scholarly journals Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0241732
Author(s):  
Michael E. C. Abundo ◽  
John M. Ngunjiri ◽  
Kara J. M. Taylor ◽  
Hana Ji ◽  
Amir Ghorbani ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Production Performance ◽  
Rrna Gene ◽  
Sequence Variant ◽  
Sample Type ◽  
Alpha And Beta Diversity ◽  
Gene Copies ◽  
Diversity Metrics

Characterization of poultry microbiota is becoming increasingly important due to the growing need for microbiome-based interventions to improve poultry health and production performance. However, the lack of standardized protocols for sampling, sample processing, DNA extraction, sequencing, and bioinformatic analysis can hinder data comparison between studies. Here, we investigated how the DNA extraction process affects microbial community compositions and diversity metrics in different chicken respiratory sample types including choanal and tracheal swabs, nasal cavity and tracheal washes, and lower respiratory lavage. We did a side-by-side comparison of the performances of Qiagen DNeasy blood and tissue (BT) and ZymoBIOMICS DNA Miniprep (ZB) kits. In general, samples extracted with the BT kit yielded higher concentrations of total DNA while those extracted with the ZB kit contained higher numbers of bacterial 16S rRNA gene copies per unit volume. Therefore, the samples were normalized to equal amounts of 16S rRNA gene copies prior to sequencing. For each sample type, all predominant bacterial taxa detected in samples extracted with one kit were present in replicate samples extracted with the other kit and did not show significant differences at the class level. However, a few differentially abundant shared taxa were observed at family and genus levels. Furthermore, between-kit differences in alpha and beta diversity metrics at the amplicon sequence variant level were statistically indistinguishable. Therefore, both kits perform similarly in terms of 16S rRNA gene-based poultry microbiome analysis for the sample types analyzed in this study.

scholarly journals Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types

2020 ◽  
Author(s):  
Michael E.C. Abundo ◽  
John M. Ngunjiri ◽  
Kara J.M. Taylor ◽  
Hana Ji ◽  
Amir Ghorbani ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Extraction Process ◽  
Production Performance ◽  
Rrna Gene ◽  
Sample Type ◽  
Alpha And Beta Diversity ◽  
Gene Copies ◽  
Diversity Metrics

AbstractCharacterization of poultry microbiota is becoming increasingly important due to the growing need for microbiome-based interventions to improve poultry health and production performance. However, the lack of standardized protocols for sampling, sample processing, DNA extraction, sequencing, and bioinformatic analysis can hinder data comparison between studies. Here, we investigated how the DNA extraction process affects microbial community compositions and diversity metrics in different chicken respiratory sample types including choanal and tracheal swabs, nasal cavity and tracheal washes, and lower respiratory lavage. We did a side-by-side comparison of the performances of Qiagen DNeasy blood and tissue (BT) and ZymoBIOMICS DNA Miniprep (ZB) kits. In general, samples extracted with the BT kit yielded higher concentrations of total DNA while those extracted with the ZB kit contained higher numbers of bacterial 16S rRNA gene copies per unit volume. Therefore, the samples were normalized to equal amounts of 16S rRNA gene copies prior to sequencing. For each sample type, all predominant taxa detected in samples extracted with one kit were present in replicate samples extracted with the other kit and did not show significant differences at the class level. Furthermore, between-kit differences in alpha and beta diversity metrics were statistically indistinguishable. Therefore, both kits perform similarly with regard to 16S rRNA gene-based poultry microbiome analysis.

scholarly journals Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals

2021 ◽  
Vol 12 ◽  
Author(s):  
Hannah E. Epstein ◽  
Alejandra Hernandez-Agreda ◽  
Samuel Starko ◽  
Julia K. Baum ◽  
Rebecca Vega Thurber
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Beta Diversity ◽  
Sequence Data ◽  
Alpha Diversity ◽  
Gene Profiling ◽  
Rrna Gene ◽  
Sequencing Platform ◽  
Alpha And Beta Diversity ◽  
Diversity Metrics

