scholarly journals The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Ingo C. Starke ◽  
Wilfried Vahjen ◽  
Robert Pieper ◽  
Jürgen Zentek
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Dna Extraction ◽  
16S Rrna Genes ◽  
Read Length ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Variable Regions ◽  
Extraction Procedures ◽  
Primer Sets

In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2×105 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

scholarly journals Quantitative and Qualitative Characterization of Plant Endo-bacteriome by Plant Dna-free Sequencing Method

2020 ◽  
Author(s):  
Lili Zhang ◽  
Liying Chen ◽  
Mengting Zhang ◽  
Da Liu ◽  
Hongbo Sun ◽  
...  
Keyword(s):  
16S Rrna ◽  
Quantitative Pcr ◽  
Endophytic Bacteria ◽  
Plant Material ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Variable Regions ◽  
Plant Dna ◽  
Primer Sets

Abstract Background: Plant endophytic bacteria colonize plants’ internal tissues and interact with plants more closely than do epiphytic or environmental bacteria. However, the community structure of such endophytic bacteria could not be efficiently deciphered, and the microbiota abundance could not be quantified through absolute quantification. Application of 16S rRNA gene sequencing to characterize the plant endophytic community is greatly hindered by the high sequence identities among bacterial 16S rRNA, plant mitochondrial 18S rRNA and chloroplast 16S rRNA genes. This makes it difficult to identify bacterial sequences among total DNAs extracted from plant material.Results: We designed PCR primer sets that are able to specifically amplify bacterial DNAs, even when there are very few bacteria colonizing the plant material. We computationally and experimentally evaluated the specificity, coverage, and accuracy of the newly designed primer sets (322F/796R and 799F/1107R) and two widely used primer sets (338F/806R and 799F/1193R). When applied to a same planting-soil community through next generation sequencing (NGS), the four different primer sets revealed similar high-abundant taxa composition with variation in taxa abundance. Primer sets amplifying the same 16S variable regions generated more comparable sequencing results. When applied to a rice endo-bacteriome, both 799F/1107R and modified 322F-Dr/796Rs (Primer pair 322F/796R with a penultimate-base substitution in 322F) produced plant DNA-free bacterial amplicon libraries. Primer 322F-A/796R was then used through NGS to decipher the rice endo-bacteriomes. The rice root and leaf endo-bacteriomes shared 66.36% OTU identity but enriched different bacterial species. Within the same host genotype and soil type, the root endo-bacteriome was more stable than the leaf endo-bacteriome across individual plants. 322F-A/796R was used through absolute quantitative PCR to quantitate the population size of leaf or root endophytes, which revealed 106–107 and 109–1010 bacteria per gram fresh weight, respectively.Conclusions: This is the first study to develop plant DNA-free bacterial 16S amplification methods. The newly designed primer sets combined with NGS deciphered the rice endo-bacteriome structure, and absolute quantitative PCR quantitated the size of the endobacterial population. The protocols developed here are suitable for various plants, will significantly advance studies on plant endo-bacteriomes.

scholarly journals Multilocus sequence analysis of Ensifer and related taxa

2007 ◽  
Vol 57 (3) ◽  
pp. 489-503 ◽  
Author(s):  
Miet Martens ◽  
Manuel Delaere ◽  
Renata Coopman ◽  
Paul De Vos ◽  
Monique Gillis ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Housekeeping Genes ◽  
16S Rrna Genes ◽  
New Combination ◽  
Rrna Genes ◽  
Multilocus Sequence Analysis ◽  
Bootstrap Support ◽  
Rrna Gene ◽  
Separate Species

