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121. J Microbiol Methods. 2013 Nov;95(2):175-81. doi: 10.1016/j.mimet.2013.08.009.

Epub 2013 Aug 30.


Efficacy of a 3rd generation high-throughput sequencing platform for analyses of

16S rRNA genes from environmental samples.


Mosher JJ(1), Bernberg EL, Shevchenko O, Kan J, Kaplan LA.


Author information:

(1)Stroud Water Research Center, Avondale, PA 19311, United States. Electronic

address: jmosher@stroudcenter.org.


Longer sequences of the bacterial 16S rRNA gene could provide greater

phylogenetic and taxonomic resolutions and advance knowledge of population

dynamics within complex natural communities. We assessed the accuracy of a

Pacific Biosciences (PacBio) single molecule, real time (SMRT) sequencing based

on DNA polymerization, a promising 3rd generation high-throughput technique, and

compared this to the 2nd generation Roche 454 pyrosequencing platform. Amplicons

of the 16S rRNA gene from a known isolate, Shewanella oneidensis MR1, and

environmental samples from two streambed habitats, rocks and sediments, and a

riparian zone soil, were analyzed. On the PacBio we analyzed ~500 bp amplicons

that covered the V1-V3 regions and the full 1500 bp amplicons of the V1-V9

regions. On the Roche 454 we analyzed the ~500 bp amplicons. Error rates

associated with the isolate were lowest with the Roche 454 method (2%), increased

by more than 2-fold for the 500 bp amplicons with the PacBio SMRT chip (4-5%),

and by more than 8-fold for the full gene with the PacBio SMRT chip (17-18%).

Higher error rates with the PacBio SMRT chip artificially inflated estimates of

richness and lowered estimates of coverage for environmental samples. The 3rd

generation sequencing technology we evaluated does not provide greater

phylogenetic and taxonomic resolutions for studies of microbial ecology.


© 2013.


DOI: 10.1016/j.mimet.2013.08.009

PMID: 23999276  [PubMed - indexed for MEDLINE]



122. J Clin Microbiol. 2014 May;52(5):1754-7. doi: 10.1128/JCM.00552-14. Epub 2014 Mar



Application of qualitative and quantitative real-time PCR, direct sequencing, and

terminal restriction fragment length polymorphism analysis for detection and

identification of polymicrobial 16S rRNA genes in ascites.


Krohn S(1), Böhm S, Engelmann C, Hartmann J, Brodzinski A, Chatzinotas A, Zeller

K, Prywerek D, Fetzer I, Berg T.


Author information:

(1)University Hospital Leipzig, Division of Gastroenterology and Hepatology,

Leipzig, Germany.


Qualitative and quantitative 16S rRNA gene-based real-time PCR and direct

sequencing were applied for rapid detection and identification of bacterial DNA

(bactDNA) in 356 ascites samples. bactDNA was detected in 35% of samples, with a

mean of 3.24 log copies ml(-1). Direct sequencing of PCR products revealed 62%

mixed chromatograms predominantly belonging to Gram-positive bacteria. Terminal

restriction fragment length polymorphism (T-RFLP) results of a sample subset

confirmed sequence data showing polymicrobial DNA contents in 67% of

bactDNA-positive ascites samples.


DOI: 10.1128/JCM.00552-14

PMCID: PMC3993693

PMID: 24622095  [PubMed - indexed for MEDLINE]



123. PLoS One. 2014 Sep 3;9(9):e106215. doi: 10.1371/journal.pone.0106215. eCollection



Anterior foregut microbiota of the glassy-winged sharpshooter explored using deep

16S rRNA gene sequencing from individual insects.


Rogers EE(1), Backus EA(1).


Author information:

(1)United States Department of Agriculture, Agricultural Research Service, San

Joaquin Valley Agricultural Sciences Center, Parlier, California, United States

of America.


