Microbial 16S rRNA Ion Tag and community metagenome sequencing using the Ion Torrent (PGM) Platform

Whiteley, Andrew S.; Jenkins, Sue; Waite, Ian; Kresoje, Nina; Payne, H.G.; Mullan, B.P.; Allcock, Richard J. N.; O'Donnell, Anthony G.

doi:10.1016/j.mimet.2012.07.008

Cited by 180 publications

(115 citation statements)

References 49 publications

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“…Each sample was subjected to a bead beating step before nucleic acid extraction using Lysing Matrix-B (MP Bio, Santa Ana, CA, USA). We amplified the bacterial 16S rRNA V3 hypervariable region using the forward primer 341F (5 0 -CCTACGG GAGGCAGCAG-3 0 ) fused with the Ion Torrent Adaptor A sequence and one of 23 unique 10-bp barcodes, and reverse primer 514R (5 0 -ATTACCGCGGCTGCTG G-3 0 ) was fused with the Ion Torrent Adaptor P1 from the saliva of each subject and time point (Whiteley et al, 2012). PCR reactions were performed using Platinum PCR SuperMix (Invitrogen) with the following cycling parameters: 94 1C for 10 min, followed by 30 cycles of 94 1C for 30 s, 53 1C for 30 s, 72 1C for 30 s and a final elongation step of 72 1C for 10 min.…”

Section: Analysis Of 16s Rrnamentioning

confidence: 99%

Human oral viruses are personal, persistent and gender-consistent

et al. 2014

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Viruses are the most abundant members of the human oral microbiome, yet relatively little is known about their biodiversity in humans. To improve our understanding of the DNA viruses that inhabit the human oral cavity, we examined saliva from a cohort of eight unrelated subjects over a 60-day period. Each subject was examined at 11 time points to characterize longitudinal differences in human oral viruses. Our primary goals were to determine whether oral viruses were specific to individuals and whether viral genotypes persisted over time. We found a subset of homologous viral genotypes across all subjects and time points studied, suggesting that certain genotypes may be ubiquitous among healthy human subjects. We also found significant associations between viral genotypes and individual subjects, indicating that viruses are a highly personalized feature of the healthy human oral microbiome. Many of these oral viruses were not transient members of the oral ecosystem, as demonstrated by the persistence of certain viruses throughout the entire 60-day study period. As has previously been demonstrated for bacteria and fungi, membership in the oral viral community was significantly associated with the sex of each subject. Similar characteristics of personalized, sex-specific microflora could not be identified for oral bacterial communities based on 16S rRNA. Our findings that many viruses are stable and individual-specific members of the oral ecosystem suggest that viruses have an important role in the human oral ecosystem.

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Section: Analysis Of 16s Rrnamentioning

confidence: 99%

Human oral viruses are personal, persistent and gender-consistent

et al. 2014

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“…2008; Whiteley et al . 2012), the needs for clinical diagnosis of infections from normally sterile sites (i.e. low complexity samples containing one to a few dozen organisms) are different.…”

Section: Discussionmentioning

confidence: 99%

16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria

et al. 2017

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AimsTest the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer.Methods and ResultsNine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1–V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture.ConclusionsSequencing the V1–V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare.Significance and Impact of the StudyWe have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.

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“…Currently, it delivers 400 bp reads in four hours. A microbial ecology sequencing platform has been recently proposed to assess bacterial and archaeal community dynamics using the PGM [174]. This platform was also used in a study which showed that diesel biodegradation was affected by modifying the microbial community structure [175].…”

Section: Ion Personal Genome Machine (Pgm)mentioning

confidence: 99%

Culture-Independent Molecular Tools for Soil and Rhizosphere Microbiology

2013

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Soil microbial communities play an important role in plant health and soil quality. Researchers have developed a wide range of methods for studying the structure, diversity, and activity of microbes to better understand soil biology and plant-microbe interactions. Functional microbiological analyses of the rhizosphere have given new insights into the role of microbial communities in plant nutrition and plant protection against diseases. In this review, we present the most commonly used traditional as well as new culture-independent molecular methods to assess the diversity and function of soil microbial communities. Furthermore, we discuss advantages and disadvantages of these techniques and provide a perspective on emerging technologies for soil microbial community profiling.

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Microbial 16S rRNA Ion Tag and community metagenome sequencing using the Ion Torrent (PGM) Platform

Cited by 180 publications

References 49 publications

Human oral viruses are personal, persistent and gender-consistent

Human oral viruses are personal, persistent and gender-consistent

16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria

Culture-Independent Molecular Tools for Soil and Rhizosphere Microbiology

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