Host-associated microbe PCR (hamPCR): accessing new biology through convenient measurement of both microbial load and community composition

Lundberg, Derek S.; Ayutthaya, Pratchaya Pramoj Na; Strauß, Annett; Shirsekar, Gautam; Lo, Wen‐Sui; Lahaye, Thomas; Weigel, Detlef

doi:10.1101/2020.05.19.103937

Cited by 6 publications

(12 citation statements)

References 83 publications

(132 reference statements)

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“…Samples had between 38 and 1,219,071 reads that aligned to the reference genome, A. rohweri Acer44 (GCA 003953955) ( Table S1). The A. rohweri reads were normalized to the total coral host reads as an intrinsic measure of microbial load [75][76][77]; using this method, reads varied according to coral host and sampling location ( Belize were 100% complete with no contamination and a larger N50 value than the reference assembly (Table 1). All A. rohweri MAGs were at least 1.21 Mbp and had a >99% Average Nucleotide Identity (ANI) in pairwise comparisons to the reference genome and one another (Table 1; Table S2).…”

Section: Metagenome Assembled Genomes (Mags) Generated From Multiple mentioning

confidence: 99%

The coral symbiontCandidatusAquarickettsia is variably abundant in threatened Caribbean acroporids and transmitted horizontally

Baker

Reich

Kitchen

et al. 2021

Preprint

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The aquatic symbiont “Candidatus Aquarickettsia rohweri” infects a diversity of non-bilaterian metazoan phyla. In the threatened coral Acropora cervicornis, Aquarickettsia proliferates in response to increased nutrient exposure, resulting in suppressed growth and increased disease susceptibility and mortality. This study evaluated the extent, as well as the ecology and evolution of Aquarickettsia infecting the Caribbean corals: Ac. cervicornis and Ac. palmata and their hybrid (‘Ac. prolifera’). The bacterial parasite Aquarickettsia was found in all acroporids, with host and sampling location impacting infection magnitude. Phylogenomic and genome-wide single nucleotide variant analysis found Aquarickettsia clustering by region, not by coral taxon. Fixation analysis suggested within coral colonies, Aquarickettsia are genetically isolated to the extent that reinfection is unlikely. Relative to other Rickettsiales, Aquarickettsia is undergoing positive selection, with Florida populations experiencing greater positive selection relative to the other Caribbean locations. This may be due to Aquarickettsia response to increased nutrient stress in Florida, as indicated by greater in situ replication rates in these corals. Aquarickettsia did not significantly codiversify with either coral animal nor algal symbiont, and qPCR analysis of gametes and juveniles from susceptible coral genotypes indicated absence in early life stages. Thus, despite being an obligate parasite, Aquarickettsia must be horizontally transmitted via coral mucocytes, an unidentified secondary host, or a yet unexplored environmentally mediated mechanism. Importantly, the prevalence of Aquarickettsia in Ac. cervicornis and high abundance in Florida populations suggests that disease mitigation efforts in the US and Caribbean should focus on preventing early infection via horizontal transmission.

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Section: Metagenome Assembled Genomes (Mags) Generated From Multiple mentioning

confidence: 99%

The coral symbiontCandidatusAquarickettsia is variably abundant in threatened Caribbean acroporids and transmitted horizontally

Baker

Reich

Kitchen

et al. 2021

Preprint

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“…In the present study, absolute gene densities (qPCR data) were significantly different between the two soils before starting the experiment, which is one of the many cases for which analyses of microbial communities based on relative abundance is unlikely to reflect actual community patterns (Stämmler et al, 2016;Zhang et al, 2017;Props et al, 2017;Vandeputte et al, 2017;Lundberg et al, 2020) and associated ecosystem processes. Here, when estimated absolute abundances were considered, we observed notable differences from the relative abundance data and new, often contradictory, trends became visible.…”

Section: Resultsmentioning

confidence: 59%

Relative and quantitative rhizosphere microbiome profiling result in distinct abundance patterns

