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Phylogenetic microbiota profiling in fecal samples depends on combination of sequencing depth and choice of NGS analysis method
Örebro University, School of Medical Sciences.
Örebro University, School of Medical Sciences.ORCID iD: 0000-0003-3887-9519
Örebro University, School of Medical Sciences.ORCID iD: 0000-0002-0362-0008
Örebro University, School of Medical Sciences.ORCID iD: 0000-0002-8391-1576
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2019 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 14, no 9, article id e0222171Article in journal (Refereed) Published
Abstract [en]

The human gut microbiota is well established as an important factor in health and disease. Fecal sample microbiota are often analyzed as a proxy for gut microbiota, and characterized with respect to their composition profiles. Modern approaches employ whole genome shotgun next-generation sequencing as the basis for these analyses. Sequencing depth as well as choice of next-generation sequencing data analysis method constitute two main interacting methodological factors for such an approach. In this study, we used 200 million sequence read pairs from one fecal sample for comparing different taxonomy classification methods, using default and custom-made reference databases, at different sequencing depths. A mock community data set with known composition was used for validating the classification methods. Results suggest that sequencing beyond 60 million read pairs does not seem to improve classification. The phylogeny prediction pattern, when using the default databases and the consensus database, appeared to be similar for all three methods. Moreover, these methods predicted rather different species. We conclude that the choice of sequencing depth and classification method has important implications for taxonomy composition prediction. A multi-method-consensus approach for robust gut microbiota NGS analysis is recommended.

Place, publisher, year, edition, pages
PLOS , 2019. Vol. 14, no 9, article id e0222171
National Category
Bioinformatics and Systems Biology
Identifiers
URN: urn:nbn:se:oru:diva-76641DOI: 10.1371/journal.pone.0222171ISI: 000532231400010PubMedID: 31527871Scopus ID: 2-s2.0-85072287908OAI: oai:DiVA.org:oru-76641DiVA, id: diva2:1353747
Funder
Knowledge Foundation
Note

Funding Agency:

Bo Rydin Foundation  F0514

Available from: 2019-09-24 Created: 2019-09-24 Last updated: 2021-06-14Bibliographically approved
In thesis
1. Metagenomic Characterization of the Gut Microbiome in Cohorts of Elderly
Open this publication in new window or tab >>Metagenomic Characterization of the Gut Microbiome in Cohorts of Elderly
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human gut microbiota plays a vital role in maintaining host health. This thesis aims to investigate the gut microbial population and function using next generation sequencing (NGS) data from faecal samples. Paper I examines the influence of sequencing depth and analysis methods in microbiota profiling using NGS whole genome sequencing (WGS) data. By subsampling the metagenomic data, the influence of varying sequencing depths on different phylogenetic classification methods is investigated. This suggests that necessary sequencing depth would be dependent on the individual research plan. This paper recommends the need for a consensus approach and an informed choice of NGS analysis method selection for a reliable prediction. Paper II relates the gut microbiota to general health, nutrient intake, physical activity, medications, and psychological distress in community-dwelling older adults and senior orienteers. A higher abundance of F. prausnitzi in the faecal microbiota of senior orienteers confirms the hypothesis that senior orienteers can be seen as a model for healthy ageing in the perspective of the microbiota. Paper III focuses on assessing the validity of function prediction using LC-MS at multiple annotation levels. Predicted and quantified protein-pathway profiles were subjected to correlation analyses, which showed statistically significant association between predicted and quantified proteins as well as predicted and quantified pathways. This study also showed a direct relation between protein abundance and correlation for predicted and quantified proteins at higher function levels. Paper IV investigates the effects of faecal microbiota transfer (FMT) on functional microbiota profiles. This study showed that allogenic FMT did not alter the metabolite profiles, but it seems to disturb the gut microbiota-metabolite interactions when compared to autologous FMT.

This thesis reiterates the need for carefully selecting prediction tools and methods for microbiome analysis. The findings of this thesis could stimulate more focused studies using NGS in medicine and aid in better understanding of host-microbe interactions.

Place, publisher, year, edition, pages
Örebro: Örebro University, 2020. p. 52
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 222
Keywords
Gut microbiota, metagenomics, High-Throughput Nucleotide Sequencing, reference sequence database, methods validation, microbial function, host-microbe interaction
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:oru:diva-85017 (URN)978-91-7529-357-8 (ISBN)
Public defence
2020-11-02, Örebro universitet, Campus USÖ, hörsal C1, Södra Grev Rosengatan 32, Örebro, 10:00 (English)
Opponent
Supervisors
Available from: 2020-08-24 Created: 2020-08-24 Last updated: 2020-10-21Bibliographically approved

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Rajan, Sukithar K.Lindqvist, Carl MårtenBrummer, Robert JanSchoultz, IdaRepsilber, Dirk

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