Multilocus sequence analysis (MLSA) was performed on representatives of Ensifer (including species previously assigned to the genus Sinorhizobium) and related taxa. Neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) phylogenies of dnaK, gltA, glnA, recA, thrC and 16S rRNA genes were compared. The data confirm that the potential for discrimination of Ensifer species is greater using MLSA of housekeeping genes than 16S rRNA genes. In incongruence-length difference tests, the 16S rRNA gene was found to be significantly incongruent with the other genes, indicating that this gene should not be used as a single indicator of relatedness in this group. Significant congruence was detected for dnaK, glnA and thrC. Analyses of concatenated sequences of dnaK, glnA and thrC genes yielded very similar NJ, MP and ML trees, with high bootstrap support. In addition, analysis of a concatenation of all six genes essentially produced the same result, levelling out potentially conflicting phylogenetic signals. This new evidence supports the proposal to unite Ensifer and Sinorhizobium in a single genus. Support for an alternative solution preserving the two genera is less strong. In view of the opinions expressed by the Judicial Commission, the name of the genus should be Ensifer, as proposed by Young [Young, J. M. (2003). Int J Syst Evol Microbiol 53, 2107–2110]. Data obtained previously and these new data indicate that Ensifer adhaerens and ‘Sinorhizobium morelense’ are not heterotypic synonyms, but represent separate species. However, transfer to the genus Ensifer is not possible at present because the species name is the subject of a pending Request for an Opinion, which would affect whether a novel species in the genus Ensifer or a new combination based on a basonym would be created.

scholarly journals Detection and Quantification of Dehalogenimonas and “Dehalococcoides” Populations via PCR-Based Protocols Targeting 16S rRNA Genes

2009 ◽  
Vol 75 (23) ◽  
pp. 7560-7564 ◽  
Author(s):  
Jun Yan ◽  
Brian A. Rash ◽  
Fred A. Rainey ◽  
William M. Moe
Keyword(s):  
16S Rrna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Superfund Site ◽  
16S Rrna Gene Sequences ◽  
Specific Primers ◽  
Variable Regions ◽  
Bacterial Groups ◽  
Detection And Quantification

ABSTRACT Members of the haloalkane dechlorinating genus Dehalogenimonas are distantly related to “Dehalococcoides” but share high homology in some variable regions of their 16S rRNA gene sequences. In this study, primers and PCR protocols intended to uniquely target Dehalococcoides were reevaluated, and primers and PCR protocols intended to uniquely target Dehalogenimonas were developed and tested. Use of the genus-specific primers revealed the presence of both bacterial groups in groundwater at a Louisiana Superfund site.

scholarly journals Molecular characterization of Mycobacterium intracellulare-related strains based on the sequence analysis of hsp65, internal transcribed spacer and 16S rRNA genes

2010 ◽  
Vol 59 (9) ◽  
pp. 1037-1043 ◽  
Author(s):  
Joo-Hee Park ◽  
Tae-Sun Shim ◽  
Seung-Ae Lee ◽  
Hyungki Lee ◽  
In-Kyung Lee ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Internal Transcribed Spacer ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Mycobacterium Intracellulare ◽  
Epidemiological Features ◽  
The 16S Rrna Gene

We investigated the molecular epidemiological features of 94 Mycobacterium intracellulare-related strains, isolated from Korean patients, using sequence analysis targeting 3 independent chronometer molecules, hsp65, the internal transcribed spacer 1 region and the 16S rRNA gene. By collective consideration of these three gene-based approaches, the 94 strains were divided into 5 groups (INT1, INT2, INT3, INT4 and INT5). The frequencies of genotype INT1, 2, 3, 4 and 5 in the 94 isolates were 57.4 % (54), 27.7 % (26), 6.4 % (6), 5.3 % (5) and 3.2 % (3), respectively. When correlations between genotypes and clinical parameters (age, sex, radiological type and the presence of a cavity) were analysed in 78 patients with non-tuberculous mycobacteria pulmonary diseases, no relationships were observed with respect to age, sex and radiological type, but genotype and the presence of a cavity tended to be related (P=0.051).

scholarly journals Identification and classification of the genus Bacteroides by multilocus sequence analysis

Microbiology ◽  
2011 ◽  
Vol 157 (12) ◽  
pp. 3388-3397 ◽  
Author(s):  
Mitsuo Sakamoto ◽  
Moriya Ohkuma
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Sequence Similarity ◽  
Single Gene ◽  
Housekeeping Genes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Multilocus Sequence Analysis ◽  
Rrna Gene