Azarbad

Tremblay

Bainard

et al. 2021

Preprint

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Next-generation sequencing is recognized as one of the most popular and cost-effective way of characterizing microbiome in multiple samples. However, most of the currently available amplicon sequencing approaches are inherently limited, as they are often presented based on the relative abundance of microbial taxa, which may not fully represent actual microbiome profiles. Here, we combined amplicon sequencing (16S rRNA gene for bacteria and ITS region for fungi) with real-time quantitative PCR (qPCR) to characterize the rhizosphere microbiome of wheat. We show that the increase in relative abundance of major microbial phyla does not necessarily result in an increase in abundance. One striking observation when comparing relative and quantitative abundances was a substantial increase in the abundance of almost all phyla associated with the rhizosphere of plants grown in soil with no history of water stress as compared with the rhizosphere of plants growing in soil with a history of water stress, which was in contradiction with the trends observed in the relative abundance data. Our results suggest that the estimated absolute abundance approach gives a different perspective than the relative abundance approach, providing complementary information that helps to better understand the rhizosphere microbiome.

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“…Thus, we recommend increasing the number of blocking cycles when following protocol A or B used here and when no prior information about bacterial loads is available. Alternatively, a linker sequence could be used as the binding site for concatenated primers in the second step 19 . This only amplifies amplicons from the first step, not left-over template DNA.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, blocking could be minimized to only a few cycles. Lundberg et al 19 did apply our ITS blocking oligos in their protocol, demonstrating that blocking oligos can be dropped into most two-step library pipelines.…”

Section: Discussionmentioning

confidence: 99%

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Obtaining deeper insights into microbiome diversity using a simple method to block host and non-targets in amplicon sequencing

Mayer

Mari

Almario

et al. 2020

Preprint

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Microbiome profiling is revolutionizing our understanding of biological mechanisms such as metaorganismal (host+microbiome) assembly, functions and adaptation. Amplicon sequencing of multiple conserved, phylogenetically informative loci is an instrumental tool for characterization of the highly diverse microbiomes of natural systems. Investigations in many study systems are hindered by loss of essential sequencing depth due to amplification of non-target DNA from hosts or overabundant microorganisms. This issue requires urgent attention to address ecologically relevant problems using high throughput, high resolution microbial profiling. Here, we introduce a simple, low cost and highly flexible method using standard oligonucleotides (“blocking oligos”) to block amplification of non-targets and an R package to aid in their design. They can be dropped into practically any two-step amplicon sequencing library preparation pipeline. We apply them in leaves, a system presenting exceptional challenges with host and non-target microbial amplification. Blocking oligos designed for use in eight target loci reduce undesirable amplification of host and non-target microbial DNA by up to 90%. In addition, 16S and 18S “universal” plant blocking oligos efficiently block most plant hosts, leading to increased microbial alpha diversity discovery without biasing beta diversity measurements. By blocking only chloroplast 16S amplification, we show that blocking oligos do not compromise quantitative microbial load information inherent to plant-associated amplicon sequencing data. Using these tools, we generated a near-complete survey of the Arabidopsis thaliana leaf microbiome based on diversity data from eight loci and discuss complementarity of commonly used amplicon sequencing regions for describing leaf microbiota. The blocking oligo approach has potential to make new questions in a variety of study systems more tractable by making amplicon sequencing more targeted, leading to deeper, systems-based insights into microbial discovery.

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Host-associated microbe PCR (hamPCR): accessing new biology through convenient measurement of both microbial load and community composition

Cited by 6 publications

References 83 publications

The coral symbiontCandidatusAquarickettsia is variably abundant in threatened Caribbean acroporids and transmitted horizontally

The coral symbiontCandidatusAquarickettsia is variably abundant in threatened Caribbean acroporids and transmitted horizontally

Relative and quantitative rhizosphere microbiome profiling result in distinct abundance patterns

Obtaining deeper insights into microbiome diversity using a simple method to block host and non-targets in amplicon sequencing

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