scholarly journals Comparing 16S rDNA amplicon sequencing and hybridization capture for pea aphid microbiota diversity analysis

2018 ◽  
Vol 11 (1) ◽  
Author(s):  
Marie Cariou ◽  
Céline Ribière ◽  
Stéphanie Morlière ◽  
Jean-Pierre Gauthier ◽  
Jean-Christophe Simon ◽  
...  
Keyword(s):  
16S Rdna ◽  
Amplicon Sequencing ◽  
Pea Aphid ◽  
Diversity Analysis ◽  
Hybridization Capture ◽  
16S Rdna Amplicon Sequencing ◽  
Microbiota Diversity

scholarly journals Composition and Diversity of the Ocular Surface Microbiota in Patients With Blepharitis in Northwestern China

2021 ◽  
Vol 8 ◽  
Author(s):  
Changhao Wang ◽  
Xiuhong Dou ◽  
Jian Li ◽  
Jie Wu ◽  
Yan Cheng ◽  
...  
Keyword(s):  
16S Rdna ◽  
Relative Abundance ◽  
Ocular Surface ◽  
Amplicon Sequencing ◽  
Northwestern China ◽  
Healthy Control Group ◽  
Control Group ◽  
Eyelid Margin ◽  
16S Rdna Amplicon Sequencing ◽  
Healthy Control

Purpose: To investigate the composition and diversity of the microbiota on the ocular surface of patients with blepharitis in northwestern China via 16S rDNA amplicon sequencing.Methods: Thirty-seven patients with blepharitis divided into groups of anterior, posterior and mixed blepharitis and twenty healthy controls from northwestern China were enrolled in the study. Samples were collected from the eyelid margin and conjunctival sac of each participant. The V3–V4 region of bacterial 16S rDNA in each sample was amplified and sequenced on the Illumina HiSeq 2500 sequencing platform, and the differences in taxonomy and diversity among different groups were compared.Results: The composition of the ocular surface microbiota of patients with blepharitis was similar to that of healthy subjects, but there were differences in the relative abundance of each bacterium. At the phylum level, the abundances of Actinobacteria, Cyanobacteria, Verrucomicrobia, Acidobacteria, Chloroflexi, and Atribacteria were significantly higher in the blepharitis group than in the healthy control group, while the relative abundance of Firmicutes was significantly lower (p < 0.05, Mann-Whitney U). At the genus level, the abundances of Lactobacillus, Ralstonia, Bacteroides, Akkermansia, Bifidobacterium, Escherichia-Shigella, Faecalibacterium, and Brevibacterium were significantly higher in the blepharitis group than in the healthy control group, while the relative abundances of Bacillus, Staphylococcus, Streptococcus, and Acinetobacter were significantly lower in the blepharitis group (p < 0.05, Mann-Whitney U). The microbiota of anterior blepharitis was similar to that of mixed blepharitis but different from that of posterior blepharitis. Lactobacillus and Bifidobacterium are biomarkers of posterior blepharitis, and Ralstonia is a biomarker of mixed blepharitis. There was no significant difference in the ocular surface microbiota between the eyelid margin and conjunctival sac with or without blepharitis.Conclusion: The ocular surface microbiota of patients with blepharitis varied among different study groups, according to 16S rDNA amplicon sequencing analysis. The reason might be due to the participants being from different environments and having different lifestyles. Lactobacillus, Bifidobacterium, Akkermansia, Ralstonia, and Bacteroides may play important roles in the pathogenesis of blepharitis.

scholarly journals Metagenomic Analysis of Slovak Bryndza Cheese Using Next-Generation 16S rDNA Amplicon Sequencing

2016 ◽  
Vol 15 (1) ◽  
pp. 23-34 ◽  
Author(s):  
Matej Planý ◽  
Tomáš Kuchta ◽  
Katarína Šoltýs ◽  
Tomáš Szemes ◽  
Domenico Pangallo ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rdna ◽  
Lactococcus Lactis ◽  
Microbial Population ◽  
Starter Culture ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Next Generation ◽  
16S Rdna Amplicon Sequencing

