Using mock communities of arbuscular mycorrhizal fungi to evaluate fidelity associated with Illumina sequencing

Egan, Cameron; Rummel, Alexii; Kokkoris, Vasilis; Klironomos, John N.; Lekberg, Ylva; Hart, Miranda M.

doi:10.1016/j.funeco.2018.01.004

Cited by 41 publications

(26 citation statements)

References 68 publications

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“…We urge further research into each step of soil sampling and processing to get a more complete understanding of how these decisions impact study results and interpretations. For example, several papers look at the impact of different bioinformatical pipelines on results (Bokulich et al, 2013; Cline, Song, Al‐Ghalith, Knights, & Kennedy, 2017; Egan et al, 2018). We are hopeful that this work will help scientists evaluate the costs and benefits of experimental approaches when studying soil bacteria and fungi.…”

Section: Discussionmentioning

confidence: 99%

Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results

Delavaux

Bever

Karppinen

et al. 2020

Ecology and Evolution

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With the advances of sequencing tools, the fields of environmental microbiology and soil ecology have been transformed. Today, the unculturable majority of soil microbes can be sequenced. Although these tools give us tremendous power and open many doors to answer important questions, we must understand how sample processing may impact our results and interpretations. Here, we test the impacts of four soil storage methods on downstream amplicon metabarcoding and qPCR analyses for fungi and bacteria. We further investigate the impact of thaw time on extracted DNA to determine a safe length of time during which this can occur with minimal impact on study results. Overall, we find that storage using standard cold packs with subsequent storage at −20°C is little different than immediate storage in liquid nitrogen, suggesting that the historical and current method is adequate. We further find evidence that storage at room temperature or with aid of RNAlater can lead to changes in community composition and in the case of RNAlater, lower gene copies. We therefore advise against these storage methods for metabarcoding analyses. Finally, we show that over 1 month, DNA extract thaw time does not impact diversity or qPCR metrics. We hope that this work will help researchers working with soil bacteria and fungi make informed decisions about soil storage and transport to ensure repeatability and accuracy of results and interpretations.

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Section: Discussionmentioning

confidence: 99%

Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results

Delavaux

Bever

Karppinen

et al. 2020

Ecology and Evolution

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“…PCR was conducted on the normalized DNA samples using forward universal eukaryotic primer WANDA 32 and reverse AMF-specific primer AML2 33 . This primer pair spans a variable 530-bp region in the SSU rRNA gene 34 . The PCR products were run on a gel to confirm the presence of DNA at 530 bp, then sequenced by Sanger sequencing at the UC Berkeley DNA Sequencing Facility.…”

Section: Molecular Methodsmentioning

confidence: 99%

Routes to Roots: Direct Evidence of Water Transport by Arbuscular Mycorrhizal Fungi to Host Plants

Kakouridis

Hagen

Kan

et al. 2020

Preprint

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Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with 80% of surveyed land plant species and are well-recognized for accessing and transferring nutrients to plants1. Yet AMF also perform other essential functions, notably improving plant-water relations2. Some research attributes the role of AMF in plant-water relations solely to enhancing plant nutrition and osmoregulation for plants partnered with AMF3,4,5, while indirect evidence suggests AMF may transport water to plants1,6,7. Here, we used isotopically-labeled water and a fluorescent dye to directly track and quantify water transport by AMF to plants in a greenhouse experiment. We specifically assessed whether AMF can access water in soil unavailable to plants and transport it across an air gap to host plants. Plants grown with AMF that had access to a physically separated 18O-labeled water source transpired twice as much, and this transpired water contained three times as much label compared to plants with AMF with no access to the separated labeled water source. We estimated that water transported by AMF could explain 46.2% of the water transpired. In addition, a fluorescent dye indicated that water was transported via an extracytoplasmic hyphal pathway.

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“…By using a strategy based on a nested PCR followed by MiSeq-Illumina sequencing and a phylogenetic pipeline that used a reference phylogenetic tree and an evolutionary placement algorithm (EPA; Berger and Stamatakis 2011; Berger et al 2011), we were able to annotate the studied AMF communities with high-throughput, at the species level. Egan et al (2018) demonstrated that MiSeq-Illumina sequencing can recapture an AMF community from a mock community, even in terms of relative abundances, suggesting a robust technique. However, they found a problem in taxonomy assignment that may be due to the gene region used which was a 500 bp fragment of the SSU rDNA region that does not provide species level resolution for many AMF (Stockinger et al 2010).…”

Section: Amf Species Delimitation Methodsmentioning

confidence: 99%

“…However, they found a problem in taxonomy assignment that may be due to the gene region used which was a 500 bp fragment of the SSU rDNA region that does not provide species level resolution for many AMF (Stockinger et al 2010). Also, the lack of type sequence representation in databases was mentioned by Egan et al (2018). Clearly, the selection of the DNA region is crucial to obtain sufficient phylogenetic resolution when characterizing sequences to the species level.…”

Section: Amf Species Delimitation Methodsmentioning

confidence: 99%

“…After sequencing, consecutive bioinformatics analyses consist of using similarity thresholds (usually 97%) to cluster reads into operational taxonomic units (OTUs) and BLAST them against public or curated databases (Drumbell et al, 2011). However, taxonomic classification to species is often impossible or unreliable due to the short read length and because the SSU rDNA region can only be reliably used to characterize genus level or above (Stockinger et al 2009;Egan et al 2018). Moreover, BLAST based approaches require reference sequences to make accurate taxonomic annotations but there are many unknown AMF species in unexplored areas (Öpik et al 2013) which are not represented in databases and therefore their correct affiliation is restricted to described species .…”

Section: Introductionmentioning

confidence: 99%

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New MiSeq based strategy exposed plant-preferential arbuscular mycorrhizal fungal communities in arid soils of Mexico

et al. 2020

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Arbuscular mycorrhizal fungi (AMF) are obligate symbionts of c. 80% of land plants, having enormous ecological and economic impact, as they often improve crop plant nutrition and yield. DNA-based identification with molecular markers is used to analyze AM fungal communities in the field, but reaching species level taxonomic resolution remains challenging. Thus, currently there is no consensus on how to analyze high-throughput sequences and assign them into species. Here, a new sequencing strategy combined with taxonomic affiliations implemented with an evolutionary placement algorithm (EPA) was established. It is based on sequencing a c. 450 bp region of the large subunit (LSU) ribosomal rRNA gene with the MiSeq-Illumina platform. The method is suitable for the discrimination of closely related AMF species and was used to study host-AMF preferences in roots of Pequin pepper, soybean and orange at one location in the arid northeast of Mexico. Twenty AM fungal species from 13 genera were detected. Phylogenetic affiliation of reads to species revealed crop preferential associations. In Pequin pepper roots, several Rhizophagus species represented most of the community, Rhizophagus clarus being the most abundant. The soybean AM fungal community was dominated by Rhizophagus irregularis and Funneliformis mosseae and that of orange by several species of Dominikia, some of them only found in this crop. Unraveling the AMF-plant preferences of important crops by an affordable and robust sequencing method, combined with phylotaxonomic AMF species resolution, is an important tool to obtain taxonomic units that are meaningful in both biological and ecological studies.

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Using mock communities of arbuscular mycorrhizal fungi to evaluate fidelity associated with Illumina sequencing

Cited by 41 publications

References 68 publications

Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results

Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results

Routes to Roots: Direct Evidence of Water Transport by Arbuscular Mycorrhizal Fungi to Host Plants

New MiSeq based strategy exposed plant-preferential arbuscular mycorrhizal fungal communities in arid soils of Mexico

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