16S rRNA gene profiling (amplicon sequencing) is a popular technique for understanding host-associated and environmental microbial communities. Most protocols for sequencing amplicon libraries follow a standardized pipeline that can differ slightly depending on laboratory facility and user. Given that the same variable region of the 16S gene is targeted, it is generally accepted that sequencing output from differing protocols are comparable and this assumption underlies our ability to identify universal patterns in microbial dynamics through meta-analyses. However, discrepant results from a combined 16S rRNA gene dataset prepared by two labs whose protocols differed only in DNA polymerase and sequencing platform led us to scrutinize the outputs and challenge the idea of confidently combining them for standard microbiome analysis. Using technical replicates of reef-building coral samples from two species, Montipora aequituberculata and Porites lobata, we evaluated the consistency of alpha and beta diversity metrics between data resulting from these highly similar protocols. While we found minimal variation in alpha diversity between platform, significant differences were revealed with most beta diversity metrics, dependent on host species. These inconsistencies persisted following removal of low abundance taxa and when comparing across higher taxonomic levels, suggesting that bacterial community differences associated with sequencing protocol are likely to be context dependent and difficult to correct without extensive validation work. The results of this study encourage caution in the statistical comparison and interpretation of studies that combine rRNA gene sequence data from distinct protocols and point to a need for further work identifying mechanistic causes of these observed differences.

scholarly journals Intragenomic and intraspecific heterogeneity in rrs may surpass interspecific variability in a natural population of Veillonella

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 2080-2091 ◽  
Author(s):  
Anne-Laure Michon ◽  
Fabien Aujoulat ◽  
Laurent Roudière ◽  
Olivier Soulier ◽  
Isabelle Zorgniotti ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Diversity ◽  
Natural Populations ◽  
Variable Region ◽  
Housekeeping Gene ◽  
Single Copy ◽  
Population Based ◽  
Rrna Gene ◽  
Gene Copies

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of Veillonella spp. of human origin and in 13 type or reference Veillonella strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5–2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene rpoB was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different rpoB clades, impaired the 16S rRNA-based identification for some Veillonella species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

scholarly journals Six species of nontuberculous mycobacteria carry non-identical 16S rRNA gene copies

2018 ◽  
Vol 155 ◽  
pp. 34-36
Author(s):  
Keita Takeda ◽  
Kinuyo Chikamatsu ◽  
Yuriko Igarashi ◽  
Yuta Morishige ◽  
Yoshiro Murase ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nontuberculous Mycobacteria ◽  
Rrna Gene ◽  
Gene Copies

DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities

1992 ◽  
Vol 100 (1-3) ◽  
pp. 59-65 ◽  
Author(s):  
Paul A. Rochelle ◽  
John C. Fry ◽  
R. John Parkes ◽  
Andrew J. Weightman
Keyword(s):  
Genetic Diversity ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Gene Analysis ◽  
Rrna Gene ◽  
16S Rrna Gene Analysis ◽  
Deep Sediment

scholarly journals Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor

2017 ◽  
Vol 28 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Anniina Rintala ◽  
Sami Pietilä ◽  
Eveliina Munukka ◽  
Erkki Eerola ◽  
Juha-Pekka Pursiheimo ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Rrna Gene ◽  
Gene Target ◽  
Target Region ◽  
Microbiota Analysis ◽  
The Impact ◽  
The 16S Rrna Gene

Heterogeneity between 16S ribosomal RNA gene copies borne by one Desulfitobacterium strain is caused by different 100-200 bp insertions in the 5´ region

2007 ◽  
Vol 53 (1) ◽  
pp. 116-128 ◽  
Author(s):  
Richard Villemur ◽  
Philippe Constant ◽  
Annie Gauthier ◽  
Martine Shareck ◽  
Réjean Beaudet
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Ribosomal Rna ◽  
16S Ribosomal Rna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Gene Copies