Abstract Knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. Aim of this study was to investigate microbial consortia composition in Slovak bryndza cheese. For this purpose, we used culture-independent approach based on 16S rDNA amplicon sequencing using next generation sequencing platform. Results obtained by the analysis of three commercial (produced on industrial scale in winter season) and one traditional (artisanal, most valued, produced in May) Slovak bryndza cheese sample were compared. A diverse prokaryotic microflora composed mostly of the genera Lactococcus, Streptococcus, Lactobacillus, and Enterococcus was identified. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. Second most abundant species, detected in all bryndza cheeses, were Lactococcus fujiensis and Lactococcus taiwanensis, independently by two different approaches, using different reference 16S rRNA genes databases (Greengenes and NCBI respectively). They have been detected in bryndza cheese samples in substantial amount for the first time. The narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. Metagenomic analysis by high-throughput sequencing using 16S rRNA genes seems to be a powerful tool for studying the structure of the microbial population in cheeses.

scholarly journals Technical note: overcoming host contamination in bovine vaginal metagenomic samples with Nanopore adaptive sequencing

2021 ◽  
Author(s):  
Chian Teng Ong ◽  
Elizabeth M Ross ◽  
Gry B Boe-Hansen ◽  
Conny Turni ◽  
Ben J Hayes ◽  
...  
Keyword(s):  
16S Rdna ◽  
Sequence Data ◽  
Amplicon Sequencing ◽  
Technical Note ◽  
Metagenomic Data ◽  
High Coverage ◽  
Long Reads ◽  
Oxford Nanopore ◽  
16S Rdna Amplicon Sequencing ◽  
Functional Profiles

Abstract Animal metagenomic studies, in which host-associated microbiomes are profiled, are an increasingly important contribution to our understanding of the physiological functions, health and susceptibility to diseases of livestock. One of the major challenges in these studies is host DNA contamination, which limits the sequencing capacity for metagenomic content and reduces the accuracy of metagenomic profiling. This is the first study comparing the effectiveness of different sequencing methods for profiling bovine vaginal metagenomic samples. We compared the new method of Oxford Nanopore Technologies (ONT) adaptive sequencing, which can be used to target or eliminate defined genetic sequences, to standard ONT sequencing, Illumina 16S rDNA amplicon sequencing, and Illumina shotgun sequencing. The efficiency of each method in recovering the metagenomic data and recalling the metagenomic profiles was assessed. ONT adaptive sequencing yielded a higher amount of metagenomic data than the other methods per 1 Gb of sequence data. The increased sequencing efficiency of ONT adaptive sequencing consequently reduced the amount of raw data needed to provide sufficient coverage for the metagenomic samples with high host-to-microbe DNA ratio. Additionally, the long reads generated by ONT adaptive sequencing retained the continuity of read information, which benefited the in-depth annotations for both taxonomical and functional profiles of the metagenome. The different methods resulted in the identification of different taxa. Genera Clostridium, which was identified at low abundances and categorised under Order “Unclassified Clostridiales” when using the 16S rDNA amplicon sequencing method, was identified to be the dominant genera in the sample when sequenced with the three other methods. Additionally, higher numbers of annotated genes were identified with ONT adaptive sequencing, which also produced high coverage on most of the commonly annotated genes. This study illustrates the advantages of ONT adaptive sequencing in improving the amount of metagenomic data derived from microbiome samples with high host-to-microbe DNA ratio and the advantage of long reads in preserving intact information for accurate annotations.

scholarly journals Sediment-associated microbial community profiling: sample pre-processing through sequential membrane filtration for 16s rDNA amplicon sequencing

2020 ◽  
Author(s):  
Joeselle M. Serrana ◽  
Kozo Watanabe
Keyword(s):  
Microbial Community ◽  
16S Rdna ◽  
Community Composition ◽  
Membrane Filtration ◽  
Amplicon Sequencing ◽  
Sediment Samples ◽  
Community Profiling ◽  
Significant Difference ◽  
16S Rdna Amplicon Sequencing ◽  
Read Abundance

