Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing

Bokulich, Nicholas A.; Subramanian, Sathish; Faith, Jeremiah J.; Gevers, Dirk; Gordon, Jeffrey I.; Knight, Rob; Mills, David A.; Caporaso, J. Gregory

doi:10.1038/nmeth.2276

Cited by 3,469 publications

(2,340 citation statements)

References 15 publications

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“…Designing primers to successfully amplify ITS amplicons with Illumina MiSeq is a recent advance and many questions about appropriate data quality control remain . Researchers are conflicted over appropriate ways to deal with uneven sequencing depth (McMurdie and Holmes, 2014), and how to use negative controls and mock communities to calibrate bioinformatic pipelines (Bokulich et al, 2013;Nguyen et al, 2014). We took great pains to account for uneven sequencing depth, mock communities and negative controls with our analyses.…”

Section: Discussionmentioning

confidence: 99%

Ectomycorrhizal fungal spore bank recovery after a severe forest fire: some like it hot

et al. 2015

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After severe wildfires, pine recovery depends on ectomycorrhizal (ECM) fungal spores surviving and serving as partners for regenerating forest trees. We took advantage of a large, severe natural forest fire that burned our long-term study plots to test the response of ECM fungi to fire. We sampled the ECM spore bank using pine seedling bioassays and high-throughput sequencing before and after the California Rim Fire. We found that ECM spore bank fungi survived the fire and dominated the colonization of in situ and bioassay seedlings, but there were specific fire adapted fungi such as Rhizopogon olivaceotinctus that increased in abundance after the fire. The frequency of ECM fungal species colonizing pre-fire bioassay seedlings, post-fire bioassay seedlings and in situ seedlings were strongly positively correlated. However, fire reduced the ECM spore bank richness by eliminating some of the rare species, and the density of the spore bank was reduced as evidenced by a larger number of soil samples that yielded uncolonized seedlings. Our results show that although there is a reduction in ECM inoculum, the ECM spore bank community largely remains intact, even after a high-intensity fire. We used advanced techniques for data quality control with Illumina and found consistent results among varying methods. Furthermore, simple greenhouse bioassays can be used to determine which fungi will colonize after fires. Similar to plant seed banks, a specific suite of ruderal, spore bank fungi take advantage of open niche space after fires.

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Section: Discussionmentioning

confidence: 99%

Ectomycorrhizal fungal spore bank recovery after a severe forest fire: some like it hot

et al. 2015

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“…A complete set of samples was not available for all subjects because of issues with logistics and subjects dropping out of the study. Processing of the biological samples, and generation of 16S rRNA sequencing data was as described in (Bokulich et al, 2013;Dogra et al, 2015). Briefly, the variable regions V4-V5-V6 (V456) of the 16S rRNA gene were used to characterise the microbiota by the barcoded-primer approach to multiplex pyrosequencing.…”

Section: S Rrna Sequencing and Qpcrmentioning

confidence: 99%

“…Taxonomy assignment of OTU representative sequences used the RDP Classifier with confidence threshold of 0.6 (Wang et al, 2007) on the Greengenes reference database v.13.8 (McDonald et al, 2012). After quality filtering (Bokulich et al, 2013), similarities between all samples were computed as Binary Jaccard (for similarity in community membership) or Bray Curtis (for similarity in community structure) distances using OTUs (the lowest discriminant units with this technology). Diversity analyses were calculated in QIIME on data rarefied at 200 reads per sample.…”

Section: S Rrna Sequencing and Qpcrmentioning

confidence: 99%

Does the maternal vaginal microbiota play a role in seeding the microbiota of neonatal gut and nose?