Multilocus sequence analysis (MLSA) was performed on representative species of the genus Bacteroides. Internal fragments of the genes selected, dnaJ, gyrB, hsp60, recA, rpoB and 16S rRNA, were amplified by direct PCR and then sequenced from 38 Bacteroides strains representing 35 species. Neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) phylogenies of the individual genes were compared. The data confirm that the potential for discrimination of Bacteroides species is greater using MLSA of housekeeping genes than 16S rRNA genes. Among the housekeeping genes analysed, gyrB was the most informative, followed by dnaJ. Analyses of concatenated sequences (4816 bp) of all six genes revealed robust phylogenetic relationships among different Bacteroides species when compared with the single-gene trees. The NJ, ML and MP trees were very similar, and almost fully resolved relationships of Bacteroides species were obtained, to our knowledge for the first time. In addition, analysis of a concatenation (2457 bp) of the dnaJ, gyrB and hsp60 genes produced essentially the same result. Ten distinct clades were recognized using the SplitsTree4 program. For the genus Bacteroides, we can define species as a group of strains that share at least 97.5 % gene sequence similarity based on the fragments of five protein-coding housekeeping genes and the 16S rRNA gene. This study demonstrates that MLSA of housekeeping genes is a valuable alternative technique for the identification and classification of species of the genus Bacteroides.

scholarly journals Phylogenetic Characterization of a Polychlorinated-Dioxin- Dechlorinating Microbial Community by Use of Microcosm Studies

2005 ◽  
Vol 71 (8) ◽  
pp. 4325-4334 ◽  
Author(s):  
Naoko Yoshida ◽  
Nobutaka Takahashi ◽  
Akira Hiraishi
Keyword(s):  
16S Rrna ◽  
Oxidative Degradation ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Aerobic Bacteria ◽  
Rrna Gene ◽  
Phylogenetic Characterization ◽  
Gene Copies ◽  
Primer Sets ◽  
Microcosm Studies

ABSTRACT Microcosms capable of reductive dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed in glass bottles by seeding them with a polluted river sediment and incubating them anaerobically with an organic medium. All of the PCDD/F congeners detected were equally reduced without the accumulation of significant amounts of less-chlorinated congeners as the intermediate or end products. Alternatively, large amounts of catechol and salicylic acid were produced in the upper aqueous phase. Thus, the dechlorination of PCDD/Fs and the oxidative degradation of the dechlorinated products seemed to take place simultaneously in the microcosm. Denaturing gel gradient electrophoresis and clone library analyses of PCR-amplified 16S rRNA genes from the microcosm showed that members of the phyla Firmicutes, Proteobacteria, and Bacteroidetes predominated. A significant number of Chloroflexi clones were also detected. Quantitative real-time PCR with specific primer sets showed that the 16S rRNA genes of a putative dechlorinator, “Dehalococcoides,” and its relatives accounted for 0.1% of the total rRNA gene copies of the microcosm. Most of the clones thus obtained formed a cluster distinct from the typical “Dehalococcoides” group. Quinone profiling indicated that ubiquinones accounted for 18 to 25% of the total quinone content, suggesting the coexistence and activity of ubiquinone-containing aerobic bacteria. These results suggest that the apparent complete dechlorination of PCDD/Fs found in the microcosm was due to a combination of the dechlorinating activity of the “Dehalococcoides”-like organisms and the oxidative degradation of the dechlorinated products by aerobic bacteria with aromatic hydrocarbon dioxygenases.

scholarly journals Effects of Experimental Choices and Analysis Noise on Surveys of the “Rare Biosphere”

2009 ◽  
Vol 75 (10) ◽  
pp. 3263-3270 ◽  
Author(s):  
Timothy J. Hamp ◽  
W. Joe Jones ◽  
Anthony A. Fodor
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Treatment Plant ◽  
Whole Genome Shotgun ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Whole Genome ◽  
Variable Regions ◽  
Rare Biosphere