ABSTRACTSequential membrane filtration as a pre-processing step for the isolation of microorganisms could provide good quality and integrity DNA that can be preserved and kept at ambient temperatures before community profiling through culture-independent molecular techniques, e.g., 16s rDNA amplicon sequencing. Here, we assessed the impact of pre-processing sediment samples by sequential membrane filtration (from 10, 5 to 0.22 μm pore size membrane filters) for 16s rDNA-based community profiling of sediment-associated microorganisms. Specifically, we examined if there would be method-driven differences between non- and pre-processed sediment samples regarding the quality and quantity of extracted DNA, PCR amplicon, resulting high-throughput sequencing reads, microbial diversity, and community composition. We found no significant difference in the quality and quantity of extracted DNA and PCR amplicons between the two methods. Although we found a significant difference in raw and quality-filtered reads, read abundance after bioinformatics processing (i.e., denoising and the chimeric-read filtering steps) were not significantly different. These results suggest that read abundance after these read processing steps were not influenced by sediment processing or lack thereof. Although the non- and pre-processed sediment samples had more unique than shared amplicon sequence variants (ASVs), we report that their shared ASVs accounted for 74% of both methods’ absolute read abundance. More so at the genus level, the final collection filter identified most of the genera (95% of the reads) captured from the non-processed samples, with a total of 51 false-negative (2%) and 59 false-positive genera (3%). Accordingly, the diversity estimates and community composition were not significantly different between the non- and pre-processed samples. We demonstrate that while there were differences in shared and unique taxa, both methods revealed comparable microbial diversity and community composition. We also suggest the inclusion of sequential filters (i.e., pre- and mid-filters) in the community profiling, given the additional taxa not detected from the non-processed and the final collection filter. Our observations highlight the feasibility of pre-processing sediment samples for community analysis and the need to further assess sampling strategies to help conceptualize appropriate study designs for sediment-associated microbial community profiling.

scholarly journals Bacterial Microbiota in Unfed Ticks (Dermacentor nuttalli) From Xinjiang Detected Through 16S rDNA Amplicon Sequencing and Culturomics

Zoonoses ◽  
2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Kai Song ◽  
Yuxin Ji ◽  
Surong Sun ◽  
Xihong Yue ◽  
Cheng Wang ◽  
...  
Keyword(s):  
16S Rdna ◽  
Bacterial Species ◽  
Amplicon Sequencing ◽  
Zoonotic Diseases ◽  
Comprehensive Understanding ◽  
Bacterial Microbiota ◽  
Western Tianshan ◽  
Rdna Sequencing ◽  
16S Rdna Amplicon Sequencing ◽  
Acinetobacter Pittii

Background: Ticks are a major arthropod vector of zoonotic diseases affecting both humans and domestic animals worldwide. Thus, studying tick microbiota would aid in understanding of the potential threats posed by ticks. Methods: Approximately 8,000 unfed ticks, identified as Dermacentor nuttalli, were collected from the sylvosteppe in the western Tianshan mountains. To investigate their potential pathogens, we divided the ticks into 36 groups of 200–300 individuals each for examination with culturomics and 16S rDNA amplicon sequencing. Results: A total of 237 bacterial genera were identified with the two methods. Culturomics identified 46 bacterial species from 23 genera, predominantly Pseudomonas, Pantoea, and Bacillus, whereas 16S rDNA sequencing identified 461 OTUs from 233 genera, predominantly Pseudomonas (53.8%), Coxiella (17.2%), and Pantoea (6.4%). Coxiella, Rickettsia, and ten other genera were discovered only by sequencing, because optimal cultivating conditions were not used for their isolation, whereas Arthrobacter and three other genera were discovered only through culturomics. Conclusions: Several of the identified bacteria, such as line-related sepsis-causing Delftia acidovorans and the pneumonia agent Acinetobacter pittii, can cause human diseases. Thus, both sequencing and culturomics methods are crucial for comprehensive understanding of the microbiota of D. nuttalli.

