Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses

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Title: Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses
Authors: Martinez-Hernandez, Francisco | Garcia-Heredia, Inmaculada | Lluesma Gómez, Mónica | Maestre-Carballa, Lucia | Martínez Martínez, Joaquín | Martinez-Garcia, Manuel
Research Group/s: Ecología Microbiana Molecular
Center, Department or Service: Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología
Keywords: Marine | ddPCR | Pelagibacter ubique | Abundance | Virus | Single-virus genomics | vSAG 37-F6 | HTVC010P
Knowledge Area: Microbiología
Issue Date: 12-Jun-2019
Publisher: Frontiers Media
Citation: Martinez-Hernandez F, Garcia-Heredia I, Lluesma Gomez M, Maestre-Carballa L, Martínez Martínez J and Martinez-Garcia M (2019) Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses. Front. Microbiol. 10:1226. doi: 10.3389/fmicb.2019.01226
Abstract: Absolute abundances of prokaryotes are typically determined by FISH. Due to the lack of a universal conserved gene among all viruses, metagenomic fragment recruitment is commonly used to estimate the relative viral abundance. However, the paucity of absolute virus abundance data hinders our ability to fully understand how viruses drive global microbial populations. The cosmopolitan marine Pelagibacter ubique is host for the highly widespread HTVC010P pelagiphage isolate and the extremely abundant uncultured virus vSAG 37-F6 recently discovered by single-virus genomics. Here we applied droplet digital PCR (ddPCR) to calculate the absolute abundance of these pelagiphage genotypes in the Mediterranean Sea and the Gulf of Maine. Abundances were between 360 and 8,510 virus mL-1 and 1,270–14,400 virus mL-1 for vSAG 37-F6 and HTVC010P, respectively. Illumina PCR-amplicon sequencing corroborated the absence of ddPCR non-specific amplifications for vSAG 37-F6, but showed an overestimation of 6% for HTVC010P from off-targets, genetically unrelated viruses. Absolute abundances of both pelagiphages, two of the most abundance marine viruses, suggest a large viral pelagiphage diversity in marine environments, and show the efficiency and power of ddPCR to disentangle the structure of marine viral communities. Results also highlight the need for a standardized workflow to obtain accurate quantification that allows cross data comparison.
Sponsor: This work has been supported by Spanish Ministry of Economy and Competitiveness (Ref. RTI2018-094248-B-I00), Generalitat Valenciana (Ref. ACOM/2015/133 and ACIF/2015/332), and Gordon and Betty Moore Foundation (Grant 5334).
URI: http://hdl.handle.net/10045/92869
ISSN: 1664-302X
DOI: 10.3389/fmicb.2019.01226
Language: eng
Type: info:eu-repo/semantics/article
Rights: © 2019 Martinez-Hernandez, Garcia-Heredia, Lluesma Gomez, Maestre-Carballa, Martínez Martínez and Martinez-Garcia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Peer Review: si
Publisher version: https://doi.org/10.3389/fmicb.2019.01226
Appears in Collections:INV - EMM - Artículos de Revistas

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