ABSTRACT When planning a survey of 16S rRNA genes from a complex environment, investigators face many choices including which primers to use and how to taxonomically classify sequences. In this study, we explored how these choices affected a survey of microbial diversity in a sample taken from the aerobic basin of the activated sludge of a North Carolina wastewater treatment plant. We performed pyrosequencing reactions on PCR products generated from primers targeting the V1-V2, V6, and V6-V7 variable regions of the 16S rRNA gene. We compared these sequences to 16S rRNA gene sequences found in a whole-genome shotgun pyrosequencing run performed on the same sample. We found that sequences generated from primers targeting the V1-V2 variable region had the best match to the whole-genome shotgun reaction across a range of taxonomic classifications from phylum to family. Pronounced differences between primer sets, however, occurred in the “rare biosphere” involving taxa that we observed in fewer than 11 sequences. We also examined the results of analysis strategies comparing a classification scheme using a nearest-neighbor approach to directly classifying sequences with a na�ve Bayesian algorithm. Again, we observed pronounced differences between these analysis schemes in infrequently observed taxa. We conclude that if a study is meant to probe the rare biosphere, both the experimental conditions and analysis choices will have a profound impact on the observed results.

scholarly journals Sequence analysis of 12S rRNA and 16S rRNA mitochondrial genes in Iranian Afshari sheep

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Mohammad Mahmoodi ◽  
Kian Pahlevan Afshari ◽  
Hamid Reza Seyedabadi ◽  
Mehran Aboozari
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Conservation Strategies ◽  
12S Rrna ◽  
Rrna Gene ◽  
Salting Out ◽  
The Rich

Phylogenetic relationships and genetic variation in Iranian Afshari sheep breed were analyzed using 12S rRNA and 16S rRNA gene sequences. The genomic DNA was isolated by salting out method and amplified 12S rRNA and 16S rRNA genes using PCR method. PCR amplification of 12S and 16S rRNA generated PCR amplicons at 859 and 1053 bp lengths, respectively. Sequence analysis was performed using BioEdit software. Phylogenetic tree was constructed using MEGA software. Phylogenetic analysis of haplotype in the combination with the sheep from GenBank showed that Iranian Afshari sheep made a close to the Australian sheep cluster. This study was found informative for establishing relationships between breeds from different parts of the world. This study may facilitate the future researchers and breeders for better understanding the genetic interactions and breed differentiation for devising future breeding and conservation strategies to preserve the rich animal genetic reservoir of the country.

scholarly journals Molecular characterization of Pseudomonas aeruginosa isolates from Sudanese patients: A cross-sectional study

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 1135
Author(s):  
Reem H. Amoon ◽  
Amna H. Abdallha ◽  
Ahmed Osman Sharif ◽  
Ehssan H. Moglad ◽  
Hisham N. Altyb ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sequence Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bacterial Disease ◽  
Clinical Specimens

Background:16S rRNA gene sequence analysis is a robust tool for characterization of new pathogens in clinical specimens with suspected bacterial disease. The aim of this study was to characterizePseudomonas aeruginosaisolated from clinical specimens by sequencing the 16S rRNA gene.Methods:Forty bacterial isolates were obtained from different clinical specimens (wound, urine and sputum) using enrichment selective media and biochemical tests to characterize and identify the bacteria asP. aeruginosa.DNA was extracted fromP. aeruginosausing the Chelex method. A universal primer was used to amplify 16S rRNA genes by a conventional PCR technique. The amplified PCR products were sequenced, and the sequences were viewed by Finch TV program version 1.4.0. The identity and similarity of the nucleotide sequence of the isolated strains was detected by comparing them with published sequences using BLASTn. Phylogenetic trees were constructed using Phylogeny.fr software.Results:Sequence analysis by BLASTn displayed high similarity and identity withP. aeruginosafrom China KX461910, Australia JN609194 and with otherP. aeruginosaisolates from the GenBank database.Conclusions:Our observation of isolates from different origin sites, further show the utility of 16s rRNA PCR amplification. This reveals the high specify of the primers and accuracy of the PCR. Thus, 16S rRNA sequencing can be used to identify genetically atypicalP. aeruginosaisolates from different origins.

Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

2015 ◽  
Vol 41 (1) ◽  
pp. 51-58
Author(s):  
Mohammad Shamimul Alam ◽  
Hawa Jahan ◽  
Rowshan Ara Begum ◽  
Reza M Shahjahan
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fish Larva ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Valuable Insight ◽  
Multiple Sequence ◽  
Pcr Rflp ◽  
Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

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