Strains of Desulfitobacterium hafniense, such as strains PCP-1, DP7, TCE1, and TCP-A, have unusual long 16S ribosomal RNA (rRNA) genes due to an insertion of approximately 100 bp in the 5' region. In this report, we analyzed the 16S rRNA genes of different Desulfitobacterium strains to determine if such an insertion is a common feature of desulfitobacteria. We amplified this region by polymerase chain reaction (PCR) from eight Desulfitobacterium strains (D. hafniense strains PCP-1, DP7, TCP-A, TCE1, and DCB-2; D. dehalogenans; D. chlororespirans; and Desulfitobacterium sp. PCE1) and resolved each PCR product by denaturing gradient gel electrophoresis (DGGE). All strains had from two to seven DGGE- migrating bands, suggesting heterogeneity in their 16S rRNA gene copies. For each strain, the 5' region of the 16S rRNA genes was amplified and a clone library was derived. Clones corresponding to most PCR–DGGE migration bands were isolated. Sequencing of representative clones revealed that the heterogeneity was generated by insertions of 100–200 bp. An insertion was found in at least one copy of the 16S rRNA gene in all examined strains. In total, we found eight different types of insertions (INS1–INS8) that varied from 123 to 193 nt in length. Two-dimensional structural analyses of transcribed sequences predicted that all insertions would form an energetically stable loop. Reverse transcriptase – PCR experiments revealed that most of the observed insertions in the Desulfitobacterium strains were excised from the mature 16S rRNA transcripts. Insertions were not commonly found in bacterial 16S rRNA genes, and having a different insertion in several 16S rRNA gene copies borne by a single bacterial species was rarely observed. The function of these insertions is not known, but their occurrence can have an important impact in deriving 16S rRNA oligonucleotidic fluorescence in situ hybridization probes, as these insertions can be excised from 16S rRNA transcripts.Key words: Desulfitobacterium, 16S ribosomal RNA genes, heterogeneity, gene insertions, fluorescence in situ hybridization.

scholarly journals Bacterial DNAemia is associated with serum zonulin levels in older subjects

2020 ◽  
Author(s):  
Giorgio Gargari ◽  
Valentina Taverniti ◽  
Cristian Del Bo’ ◽  
Stefano Bernardi ◽  
Cristina Andres-Lacueva ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Serum Levels ◽  
Gene Profiling ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Blood Samples ◽  
Gene Copies ◽  
Older Subjects ◽  
Pcr Targeting

AbstractThe increased presence of bacteria in blood is a plausible contributing factor in the development and progression of aging-associated diseases. In this context, we performed the quantification and the taxonomic profiling of the bacterial DNA in blood samples collected from a group of forty-three older subjects enrolled in a nursing home. Quantitative PCR targeting the 16S rRNA gene revealed that all the older volunteers contained detectable amounts of bacterial DNA in their blood. The total amount of 16S rRNA gene copies varied considerably between subjects. Correlation analyses revealed that the bacterial DNAemia (expressed as concentration of 16S rRNA gene copies in blood) significantly correlated with the serum levels of zonulin, an emerging marker of intestinal permeability. This result was confirmed by the analysis of a second set of blood samples collected after approximately four months from the same subjects. Analyses of 16S rRNA gene profiling revealed that most of the bacterial DNA detected in blood was ascribable to the phylum Proteobacteria with a predominance of Pseudomonadaceae and Enterobacteriaceae. Several control samples were also analyzed to assess the influence exerted by contaminant bacterial DNA potentially originating from reagents and materials. The date reported here suggest that para-cellular permeability of epithelial (and potentially also endothelial) cell layers may play an important role in bacterial migration into the bloodstream. Bacterial DNAemia is likely to impact on several aspects of host physiology and could underpin the development and prognosis of various diseases in older subjects.

scholarly journals Evaluation of established methods for DNA extraction and primer pairs targeting 16S rRNA gene for bacterial microbiome profiling of olive xylem sap

