Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses

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.

Diel cycling of the cosmopolitan abundant Pelagibacter virus 37‐F6: one of the most abundant viruses on earth

“…A follow-up study showed that 37-F6 virus and relatives were present in sorted single Pelagibacter cells (Martinez-Hernandez et al, 2019), while recent transcriptomic data provided first evidence of high in situ activity in coastal temperate waters of the NE Atlantic (Alonso-Sáez et al, 2018). Recently, droplet digital PCR (dPCR) estimated that the absolute abundances of free viral particles of virus 37-F6 in the Mediterranean Sea and the Gulf of Maine ranged between hundreds to thousands (360-8,510) viruses mL-1 seawater; within the abundance range of the pelagiphage isolate HTVC010P (Martinez-Hernandez, et al, 2019b). dPCR data also showed that the number of infecting 37-F6 viruses in coastal bacterioplankton (i.e.…”

Absolute quantification of infecting viral particles by chip‐based digital polymerase chain reaction

“…For the TaqMan chip‐based dPCR assays, a previous validated probe and primer set named ddSeq4 (size of PCR amplicon band ≈100; Supporting Information Fig. S3A) was employed (Martinez‐Hernandez, et al ., ). First, we ensured by real‐time PCR that primer sets Seq6 and ddSeq4 used for TaqMan and SYBR chemistry assays had similar efficiencies and reproducibility for monitoring abundances of virus 37‐F6 genome (Supporting Information Fig.…”

“…dPCR comparison of SYBR Green I chip‐based dPCR chemistry versus TaqMan chip‐based dPCR with an environmental DNA template (bacterioplankton cell fraction) collected at Cape Huertas (Mediterranean Sea). Total number of infecting viruses per mL of seawater is indicated according to dPCR data correction by (Martinez‐Hernandez et al ., ).…”

“…However, regardless of the deviation of the 37‐F6 burst size value from the reported mean value (Parada et al ., ) and the consideration of virus 37‐F6 being ubiquitous and abundant in all Tara samples collected from tropical and subtropical oceans (Martinez‐Hernandez et al ., ), an enormous amount of C could be transformed by this virus through the viral shunt. Recently, using ddPCR assay, which requires a more sophisticated and costly equipment, we estimated that the absolute number of free viral particles of virus vSAG 37‐F6 (standing stock) in seawater ranged from 360 to 8510 per mL (Martinez‐Hernandez et al ., ). In the case of the isolated pelagiphage HTVC010P (Zhao et al ., ), qPCR data in different samples collected from the open Atlantic Ocean (Eggleston and Hewson, ) showed abundance values within the order of 10 3 –10 4 viruses/mL.…”

Recent advances in lab-on-a-chip technologies for viral diagnosis

“…(Hewa et al, 2009;Jung et al, 2015a) antibody production resulting in false negative results using immunoassays, which can be up as long as 35-45 days for first generation HIV testing (Cornett and Kirn, 2013). Currently, dPCR is gaining popularity due to its ability to detect either DNA or RNA, with absolute gene quantification being more immune to background noise than conventional qPCR (Martinez-Hernandez et al, 2019). NA-based detection methods have revolutionized virus-related diagnostics (Roy et al, 2017) having the false negative window period HIV between 10 and 15 days (Branson and Stekler, 2011).…”