Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data

Palmer, Jonathan M.; Jusino, Michelle A.; Banik, Mark T.; Lindner, Daniel L.

doi:10.7717/peerj.4925

Cited by 204 publications

(226 citation statements)

References 66 publications

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“…Analysis for the identification of trypanosome species in all the samples had been conducted in an earlier study using a new approach utilizing internal transcribed spacer 1 (ITS1) PCR and next-generation amplicon sequencing 38 . For mammalian species analysis, pre-processing of reads was done using the amplicon toolkit (AMPtk, version 1.4.0) pipeline 39 . The pre-processing involved trimming of primers, removal of sequences less than 200 bp, and merging pair-end reads.…”

Section: Paired-end Library Preparationmentioning

confidence: 99%

Blood meal sources and bacterial microbiome diversity in wild-caught tsetse flies

Gaithuma

Yamagishi

Hayashida

et al. 2020

Sci Rep

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Tsetse flies are the vectors of African trypanosomiasis affecting 36 sub-Saharan countries. Both wild and domestic animals play a crucial role in maintaining the disease-causing parasites (trypanosomes). Thus, the identification of animal reservoirs of trypanosomes is vital for the effective control of African trypanosomiasis. Additionally, the biotic and abiotic factors that drive gut microbiome diversity in tsetse flies are primarily unresolved, especially under natural, field conditions. In this study, we present a comprehensive DNA metabarcoding approach for individual tsetse fly analysis in the identification of mammalian blood meal sources and fly bacterial microbiome composition. We analyzed samples from two endemic foci, Kafue, Zambia collected in June 2017, and Hurungwe, Zimbabwe sampled in April 2014 (pilot study) and detected DNA of various mammals including humans, wild animals, domestic animals and small mammals (rat and bat). The bacterial diversity was relatively similar in flies with different mammalian species DNA, trypanosome infected and uninfected flies, and female and male flies. This study is the first report on bat DNA detection in wild tsetse flies. This study reveals that small mammals such as bats and rats are among the opportunistic blood meal sources for tsetse flies in the wild, and the implication on tsetse biology and ecology needs to be studied. also detected some that are commonly found in soil and water, indicating surface contaminating bacteria (from the environment). These bacteria, however, accounted for less than 0.1% of the total read count. Owing to the low read numbers obtained for non-symbiotic bacteria, and limited samples, we could not correlate any genus with blood meal source, sex, or status of trypanosome infection. Our results seem to indicate that blood meal sources from different hosts have little effect on the general gut microbiome composition of tsetse flies. However, the imposing abundance of the three symbiotic bacteria hampers the accurate detection and analysis of other bacteria; thus, the difficulty in identifying any significant associations. Studies on other vectors indicate changes in bacterial flora with different blood meals, as has been demonstrated recently in mosquitos (Aedes aegypti) 55 . Future studies should focus on more sensitive detection and analysis of non-symbiotic bacteria in tsetse flies to shed light on the relationship between bacterial microbiome, trypanosome infection, blood meal source, and other factors and their importance in aspects such as vector competence, immunity, reproduction, etc.

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Section: Paired-end Library Preparationmentioning

confidence: 99%

Blood meal sources and bacterial microbiome diversity in wild-caught tsetse flies

Gaithuma

Yamagishi

Hayashida

et al. 2020

Sci Rep

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“…The abundance corrected method introduced by Bittinger et al [71] moves toward gaining a more informative analysis. Moreover, spike-in controls have been suggested as a method to improve the accuracy of ITS sequencing data; however, the authors note that due to the limitations of ITS, researchers should not solely rely on read numbers to determine relative abundance [88]. Therefore, variation in the copy number of ribosomal RNA regions discussed earlier remains a confounding factor in mycobiome studies.…”

Section: Sequencing Methodsologiesmentioning

confidence: 99%

The Human Lung Mycobiome in Chronic Respiratory Disease: Limitations of Methods and Our Current Understanding

Weaver

Gago

Bromley

et al. 2019

Curr Fungal Infect Rep

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Purpose of the Review This review summarises the characteristics of the lung mycobiome in patients with chronic respiratory diseases and fungal lung diseases. We have also reviewed the limitations of the current methods in mycobiome studies. Recent Findings Available studies in the impacts of the mycobiome in chronic and fungal lung diseases are scarce and comparison of the available studies is hindered by heterogeneity in the sample sizes, methods and patient selection. Summary The impact of the diversity and composition of the lung mycobiome in chronic and fungal lung diseases is poorly understood. Most studies involve detection of fungi in respiratory samples by culture. However, such methods lack sensitivity and the emergence of next-generation sequencing technologies is an important advance. However, differences in the sequencing methodologies limit study comparisons. Well-designed methodological approaches and large cohort studies are needed to evaluate the impact of the lung mycobiome in respiratory diseases.

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“…Two reads of Trichromis salvini were recovered from a negative control on the S5, and two reads of Mayaheros urophthalmus were obtained from a negative control on the PGM. Because both cases involved a species common in samples from the same run, they likely reflect tag-switching due to a misread UMI [57,58] or cross-contamination. Given their rarity, it is unlikely such analytical errors significantly impacted our overall conclusions.…”

Section: Methodsmentioning

confidence: 99%

Using eDNA to biomonitor the fish community in a tropical oligotrophic lake

Valdéz-Moreno

Ivanova

Elías‐Gutiérrez

et al. 2018

Preprint

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Environmental DNA (eDNA) is an effective approach for detecting vertebrates and plants, especially in aquatic ecosystems, but prior studies have largely examined eDNA in cool temperate settings. By contrast, this study employs eDNA to survey the fish fauna in tropical Lake Bacalar (Mexico) with the additional goal of assessing the possible presence of invasive fishes, such as Amazon sailfin catfish. Sediment and water samples were collected from eight stations in Lake Bacalar on three occasions over a 4-month interval. Each sample was stored in the presence or absence of lysis buffer to compare eDNA recovery. Short fragments (184-187 bp) of the cytochrome c oxidase I (COI) gene were amplified using fusion primers and then sequenced on Ion Torrent PGM and S5 before their source species were determined using a custom reference sequence database constructed on BOLD. In total, eDNA sequences were recovered from 75 species of vertebrates including 47 fishes, 15 birds, 7 mammals, 5 reptiles, and 1 amphibian. Although all species are known from this region, 6 fish species represent new records for the study area, while 2 require verification. Sequences for five species (2 birds, 2 mammals, 1 reptile) were only detected from sediments, while sequences from 52 species were only recovered from water. Because DNA from the Amazon sailfin catfish was not detected, we used a mock eDNA experiment to confirm our methods were appropriate for its detection. We developed protocols that enabled the recovery of eDNA from tropical oligotrophic aquatic ecosystems, and confirmed their effectiveness in detecting diverse species of vertebrates including an invasive species of Amazon catfish.

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Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data

Cited by 204 publications

References 66 publications

Blood meal sources and bacterial microbiome diversity in wild-caught tsetse flies

Blood meal sources and bacterial microbiome diversity in wild-caught tsetse flies

The Human Lung Mycobiome in Chronic Respiratory Disease: Limitations of Methods and Our Current Understanding

Using eDNA to biomonitor the fish community in a tropical oligotrophic lake

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