Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

Nossa, Carlos W.; Oberdorf, William E.; Yang, Liying; Jørn, A.; Paster, Bruce J.; DeSantis, Todd Z.; Brodie, Eoin L.; Malamud, Daniel; Poles, Michael A.; Pei, Zhiheng

doi:10.3748/wjg.v16.i33.4135

Cited by 424 publications

(310 citation statements)

References 30 publications

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“…Second, biases are also likely associated with the DNA extraction, although the optimal extraction kit has been used in this study after comparing five kits. Additionally, the length of the 16S rRNA gene amplicons obtained in this study was short, about 207 bps (excluding primer sequences), although this length and region may have taxonomic classification accuracy of B85% (Nossa et al, 2010). Finally, the rarefaction curves (Supplementary Figure S2) suggest that some samples, such as that from Shek-Wu-Hui in Hong Kong, were still under-sampled even with 16 489 effective sequences.…”

Section: Pyrosequencing Reveals Bacterial Diversity Of As T Zhang Et Almentioning

confidence: 95%

454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants

2011

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Activated sludge (AS) contains highly complex microbial communities. In this study, PCR-based 454 pyrosequencing was applied to investigate the bacterial communities of AS samples from 14 sewage treatment plants of Asia (mainland China, Hong Kong, and Singapore), and North America (Canada and the United States). A total of 259 K effective sequences of 16S rRNA gene V4 region were obtained from these AS samples. These sequences revealed huge amount of operational taxonomic units (OTUs) in AS, that is, 1183-3567 OTUs in a sludge sample, at 3% cutoff level and sequencing depth of 16 489 sequences. Clear geographical differences among the AS samples from Asia and North America were revealed by (1) cluster analyses based on abundances of OTUs or the genus/family/order assigned by Ribosomal Database Project (RDP) and (2) the principal coordinate analyses based on OTUs abundances, RDP taxa abundances and UniFrac of OTUs and their distances. In addition to certain unique bacterial populations in each AS sample, some genera were dominant, and core populations shared by multiple samples, including two commonly reported genera of Zoogloea and Dechloromonas, three genera not frequently reported (i.e., Prosthecobacter, Caldilinea and Tricoccus) and three genera not well described so far (i.e., Gp4 and Gp6 in Acidobacteria and Subdivision3 genera incertae sedis of Verrucomicrobia). Pyrosequencing analyses of multiple AS samples in this study also revealed the minority populations that are hard to be explored by traditional molecular methods and showed that a large proportion of sequences could not be assigned to taxonomic affiliations even at the phylum/class levels.

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Section: Pyrosequencing Reveals Bacterial Diversity Of As T Zhang Et Almentioning

confidence: 95%

454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants

2011

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“…For the purpose of biodiversity studies, metagenomics can focus on one common gene shared by all members of the community. The most commonly used culture-independent method relies on amplification and analysis of the 16S rRNA genes in a microbiota [38].…”

Section: Exploring Bacterial Communities By 16s Pcr-ttgementioning

confidence: 99%

“…In biodiversity studies, the different 16S rRNA genes representative of the community are amplified by PCR and then separated and identified either by cloning and Sanger sequencing or by direct pyro-sequencing [38]. Tools for sequence-specific separation after bulk PCR amplification, such as T-RFLP (Terminal-Restriction Fragment Length Polymorphism) [49], D-HPLC (Denaturing High Performance Liquid Chromatography) [50], CDCE (Constant Denaturing Capillary Electrophoresis) [51], SSCP (Single Stranded Conformation Polymorphism) [52], DGGE (Denaturing Gradient Gel Electrophoresis) [53], TGGE (Temperature Gradient Gel Electrophoresis), [48] and TTGE (Temporal Temperature Gradient Gel Electrophoresis) [47], can also be used.…”

Section: Anophmentioning

confidence: 99%

Bacterial Biodiversity in Midguts of Anopheles Mosquitoes, Malaria Vectors in Southeast Asia

Manguin¹,

Ngo²,

Tainchum³

et al. 2013

Anopheles Mosquitoes - New Insights Into Malaria Vectors

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“…This would be especially beneficial for next-generation sequencing (NGS) technologies, such as pyrosequencing, because the length restriction of reads is rather stringent due to technical limitations. Furthermore, selecting target regions from pyrosequencing is much more difficult compared to conventional sequencing methodologies (Mashayekhi and Ronaghi, 2007;Nossa et al, 2010). Nevertheless, priming sites that include the most informative region with a moderate length can be easily selected if the SPAT protocol is used when designing the pyrosequencing primers.…”

Section: Discussionmentioning

confidence: 99%

Development of Single-Nucleotide Polymorphism-Based Phylum-Specific PCR Amplification Technique: Application to the Community Analysis Using Ciliates as a Reference Organism

Jung

Kim

Ryu

et al. 2012

Molecules and Cells

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Despite recent advance in mass sequencing technologies such as pyrosequencing, assessment of culture-independent microbial eukaryote community structures using universal primers remains very difficult due to the tremendous richness and complexity of organisms in these communities. Use of a specific PCR marker targeting a particular group would provide enhanced sensitivity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We discovered that many phylum-or groupspecific single-nucleotide polymorphisms (SNPs) exist in small subunit ribosomal RNA (SSU rRNA) genes from diverse eukaryote groups. By applying this discovery to a known simple allele-discriminating (SAP) PCR method, we developed a technique that enables the identification of organisms belonging to a specific higher taxonomic group (or phylum) among diverse types of eukaryotes. We performed an assay using two complementary methods, pyrosequencing and clone library screening. In doing this, specificities for the group (ciliates) targeted in this study in bulked environmental samples were 94.6% for the clone library and 99.2% for pyrosequencing, respectively. In particular, our novel technique showed high selectivity for rare species, a feature that may be more important than the ability to identify quantitatively predominant species in community structure analyses. Additionally, our data revealed that a target-specific library (or ciliate-specific one for the present study) can better explain the ecological features of a sampling locality than a universal library.

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Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

Abstract: 347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.

Cited by 424 publications

References 30 publications

454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants

454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants

Bacterial Biodiversity in Midguts of Anopheles Mosquitoes, Malaria Vectors in Southeast Asia

Development of Single-Nucleotide Polymorphism-Based Phylum-Specific PCR Amplification Technique: Application to the Community Analysis Using Ciliates as a Reference Organism

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