Slowing PCR ramp speed reduces chimera formation from environmental samples

Stevens, Julia L.; Jackson, Ronneshia L.; Olson, Julie B.

doi:10.1016/j.mimet.2013.03.013

Cited by 51 publications

(42 citation statements)

References 14 publications

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“…The ramp rate was set to 1 °C/s to prevent the generation of chimeric sequences (Stevens et al . ). Illumina sequencing adaptors were then added in the subsequent PCR using the forward primers consisting of the P5 Illumina adaptor, 8‐mer index sequences for sample identification (Hamady et al .…”

Section: Methodsmentioning

confidence: 97%

Ericaceous plant–fungus network in a harsh alpine–subalpine environment

2016

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In terrestrial ecosystems, plant species and diverse root-associated fungi form complex networks of host-symbiont associations. Recent studies have revealed that structures of those below-ground plant-fungus networks differ between arbuscular mycorrhizal and ectomycorrhizal symbioses. Nonetheless, we still remain ignorant of how ericaceous plant species, which dominate arctic and alpine tundra, constitute networks with their root-associated fungi. Based on a high-throughput DNA sequencing data set, we characterized the statistical properties of a network involving 16 ericaceous plant species and more than 500 fungal taxa in the alpine-subalpine region of Mt. Tateyama, central Japan. While all the 16 ericaceous species were associated mainly with fungi in the order Helotiales, they varied remarkably in association with fungi in other orders such as Sebacinales, Atheliales, Agaricales, Russulales and Thelephorales. The ericaceous plant-fungus network was characterized by high symbiont/host preferences. Moreover, the network had a characteristic structure called 'anti-nestedness', which has been previously reported in ectomycorrhizal plant-fungus networks. The results lead to the hypothesis that ericaceous plants in harsh environments can host unexpectedly diverse root-associated fungal taxa, constituting networks whose structures are similar to those of previously reported ectomycorrhizal networks but not to those of arbuscular mycorrhizal ones.

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Section: Methodsmentioning

confidence: 97%

Ericaceous plant–fungus network in a harsh alpine–subalpine environment

2016

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“…The PCR reaction was conducted using the buffer and DNA polymerase system of Ampdirect Plus (Shimazu) with a temperature profile of 95°C for 10 min, followed by 37 cycles at 94°C for 30 s, 50°C for 60 s, 72°C for 60 s, and a final extension at 72°C for 7 min. The ramp rate was set to 1°C/sec to prevent the generation of chimeric amplicons [48]. P5/P7 Illumina adaptors were then added in the subsequent PCR using fusion primers with 8-mer index sequences for sample identification [49] (forward, ; reverse, ).…”

Section: Methodsmentioning

confidence: 99%

Networks Depicting the Fine-Scale Co-Occurrences of Fungi in Soil Horizons

et al. 2016

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Fungi in soil play pivotal roles in nutrient cycling, pest controls, and plant community succession in terrestrial ecosystems. Despite the ecosystem functions provided by soil fungi, our knowledge of the assembly processes of belowground fungi has been limited. In particular, we still have limited knowledge of how diverse functional groups of fungi interact with each other in facilitative and competitive ways in soil. Based on the high-throughput sequencing data of fungi in a cool-temperate forest in northern Japan, we analyzed how taxonomically and functionally diverse fungi showed correlated fine-scale distributions in soil. By uncovering pairs of fungi that frequently co-occurred in the same soil samples, networks depicting fine-scale co-occurrences of fungi were inferred at the O (organic matter) and A (surface soil) horizons. The results then led to the working hypothesis that mycorrhizal, endophytic, saprotrophic, and pathogenic fungi could form compartmentalized (modular) networks of facilitative, antagonistic, and/or competitive interactions in belowground ecosystems. Overall, this study provides a research basis for further understanding how interspecific interactions, along with sharing of niches among fungi, drive the dynamics of poorly explored biospheres in soil.

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“…The multiplex PCR of the 16S and ITS regions was conducted using the MyTaq HS DNA polymerase Mastermix (Bioline) with a temperature profile of 95ºC for 2 min, followed by 37 cycles at 95ºC for 20 s, 50ºC for 20 s, 72ºC for 50 s, and a final extension at 72ºC for 10 min. The ramp rate was set to 1ºC s -1 to prevent the generation of chimeric amplicons (Stevens et al 2013). P5/P7 Illumina adaptors were then added in the subsequent PCR using fusion primers with 8-mer index sequences for sample identification (Hamady et al 2008) (forward, 5-AAT GAT ACG GCG ACC ACC GAG ATC TAC AC -[8-mer tag] -TCG TCG GCA GCG TC -3; reverse, 5-CAA GCA GAA GAC GGC ATA CGA GAT -[8-mer tag] -GTC TCG TGG GCT CGG -3).…”

Section: Illumina Sequencing Of Bacteria and Fungimentioning

confidence: 99%

Priority effects can persist across floral generations in nectar microbial metacommunities

Toju

Vannette

Gauthier

et al. 2017

Oikos

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The order of species arrival can influence how species interact with one another and, consequently, which species may coexist in local communities. This phenomenon, called priority effects, has been observed in various types of communities, but it remains unclear whether priority effects persist over the long term spanning multiple generations of local communities in metacommunities. Focusing on bacteria and yeasts that colonize floral nectar of the sticky monkey flower, Mimulus aurantiacus, via hummingbirds and other flower‐visiting animals, we experimentally manipulated initial microbial dominance on plants (regarded as metacommunities) to examine whether its effects persisted across multiple generations of flowers (regarded as local microbial habitats). The experimental introduction of Neokomagataea (= Gluconobacter) bacteria and Metschnikowia yeasts into wild flowers showed that the effects of initial dominance were observable across multiple floral generations. Three weeks after introduction, corresponding approximately to three floral generations, Neokomagataea introduction led to exclusion of yeasts, whereas Metschnikowia introduction did not result in the exclusion of Neokomagataea. Our results suggest that, even when local habitats are ephemeral, priority effects may influence multiple generations of local communities within metacommunities.

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Slowing PCR ramp speed reduces chimera formation from environmental samples

Cited by 51 publications

References 14 publications

Ericaceous plant–fungus network in a harsh alpine–subalpine environment

Ericaceous plant–fungus network in a harsh alpine–subalpine environment

Networks Depicting the Fine-Scale Co-Occurrences of Fungi in Soil Horizons

Priority effects can persist across floral generations in nectar microbial metacommunities

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