The glassy-winged sharpshooter (GWSS) is an invasive insect species that

transmits Xylella fastidiosa, the bacterium causing Pierce's disease of grapevine

and other leaf scorch diseases. X. fastidiosa has been shown to colonize the

anterior foregut (cibarium and precibarium) of sharpshooters, where it may

interact with other naturally-occurring bacterial species. To evaluate such

interactions, a comprehensive list of bacterial species associated with the

sharpshooter cibarium and precibarium is needed. Here, a survey of microbiota

associated with the GWSS anterior foregut was conducted. Ninety-six individual

GWSS, 24 from each of 4 locations (Bakersfield, CA; Ojai, CA; Quincy, FL; and a

laboratory colony), were characterized for bacteria in dissected sharpshooter

cibaria and precibaria by amplification and sequencing of a portion of the 16S

rRNA gene using Illumina MiSeq technology. An average of approximately 150,000

sequence reads were obtained per insect. The most common genus detected was

Wolbachia; sequencing of the Wolbachia ftsZ gene placed this strain in supergroup

B, one of two Wolbachia supergroups most commonly associated with arthropods. X.

fastidiosa was detected in all 96 individuals examined. By multilocus sequence

typing, both X. fastidiosa subspecies fastidiosa and subspecies sandyi were

present in GWSS from California and the colony; only subspecies fastidiosa was

detected in GWSS from Florida. In addition to Wolbachia and X. fastidiosa, 23

other bacterial genera were detected at or above an average incidence of 0.1%;

these included plant-associated microbes (Methylobacterium, Sphingomonas,

Agrobacterium, and Ralstonia) and soil- or water-associated microbes

(Anoxybacillus, Novosphingobium, Caulobacter, and Luteimonas). Sequences

belonging to species of the family Enterobacteriaceae also were detected but it

was not possible to assign these to individual genera. Many of these species

likely interact with X. fastidiosa in the cibarium and precibarium.


DOI: 10.1371/journal.pone.0106215

PMCID: PMC4153580

PMID: 25184624  [PubMed - indexed for MEDLINE]



124. Genet Mol Res. 2015 Aug 19;14(3):9703-21. doi: 10.4238/2015.August.19.3.


In silico analysis of the 16S rRNA gene of endophytic bacteria, isolated from the

aerial parts and seeds of important agricultural crops.


Bredow C(1), Azevedo JL(2), Pamphile JA(1), Mangolin CA(1), Rhoden SA(3).


Author information:

(1)Departamento de Biotecnologia, Genética e Biologia Celular, Universidade

Estadual de Maringá, PR, Brasil. (2)Laboratório de Genética de Microrganismos

"Prof. Dr. João Lúcio de Azevedo", Departamento de Genética (LGN). (3)Intituto

Federal Catarinense, São Francisco do Sul, SC, Brasil.


Because of human population growth, increased food production and alternatives to

conventional methods of biocontrol and development of plants such as the use of

endophytic bacteria and fungi are required. One of the methods used to study

microorganism diversity is sequencing of the 16S rRNA gene, which has several

advantages, including universality, size, and availability of databases for

comparison. The objective of this study was to analyze endophytic bacterial

diversity in agricultural crops using published papers, sequence databases, and

phylogenetic analysis. Fourteen papers were selected in which the ribosomal 16S

rRNA gene was used to identify endophytic bacteria, in important agricultural

crops, such as coffee, sugar cane, beans, corn, soybean, tomatoes, and grapes,

located in different geographical regions (America, Europe, and Asia). The

corresponding 16S rRNA gene sequences were selected from the NCBI database,

aligned using the Mega 5.2 program, and phylogenetic analysis was undertaken. The

most common orders present in the analyzed cultures were Bacillales,

Enterobacteriales, and Actinomycetales and the most frequently observed genera

were Bacillus, Pseudomonas, and Microbacterium. Phylogenetic analysis showed that

only approximately 1.56% of the total sequences were not properly grouped,

demonstrating reliability in the identification of microorganisms. This study

identified the main genera found in endophytic bacterial cultures from plants,

providing data for future studies on improving plant agriculture, biotechnology,

endophytic bacterium prospecting, and to help understand relationships between

endophytic bacteria and their interactions with plants.


DOI: 10.4238/2015.August.19.3

PMID: 26345903  [PubMed - indexed for MEDLINE]

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