa ◽  
Grzegorz Tomczyk ◽  
Anna Sawicka-Durkalec ◽  
Aleksandra Giza ◽  
Magdalena Słomiany-Szwarc
Keyword(s):  
16S Rrna ◽  
Respiratory Tract ◽  
Pathogenic Bacteria ◽  
Rrna Gene ◽  
Upper Respiratory Tract ◽  
Bacterial Phyla ◽  
Sequencing Technologies ◽  
Upper Respiratory ◽  
Commercial Turkey

Abstract Background: The respiratory tracts of turkeys are the main route of infection therefore plays important roles in the overall health and performance of the birds. Understanding the poultry microbiome has the potential to offer better diagnosis and rational management of many poultry diseases. Characterization of microbial communities in the upper respiratory tract of turkeys could help better understand the role of pathogenic bacteria and other commensal or symbiotic microorganisms in the infection. The aim of this study was microbiome characterization of upper respiratory tracks of commercial turkeys using next-generation sequencing technologies. Results: The microbiome from samples collected from commercial turkey flocks was determined using 16S rRNA metagenomic approach. Taxonomic analysis of the microbiome was done by of the V3 and V4 regions of 16S rRNA gene (MiSeq, Illumina) amplification. The phylogenetic analysis identified the 10 bacterial phyla in turkey, the most abundant were phyla Firmicutes and Proteobacteria, accounting for >99% of all the sequences. The turkey sequences represent 144 established bacterial genera. Differences between bacterial abundances were found at the family and genus level. Several defining markers of microbiome succession were identified, including the presence of Ornithobacterium and Mycoplasma. Conclusions: Understanding the turkey’s respiratory microbiome is very important. Unique informations about microbiome representing members of the four major phyla of the respiratory tract in turkeys was assembled. These results obtained in this study supply information about turkey microbiome and can be useful in controlling, diagnosing and treating commercial turkey flocks. Our study significantly broaden the knowledge of the upper respiratory tract microbiome of turkeys.

Sequence-based Identification and Classification of Fungi.

2021 ◽  
pp. 198-216
Author(s):  
Andrew M. Borman ◽  
Elizabeth M. Johnson
Keyword(s):  
Dna Sequences ◽  
Its Region ◽  
Pcr Primers ◽  
Taxonomic Resolution ◽  
Its2 Region ◽  
Rrna Gene ◽  
5.8S Rrna Gene ◽  
Fungal Identification ◽  
Identification Of Fungi ◽  
Fungal Dna

Abstract This book chapter describes the advantages and limitations of the ITS Region as a universal barcode for fungal identification. The ITS region offers several practical advantages as a universal fungal barcode region. The region encompasses segments that permit resolution at different taxonomic levels as it includes the highly conserved 5.8S rRNA gene, the moderately rapidly evolving ITS2 region and the rapidly evolving ITS1 region, flanked by the highly conserved SSU and LSU genes which permit design of PCR primers that are almost panfungal. Over the last two decades the sequence-based identification of fungi has certainly come of age. The ITS region is universally accepted as the primary fungal barcoding region owing to the high barcode gap with the locus for many groups of fungi. Since the species-resolution power of ITS is poor for certain groups of fungi, and higher-level taxonomic resolution is greater with proteincoding genes, the TEF1α locus has been proposed as the universal secondary barcode region. In addition, the historical problems surrounding the reliability of fungal DNA sequences in centralized repositories are slowly being resolved by the development of an increasing number of publicly accessible, curated databases.

scholarly journals Characterization of the bacterial community structure of Sydney Tar Ponds sediment

2011 ◽  
Vol 57 (6) ◽  
pp. 493-503 ◽  
Author(s):  
C. William Yeung ◽  
Monica Woo ◽  
Kenneth Lee ◽  
Charles W. Greer
Keyword(s):  
Clone Library ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Toxic Waste ◽  
Rrna Gene ◽  
Dgge Analysis ◽  
Gradient Gel Electrophoresis ◽  
Primer Sets ◽  
V3 Region ◽  
Mycobacterium Spp

The Sydney Tar Ponds is one of the largest toxic waste sites in Canada. The bacterial diversity and abundance in the Sydney Tar Ponds sediment was examined using a 16S rRNA gene clone library and denaturing gradient gel electrophoresis (DGGE) with four different primer sets. The clone library was grouped into 19 phylotypes that could be divided into five phyla: Proteobacteria (56.9%), Actinobacteria (35%), Acidobacteria (4.9%), Firmicutes (2.4%), and Verrucomicrobia (0.8%). Members of the phyla Actinobacteria (represented mainly by Mycobacterium spp.) and Alphaproteobacteria (represented by Acidocella spp.) comprised the majority of the clone library. This study also revealed that the phylogenetic results obtained from clone library analysis and from DGGE analysis, with all the primer sets, showed some variability. However, similar Mycobacterium spp. and Acidocella spp. were found in all the different DGGE analyses, again suggesting that these two genera are dominant in the Sydney Tar Ponds sediment. In addition, DGGE analysis indicated that primer sets targeting the V3 region produced results that were the most similar to those obtained with the clone library.

scholarly journals Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia

2021 ◽  
Author(s):  
Masayuki K. Sakata ◽  
Mone U. Kawata ◽  
Atsushi Kurabayashi ◽  
Takaki Kurita ◽  
Masatoshi Nakamura ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Environmental Dna ◽  
Pcr Primers ◽  
Taxonomic Resolution ◽  
Rrna Gene ◽  
Biodiversity Monitoring ◽  
Natural Ecosystems ◽  
Universal Primer ◽  
Primer Sets

Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians ecological traits (e.g., nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding-analysis of extra-organismal DNA released into the environment-allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160-311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata, and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set Amph16S had the highest resolution among the tested sets. Finally, we applied Amph16S to actual metabarcoding and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.

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