Quantitative Assessment of Shotgun Metagenomics and 16S rDNA Amplicon Sequencing in the Study of Human Gut Microbiome

2018 ◽  
Vol 22 (4) ◽  
pp. 248-254 ◽  
Author(s):  
Ilaria Laudadio ◽  
Valerio Fulci ◽  
Francesca Palone ◽  
Laura Stronati ◽  
Salvatore Cucchiara ◽  
...  
Keyword(s):  
16S Rdna ◽  
Gut Microbiome ◽  
Quantitative Assessment ◽  
Amplicon Sequencing ◽  
Human Gut ◽  
Shotgun Metagenomics ◽  
Human Gut Microbiome ◽  
16S Rdna Amplicon Sequencing

scholarly journals Bovine Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization

2021 ◽  
Vol 8 ◽  
Author(s):  
Godelind Alma Wolf-Jäckel ◽  
Mikael Lenz Strube ◽  
Kirstine Klitgaard Schou ◽  
Christiane Schnee ◽  
Jørgen S. Agerholm ◽  
...  
Keyword(s):  
16S Rdna ◽  
Pathogenic Bacteria ◽  
Amplicon Sequencing ◽  
Diagnostic Methods ◽  
Molecular Methods ◽  
Economic Losses ◽  
Growth Media ◽  
Microbial Culture ◽  
16S Rdna Amplicon Sequencing

Abortion in cattle causes significant economic losses for cattle farmers worldwide. The diversity of abortifacients makes abortion diagnostics a complex and challenging discipline that additionally is restrained by time and economy. Microbial culture has traditionally been an important method for the identification of bacterial and mycotic abortifacients. However, it comes with the inherent bias of favoring the easy-to-culture species, e.g., those that do not require cell culture, pre-enrichment, a variety of selective growth media, or different oxygen levels for in vitro growth. Molecular methods such as polymerase chain reaction (PCR) and next-generation sequencing have been established as alternatives to traditional microbial culturing methods in several diagnostic fields including abortion diagnostics. Fluorescence in situ hybridization (FISH), a bridging microscopy technique that combines molecular accuracy with culture independence, and spatial resolution of the pathogen-lesion relation, is also gaining influence in several diagnostic fields. In this study, real-time quantitative PCR (qPCR), 16S rDNA amplicon sequencing, and FISH were applied separately and in combination in order to (i) identify potentially abortifacient bacteria without the bias of culturability, (ii) increase the diagnostic rate using combined molecular methods, (iii) investigate the presence of the difficult-to-culture zoonotic agents Coxiella burnetii, Chlamydia spp., and Leptospira spp. in bovine abortions in Denmark. Tissues from 162 aborted or stillborn bovine fetuses and placentas submitted for routine diagnostics were screened for pathogenic bacteria using 16S rDNA amplicon sequencing. Lesion association of fungal elements, as well as of selection of bacterial abortifacients, was assessed using specific FISH assays. The presence of Chlamydia spp. and chlamydia-like organisms was assessed using qPCR. The study focused on bacterial and fungal abortifacients, because Danish cattle is free from most viral abortifacients. The 16S rDNA amplicon sequencing–guided FISH approach was suitable for enhancing abortion diagnostics, i.e., the diagnostic rate for cases with tissue lesions (n = 115) was increased from 46 to 53% when compared to routine diagnostic methods. Identification of Bacillus licheniformis, Escherichia coli, and Trueperella pyogenes accounted for the majority of additional cases with an established etiology. No evidence for emerging or epizootic bacterial pathogens was found. The difficult-to-culture abortifacients were either not detected or not identified as abortifacients.

scholarly journals Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Sandra Reitmeier ◽  
Thomas C. A. Hitch ◽  
Nicole Treichel ◽  
Nikolaos Fikas ◽  
Bela Hausmann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Diversity Analysis ◽  
Careful Attention ◽  
Operational Taxonomic Units ◽  
Basic Concepts ◽  
Gnotobiotic Mice ◽  
Mock Communities

Abstract16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

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