Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses
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...
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doaj-cf940396da3a4331a02c647c23153ac92020-11-24T21:43:12ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-06-011010.3389/fmicb.2019.01226461852Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter VirusesFrancisco Martinez-Hernandez0Inmaculada Garcia-Heredia1Monica Lluesma Gomez2Lucia Maestre-Carballa3Joaquín Martínez Martínez4Manuel Martinez-Garcia5Department of Physiology, Genetics, and Microbiology, University of Alicante, Alicante, SpainDepartment of Physiology, Genetics, and Microbiology, University of Alicante, Alicante, SpainDepartment of Physiology, Genetics, and Microbiology, University of Alicante, Alicante, SpainDepartment of Physiology, Genetics, and Microbiology, University of Alicante, Alicante, SpainMarine Virology Laboratory, Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, United StatesDepartment of Physiology, Genetics, and Microbiology, University of Alicante, Alicante, SpainAbsolute 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.https://www.frontiersin.org/article/10.3389/fmicb.2019.01226/fullmarineddPCRPelagibacter ubiqueabundancevirussingle-virus genomics |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Francisco Martinez-Hernandez Inmaculada Garcia-Heredia Monica Lluesma Gomez Lucia Maestre-Carballa Joaquín Martínez Martínez Manuel Martinez-Garcia |
spellingShingle |
Francisco Martinez-Hernandez Inmaculada Garcia-Heredia Monica Lluesma Gomez Lucia Maestre-Carballa Joaquín Martínez Martínez Manuel Martinez-Garcia Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses Frontiers in Microbiology marine ddPCR Pelagibacter ubique abundance virus single-virus genomics |
author_facet |
Francisco Martinez-Hernandez Inmaculada Garcia-Heredia Monica Lluesma Gomez Lucia Maestre-Carballa Joaquín Martínez Martínez Manuel Martinez-Garcia |
author_sort |
Francisco Martinez-Hernandez |
title |
Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses |
title_short |
Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses |
title_full |
Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses |
title_fullStr |
Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses |
title_full_unstemmed |
Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses |
title_sort |
droplet digital pcr for estimating absolute abundances of widespread pelagibacter viruses |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Microbiology |
issn |
1664-302X |
publishDate |
2019-06-01 |
description |
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. |
topic |
marine ddPCR Pelagibacter ubique abundance virus single-virus genomics |
url |
https://www.frontiersin.org/article/10.3389/fmicb.2019.01226/full |
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