148. Single-amplicon, Multiplex Real-time RT-PCR with Tiled Probes to Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern

Background Detection and surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is of great public health importance. Broadly accessible and inexpensive assays are needed to enhance variant surveillance and detection globally. We developed and validated a single-reaction multiplex real-time RT-PCR (the Spike SNP assay) to detect specific mutations associated with variants of concern (VOC). Methods A single primer pair was designed to amplify a 348 bp region of spike. Probes were initially designed with locked nucleic acids (LNAs) to increase probe melting temperature, shorten probe length, and specifically detect 417K, E484K, and N501Y (Figure). The assay was optimized and evaluated using characterized variant sample pools. Clinical evaluation was performed on a convenience set of residual nasopharyngeal swabs, and variant calls were confirmed by SARS-CoV-2 genomic sequencing in a subset of samples. Following the initial evaluation, unmodified probes (without LNAs) were designed to detect L452R, L452Q, and E484Q. Figure. Spike SNP distinguishes mutations occurring in different lineages (A-C). Representative results of variant detection a single Spike SNP run are shown for mutations in the codons for 4177K (A) and mutations that encode 484K (B) and 501Y (C). Curves show dilutions of the following variants: blue, BEI 52286 (wild type); pink B.1.1.7; purple, B1.525; and green, P.1. Variant pools were used for B.1.17, B.1.525, and P.1 strains. Curves are displayed for a given dilution in each channel and result interpretation is shown (D). Results The lower limit of 95% detection was 2.46 to 2.48 log10 GE/mL for the three targets (~1-2 GE/reaction). Among 253 nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%), including all samples with Ct values < 30 (220/220) for the N2 target and 18/33 samples with N2 Ct values ≥ 30. Results were confirmed by SARS-CoV-2 genomic sequencing in 50/50 samples (100%). Subsequent addition of the 452R probe did not affect performance for the original targets, and probes for 452Q and 484Q performed similarly to LNA-modified probes. Conclusion The Spike SNP assay provides fast, inexpensive and sensitive detection of specific mutations associated with SARS-CoV-2 VOCs, and the assay can be quickly modified to detect new mutations in the receptor binding domain. Similar analytical performance of LNA-modified and unmodified probes presents options for future assay customization that balance the shorter probe length (LNAs) and increased accessibility (unmodified). The Spike SNP assay, if implemented across laboratories offering SARS-CoV-2 testing, could greatly increase capacity for variant detection and surveillance globally. Disclosures Colleen S. Kraft, MD, MSc, Rebiotix (Individual(s) Involved: Self): Advisor or Review Panel member

Single-amplicon, multiplex real-time RT-PCR with tiled probes to detect SARS-CoV-2 spike mutations associated with variants of concern

To provide an accessible and inexpensive method to surveil for SARS-CoV-2 mutations, we developed a multiplex real-time RT-PCR (the Spike SNP assay) to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348 bp region of spike , and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log 10 GE/mL for the three initial targets (∼1-2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with Ct values < 30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 Ct values ≥ 30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in receptor binding domain, and it can be quickly modified to detect new mutations that emerge.

Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition

The ratio of microbial population size relative to the amount of host tissue, or 'microbial load', is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because existing methods to determine load, such as serial dilution plating, quantitative PCR, and whole metagenome sequencing, add substantial cost and/or experimental burden, they are only rarely paired with amplicon sequencing. We introduce host-associated microbe PCR (hamPCR), a robust strategy to both quantify microbial load and describe interkingdom microbial community composition in a single amplicon library. We demonstrate its accuracy across multiple study systems, including nematodes and major crops, and further present a cost-saving technique to reduce host overrepresentation in the library prior to sequencing. Because hamPCR provides an accessible experimental solution to the well-known limitations and statistical challenges of compositional data, it has far-reaching potential in culture-independent microbiology.

SARS-CoV-2 detection status associates with bacterial community composition in patients and the hospital environment

