Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the  rrn operon

F1000Research
Anna CuscóOlga Francino

Abstract

Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities and two pools of low-biomass samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with rrn database. Consensus sequences of the 16S-ITS-23S genetic marker were obtained using canu. Results: The full-length 16S rRNA and the 16S-ITS-23S region of the rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct ba...Continue Reading

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Citations

Sep 26, 2019·Canadian Journal of Microbiology·Maria Ludovica SaccàSalvatore Frisullo
Nov 13, 2019·The ISME Journal·Tess E BrewerNoah Fierer
Apr 17, 2020·GigaScience·Olufunmilola IbironkeLee J Kerkhof
Feb 23, 2020·Scientific Reports·Ana Teresa GonçalvesCristian Gallardo-Escárate
Aug 28, 2020·Frontiers in Cellular and Infection Microbiology·Mathys J RedelinghuysMarleen M Kock
Jan 20, 2021·ELife·Lara UrbanMaximilian R Stammnitz
Nov 3, 2020·Biology Methods and Protocols·Adriel Latorre-PérezCristina Vilanova
Feb 26, 2021·PeerJ·Liying LowUNKNOWN West Midlands Collaborative Ophthalmology Network for Clinical Effectiveness & Research by Trainees (WM CONCERT)
Jan 15, 2021·FEMS Microbiology Ecology·Lee J Kerkhof
Apr 1, 2021·World Journal of Microbiology & Biotechnology·Veena Gayathri KrishnaswamyKabilan Mani
Feb 26, 2021·MSphere·Isabel Abellan-SchneyderKlaus Neuhaus
Jun 16, 2021·MSystems·Longxian LvYing Yu
Sep 14, 2021·Frontiers in Public Health·Guilherme Marcelino Viana de SiqueiraMaría-Eugenia Guazzaroni

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Software Mentioned

SpotON
R
Ampvis2
EPI2ME
Porechop
yacrd
BLAST
Albacore
[UNK]
Phyloseq

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