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ABSTRACT Since the discovery of complete ammonia oxidizers (comammox) within the genus Nitrospira, their distribution and abundance across habitats have been intensively studied to better understand their ecological significance. Many primers targeting their ammonia monooxygenase subunit A gene (amoA) have been designed to detect and quantify comammox bacteria and to describe their community structure. We identified 38 published primers, but only few had high coverage and specificity for all known comammox Nitrospira or one of the two described subclades. For each target group, we comprehensively evaluated selected primer pairs using in silico analyses, endpoint PCRs, qPCRs, and amplicon sequencing on samples from various environments. Endpoint PCRs and qPCRs showed that the most commonly used primer pairs (comaA-244F/659R, comaB-244F/659R, and Ntsp-amoA162F/359R) produced several bands, which likely inflated quantifications via qPCR. In contrast, the recently published primer combinations CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R resulted mostly in a single band. Furthermore, amplicon sequencing demonstrated that these primer combinations also captured the highest richness of comammox Nitrospira. Taken together, our results indicate that few existing comammox amoA primer combinations have both high specificity and coverage and that the choice of these high-specificity and high-coverage primer pairs substantially impacts the accurate detection, quantification, and community description of comammox bacteria. We, therefore, recommend using the CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R primer pairs.IMPORTANCEBacteria that can fully convert ammonia via nitrite to nitrate, the complete ammonia oxidizers (comammox), were recently discovered and are found in many natural and engineered environments. PCR-based tools to study their abundance and diversity were rapidly developed, resulting in a plethora of primers available, many of which are widely used. The presence of comammox bacteria in an environment can, however, only be correctly determined if the used primers detect all members of this group while not detecting any other guilds. This study assesses the coverage and specificity of existing primers targeting comammox bacteria using both computational and standard molecular techniques, revealing large differences in their performance. The uniform usage of well-performing primers across studies could aid in generating comparable and generalizable data to better understand the importance of comammox bacteria in the environment.

ABSTRACT The ARTIC Network provides a common resource of PCR primer sequences and recommendations for amplifying SARS-CoV-2 genomes. The initial tiling strategy was developed with the reference genome Wuhan-01, and subsequent iterations have addressed areas of low amplification and sequence drop out. Recently, a new version (V4) was released, based on new variant genome sequences, in response to the realization that some V3 primers were located in regions with key mutations. Herein, we compare the performance of the ARTIC V3 and V4 primer sets with a matched set of 663 SARS-CoV-2 clinical samples sequenced with an Illumina NovaSeq 6000 instrument. We observe general improvements in sequencing depth and quality, and improved resolution of the SNP causing the D950N variation in the spike protein. Importantly, we also find nearly universal presence of spike protein substitution G142D in Delta-lineage samples. Due to the prior release and widespread use of the ARTIC V3 primers during the initial surge of the Delta variant, it is likely that the G142D amino acid substitution is substantially underrepresented among early Delta variant genomes deposited in public repositories. In addition to the improved performance of the ARTIC V4 primer set, this study also illustrates the importance of the primer scheme in downstream analyses. IMPORTANCE ARTIC Network primers are commonly used by laboratories worldwide to amplify and sequence SARS-CoV-2 present in clinical samples. As new variants have evolved and spread, it was found that the V3 primer set poorly amplified several key mutations. In this report, we compare the results of sequencing a matched set of samples with the V3 and V4 primer sets. We find that adoption of the ARTIC V4 primer set is critical for accurate sequencing of the SARS-CoV-2 spike region. The absence of metadata describing the primer scheme used will negatively impact the downstream use of publicly available SARS-Cov-2 sequencing reads and assembled genomes.

Complete ammonia-oxidizing (comammox) bacteria play key roles in environmental nitrogen cycling and all belong to the genus Nitrospira, which was originally believed to include only strict nitrite-oxidizing bacteria (sNOB). Thus, differential estimation of sNOB abundance from that of comammox Nitrospira has become problematic, since both contain nitrite oxidoreductase genes that serve as common targets for sNOB detection. Herein, we developed novel comammox Nitrospira clade A- and B-specific primer sets targeting the -subunit of the ammonia monooxygenase gene (amoA) and a sNOB-specific primer set targeting the cyanase gene (cynS) for quantitative PCR (qPCR). The high coverage and specificity of these primers were checked by use of metagenome and metatranscriptome data sets. Efficient and specific amplification with these primers was demonstrated using various environmental samples. Using the newly designed primers, we successfully estimated the abundances of comammox Nitrospira and sNOB in samples from two chloramination-treated drinking water systems and found that, in most samples, comammox Nitrospira clade A was the dominant type of Nitrospira and also served as the primary ammonia oxidizer. Compared with other ammonia oxidizers, comammox Nitrospira had a higher abundance in process water samples in these two drinking water systems. We also demonstrated that sNOB can be readily misrepresented by an earlier method, calculated by subtracting the comammox Nitrospira abundance from the total Nitrospira abundance, especially when the comammox Nitrospira proportion is relatively high. The new primer sets were successfully applied to comammox Nitrospira and sNOB quantification, which may prove useful in understanding the roles of Nitrospira in nitrification in various ecosystems. [ABSTRACT FROM AUTHOR]