Sakwińska

Foata

Berger

et al. 2017

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The acquisition and early maturation of infant microbiota is not well understood despite its likely influence on later health. We investigated the contribution of the maternal microbiota to the microbiota of infant gut and nose in the context of mode of delivery and feeding. Using 16S rRNA sequencing and specific qPCR, we profiled microbiota of 42 mother-infant pairs from the GUSTO birth cohort, at body sites including maternal vagina, rectum and skin; and infant stool and nose. In our study, overlap between maternal vaginal microbiota and infant faecal microbiota was minimal, while the similarity between maternal rectal microbiota and infant microbiota was more pronounced. However, an infant's nasal and gut microbiota were no more similar to that of its own mother, than to that of unrelated mothers. These findings were independent of delivery mode. We conclude that the transfer of maternal vaginal microbes play a minor role in seeding infant stool microbiota. Transfer of maternal rectal microbiota could play a larger role in seeding infant stool microbiota, but approaches other than the generally used analyses of community similarity measures are likely to be needed to quantify bacterial transmission. We confirmed the clear difference between microbiota of infants born by Caesarean section compared to vaginally delivered infants and the impact of feeding mode on infant gut microbiota. Only vaginally delivered, fully breastfed infants had gut microbiota dominated by Bifidobacteria. Our data suggest that reduced transfer of maternal vaginal microbial is not the main mechanism underlying the differential infant microbiota composition associated with Caesarean delivery. The sources of a large proportion of infant microbiota could not be identified in maternal microbiota, and the sources of seeding of infant gut and nasal microbiota remain to be elucidated.

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“…The FastTree program (Price et al 2009) was used to construct a phylogeny based on the representative OTU sequences. In order to ensure the accuracy of the results, OTUs with an abundance value <0.001% of the total number of sequences were removed (Bokulich et al 2012). The ACE index was used to measure community richness (Pitta et al 2010), and the Shannon index was used to evaluate microbial diversity in soil samples (Shannon and Weaver 1949).…”

Section: Clustering and Annotation Of Operational Taxonomic Unitsmentioning

confidence: 99%

Effects of vermicompost amendment as a basal fertilizer on soil properties and cucumber yield and quality under continuous cropping conditions in a greenhouse

Zhao

Zhang

et al. 2017

J Soils Sediments

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Purpose We examined the effects of vermicompost application as a basal fertilizer on the properties of a sandy loam soil used for growing cucumbers under continuous cropping conditions when compared to inorganic or organic fertilizers. Materials and methods A commercial cucumber (Cucumis sativus L.) variety was grown on sandy loam soil under four soil amendment conditions: inorganic compound fertilizer (750 kg/ha,), replacement of 150 kg/ha of inorganic compound fertilizer with 3000 kg/ha of organic fertilizer or vermicompost, and untreated control. Experiments were conducted in a greenhouse for 4 years, and continuous planting resulted in seven cucumber crops. The yield and quality of cucumber fruits, basic physical and chemical properties of soil, soil nutrient characteristics, and the soil fungal community structure were measured and evaluated. Results and discussion Continuous cucumber cropping decreased soil pH and increased electrical conductivity. However, application of vermicompost significantly improved several soil characteristics and induced a significant change in the rhizosphere soil fungal community compared to the other treatments. Notably, the vermicompost amendments resulted in an increase in the relative abundance of Ascomycota, Chytridiomycota, Sordariomycetes, Eurotiomycetes, and Saccharomycetes, and a decrease in Glomeromycota, Zygomycota, Dothideomycetes, Agaricomycetes, and Incertae sedis. Compared to the organic fertilizer treatment, vermicompost amendment increased the relative abundance of beneficial fungi and decreased those of pathogenic fungi. Cucumber fruit yield decreased yearly under continuous cropping conditions, but both inorganic and organic fertilizer amendments increased yields. Vermicompost amendment maintained higher fruit yield and quality under continuous cropping conditions. Conclusions Continuous cropping decreased cucumber yield in a greenhouse, but basic fertilizer amendment reduced this decline. Moreover, basal fertilizer amendment decreased beneficial and pathogenic fungi, and the use of vermicompost amendment in the basic fertilizer had a positive effect on the health of the soil fungal community.

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Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing

Cited by 3,469 publications

References 15 publications

Ectomycorrhizal fungal spore bank recovery after a severe forest fire: some like it hot

Ectomycorrhizal fungal spore bank recovery after a severe forest fire: some like it hot

Does the maternal vaginal microbiota play a role in seeding the microbiota of neonatal gut and nose?

Effects of vermicompost amendment as a basal fertilizer on soil properties and cucumber yield and quality under continuous cropping conditions in a greenhouse

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