2020 ◽  
Author(s):  
CARMEN HARO ◽  
MANUEL ANGUITA-MAESO ◽  
Madis Metsis ◽  
JUAN A NAVAS-CORTES ◽  
BLANCA LANDA
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Bacterial Communities ◽  
Methodological Approach ◽  
Xylem Sap ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Community Assessment ◽  
Woody Crop

Next Generation Sequencing has revolutionized our ability to investigate the microbiota composition of diverse and complex environments. However, a number of factors can affect the accuracy of microbial community assessment, such as the DNA extraction method, the hypervariable region of 16S rRNA gene targeted or the PCR primers used for amplification. The aim of this study was to assess the influence of commercially available DNA extraction kits and different primer pairs to provide a nonbiased vision of the composition of bacterial communities present in olive xylem sap. For that purpose, branches from 'Picual' and 'Arbequina' olive cultivars were used for xylem sap extraction using a Scholander chamber device. The DNA extraction protocol significantly affected xylem sap bacterial community assessment. That resulted in significant differences in alpha (Richness) and beta diversity (UNIFRAC distances) metrics among DNA extraction protocols, with the 12 DNA extraction kits evaluated being clustered in four groups behaving differently. Although the core number of taxa detected by all DNA extraction kits included four phyla, seven classes, 12 orders, and 16 or 21 families, and 12 or 14 genera when using the Greengenes or Silva database for taxonomic assignation, respectively, some taxa, particularly those identified at low frequency, were detected by some DNA extraction kits only. The most accurate depiction of a bacterial mock community artificially inoculated on sap samples was generated when using the PowerPlant DNA extraction Kit, the combination of 799F/1193R primers amplifying the hypervariable V5-V7 region and the Silva 132 database for taxonomic assignation. The DESeq2 analysis displayed significant differences among genera abundance between the different PCR primer pairs tested. Thus, Enterobacter, Granulicatella, Prevotella and Brevibacterium presented a significant higher abundance in all PCR protocols when compared with primer pair 799F/1193R, while the opposite was true for Pseudomonas and Pectobacterium. The methodological approach followed in this study can be useful to optimize plant-associated microbiome analysis, especially when exploring new plant niches. Some of the DNA extraction kits and PCR primers selected in this study will contribute to better characterize bacterial communities inhabiting within the xylem sap of olives or other woody crop species.

scholarly journals Six species of non-tuberculosis mycobacteria carry non-identical 16S rRNA gene copies

2018 ◽  
Author(s):  
Keita Takeda ◽  
Kinuyo Chikamatsu ◽  
Yuriko Igarashi ◽  
Yuta Morishige ◽  
Yoshiro Murase ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Target Sequence ◽  
Nucleotide Polymorphisms ◽  
16S Rrna Gene Sequences ◽  
Cloning And Sequencing ◽  
Single Nucleotide ◽  
Gene Copies ◽  
The 16S Rrna Gene

AbstractNon-tuberculosis mycobacteria (NTM) can carry two or more 16S rRNA gene copies that are, in some instances, non-identical. In this study, we used a combined cloning and sequencing approach to analyze the 16S rRNA gene sequences of six NTM species,Mycobacterium cosmeticum, M. pallens, M. hodleri, M. crocinum, M. flavescens, andM. xenopi. The approach facilitated the identification of two distinct gene copies in each species. The twoM. cosmeticumgenes had a single nucleotide difference, whereas two nucleotide polymorphisms were identified inM. hodleri, M. flavescens, andM. xenopi. M. pallenshad a difference in four nucleotides andM. crocinumin 23. Hence, we showed that the six NTM species possess at least two non-identical 16S rRNA gene copies.ImportanceThe presence of multiple 16S rRNA gene copies with nucleotide polymorphisms represents a challenge for species identification using 16S rRNA as the target sequence. Our analysis was focused on six NTM species,M. cosmeticum, M. pallens, M. hodleri, M. crocinum, M. flavescens, andM. xenopi. As a result, we generated the full-length sequences of two non-identical 16S rRNA copies for each NTM species. The data will be helpful for the sequence analysis of specimens or other samples.

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