Denitrifying Epsilonproteobacteria may dominate nitrogen loss processes in marine habitats with intense redox gradients, but assessment of their importance is limited by the currently available primers for nitrite reductase genes. Nine new primers targeting the nirS gene of denitrifying Epsilonproteobacteria were designed and tested for use in sequencing and quantitative PCR on two microbial mat samples (vent 2 and vent 4) from the Calypso hydrothermal vent field, Bay of Plenty, New Zealand. Commonly used nirS and nirK primer sets nirS1F/nirS6R, cd3aF/R3cd, nirK1F/nirK5R, and F1aCu/R3Cu were also tested to determine what may be missed by the common single-primer approach to assessing denitrifier diversity. The relative importance of Epsilonproteobacteria in these samples was evaluated by 16S rRNA gene sequencing. Epsilonproteobacteria represented up to 75.6% of 16S rRNA libraries, but nirS genes from this group were not found with commonly used primers. Pairing of the new primer EPSnirS511F with either EPSnirS1100R or EPSnirS1105R recovered nirS sequences from members of the genera Sulfurimonas, Sulfurovum, and Nitratifractor. The new quantitative PCR primers EPSnirS103F/EPSnirS530R showed dominance of denitrifying Epsilonproteobacteria in vent 4 compared to vent 2, which had greater representation by "standard" denitrifiers measured with the cd3aF/R3cd primers. Limited results from commonly used nirK primers suggest biased amplification between primers. Future application of multiple nirS and nirK primers, including the new epsilonproteobacterial nirS primers, will improve the detection of denitrifier diversity and the capability to identify changes in dominant denitrifying communities. IMPORTANCE: Estimating the potential for increasing nitrogen limitation in the changing global ocean is reliant on understanding the microbial community that removes nitrogen through the process of denitrification. This process is favored under oxygen limitation, which is a growing global-ocean phenomenon. Current methods use the nitrite reductase genes nirS and nirK to assess denitrifier diversity and abundance using primers that target only a few known denitrifiers and systematically exclude denitrifying Epsilonproteobacteria, a group known to dominate in reducing environments, such as hydrothermal vents and anoxic basins. As oxygen depletion expands in the oceans, it is important to study denitrifier community dynamics within those areas to predict future global ocean changes. This study explores the design and testing of new primers that target epsilonproteobacterial nirS and reveals the varied success of existing primers, leading to the recommendation of a multiple-primer approach to assessing denitrifier diversity. [ABSTRACT FROM AUTHOR]

ABSTRACT Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.

The objective of this study was to design a pangenotypic PCR-based assay for the detection and quantification of hepatitis E virus (HEV) RNA from across the entire spectrum of described genotypes belonging to the Orthohepevirus A genus. The optimal conditions and the performance of the assay were determined by testing the WHO standard strain (6219/10) and the WHO HEV panel (8578/13). Similarly, performance comparisons were made with two commercial assays (Real Star HEV RT-PCR 2.0 and ampli Cube HEV 2.0 Quant) to detect HEV RNA at concentrations below 1,000 IU/ml with viral strains from the WHO and to test samples from 54 patients with acute hepatitis. The assay presented in this study was able to detect the entire spectrum of described genotypes belonging to the Orthohepevirus A genus, demonstrating better performance than both commercial kits. This procedure may represent a significant improvement in the molecular diagnosis of HEV infection., (Copyright © 2021 American Society for Microbiology.)

Complete ammonia-oxidizing (comammox) bacteria play key roles in environmental nitrogen cycling and all belong to the genus Nitrospira , which was originally believed to include only strict nitrite-oxidizing bacteria (sNOB). Thus, differential estimation of sNOB abundance from that of comammox Nitrospira has become problematic, since both contain nitrite oxidoreductase genes that serve as common targets for sNOB detection. Herein, we developed novel comammox Nitrospira clade A- and B-specific primer sets targeting the α-subunit of the ammonia monooxygenase gene ( amoA ) and a sNOB-specific primer set targeting the cyanase gene ( cynS ) for quantitative PCR (qPCR). The high coverage and specificity of these primers were checked by use of metagenome and metatranscriptome data sets. Efficient and specific amplification with these primers was demonstrated using various environmental samples. Using the newly designed primers, we successfully estimated the abundances of comammox Nitrospira and sNOB in samples from two chloramination-treated drinking water systems and found that, in most samples, comammox Nitrospira clade A was the dominant type of Nitrospira and also served as the primary ammonia oxidizer. Compared with other ammonia oxidizers, comammox Nitrospira had a higher abundance in process water samples in these two drinking water systems. We also demonstrated that sNOB can be readily misrepresented by an earlier method, calculated by subtracting the comammox Nitrospira abundance from the total Nitrospira abundance, especially when the comammox Nitrospira proportion is relatively high. The new primer sets were successfully applied to comammox Nitrospira and sNOB quantification, which may prove useful in understanding the roles of Nitrospira in nitrification in various ecosystems. IMPORTANCE Nitrospira prevents the use of previously identified primers targeting the Nitrospira prevents the use of previously identified primers targeting the Nitrospira 16S rRNA gene or nitrite oxidoreductase ( nxr ) gene for differential determination of strict nitrite-oxidizing bacteria (sNOB) in the genus Nitrospira and among comammox bacteria in this genus. We designed three novel primer sets that enabled quantification of comammox Nitrospira clades A and B and sNOB with high coverage, specificity, and accuracy in various environments. With the designed primer sets, sNOB and comammox Nitrospira and sNOB by use of the newly designed primers will provide essential information for evaluating the contribution of Nitrospira and sNOB by use of the newly designed primers will provide essential information for evaluating the contribution of Nitrospira to nitrification in various ecosystems., (Copyright © 2020 American Society for Microbiology.)

Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5' end, allowing for a range of different 3' primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.