Background SARS-CoV-2 is an RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Viruses exist in complex microbial environments, and recent studies have revealed both synergistic and antagonistic effects of specific bacterial taxa on viral prevalence and infectivity. We set out to test whether specific bacterial communities predict SARS-CoV-2 occurrence in a hospital setting. Methods We collected 972 samples from hospitalized patients with COVID-19, their health care providers, and hospital surfaces before, during, and after admission. We screened for SARS-CoV-2 using RT-qPCR, characterized microbial communities using 16S rRNA gene amplicon sequencing, and used these bacterial profiles to classify SARS-CoV-2 RNA detection with a random forest model. Results Sixteen percent of surfaces from COVID-19 patient rooms had detectable SARS-CoV-2 RNA, although infectivity was not assessed. The highest prevalence was in floor samples next to patient beds (39%) and directly outside their rooms (29%). Although bed rail samples more closely resembled the patient microbiome compared to floor samples, SARS-CoV-2 RNA was detected less often in bed rail samples (11%). SARS-CoV-2 positive samples had higher bacterial phylogenetic diversity in both human and surface samples and higher biomass in floor samples. 16S microbial community profiles enabled high classifier accuracy for SARS-CoV-2 status in not only nares, but also forehead, stool, and floor samples. Across these distinct microbial profiles, a single amplicon sequence variant from the genus Rothia strongly predicted SARS-CoV-2 presence across sample types, with greater prevalence in positive surface and human samples, even when compared to samples from patients in other intensive care units prior to the COVID-19 pandemic. Conclusions These results contextualize the vast diversity of microbial niches where SARS-CoV-2 RNA is detected and identify specific bacterial taxa that associate with the viral RNA prevalence both in the host and hospital environment.

In silico characterization of laccase gene isoforms of edible and medicinal basidiomycetes

Laccases are part of the family of ligninolytic enzymes and have played essential roles in several biological filamentous fungi processes, including fruiting body formation and lignin degradation. This study aimed to identify and characterize laccase genes in silico of several basidiomycete strains. The applied guaiacol oxidation test allowed the selection of seven out of 11 strains with ligninase activity, which were used for DNA extraction and amplification of the copper-binding region. A single amplicon of approximately 450 bp, was produced by all selected strains and they were further sequenced. Sequence analysis has suggested the presence of a new subdivision of the laccase genes. Clustering analysis confirmed the existence of two groups: cluster A with six strains and singleton B with U8-11 strain. The structural predictions of the U8-11 protein were dissimilar compared to other proteins described in our study due to the absence of the ALAVIN motif and, therefore, the U8-11 amino acid sequence was separated in a different cluster.

High quality single amplicon sequencing method for illumina platforms using ‘N’ (0-10) spacer primer pool without PhiX spik-in

Illumina sequencing platform requires base diversity in initial 11 cycles for efficient cluster identification and color matrix estimation. This limitation yields low quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but overall reduces the number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms we developed high throughput single amplicon sequencing method by introducing ‘N’ spacers in target gene amplification primers that are pooled for simple handling. We evaluated the efficiency of ‘N’ spacer primers by targeting bacterial 16S V3-V4 region, demonstrating heterogonous base library construction. Addition of ‘N’ spacer causes sequencing frame shift at every base that leads to base diversity and produces heterogenous high quality reads within single amplicon library. We have written a python script “MetReTrim” to trim the heterogenous ‘N’ spacers from the pre-processed reads. This method terminates the need for PhiX spike-in and allows for multiplexing of multiple samples, greatly reducing the overall cost and yields improved sequence quality.

Towards an in-depth characterization of Symbiodiniaceae in tropical giant clams via metabarcoding of pooled multi-gene amplicons

High-throughput sequencing is revolutionizing our ability to comprehensively characterize free-living and symbiotic Symbiodiniaceae, a diverse dinoflagellate group that plays a critical role in coral reef ecosystems. Most studies however, focus on a single marker for metabarcoding Symbiodiniaceae, potentially missing important ecological traits that a combination of markers may capture. In this proof-of-concept study, we used a small set of symbiotic giant clam (Tridacna maxima) samples obtained from nine French Polynesian locations and tested a dual-index sequence library preparation method that pools and simultaneously sequences multiple Symbiodiniaceae gene amplicons per sample for in-depth biodiversity assessments. The rationale for this approach was to allow the metabarcoding of multiple genes without extra costs associated with additional single amplicon dual indexing and library preparations. Our results showed that the technique effectively recovered very similar proportions of sequence reads and dominant Symbiodiniaceae clades among the three pooled gene amplicons investigated per sample, and captured varying levels of phylogenetic resolution enabling a more comprehensive assessment of the diversity present. The pooled Symbiodiniaceae multi-gene metabarcoding approach described here is readily scalable, offering considerable analytical cost savings while providing sufficient phylogenetic information and sequence coverage.

Towards an in-depth characterization of Symbiodiniaceae in tropical giant clams via metabarcoding of pooled multi-gene amplicons

High-throughput sequencing is revolutionizing our ability to comprehensively characterize free-living and symbiotic Symbiodiniaceae, a diverse dinoflagellate group that plays a critical role in coral reef ecosystems. Most studies however, focus on a single marker for metabarcoding Symbiodiniaceae, potentially missing important ecological traits that a combination of markers may capture. In this proof-of-concept study, we used a small set of symbiotic giant clam (Tridacna maxima) samples obtained from nine French Polynesian locations and tested a dual-index sequence library preparation method that pools and simultaneously sequences multiple Symbiodiniaceae gene amplicons per sample for in-depth biodiversity assessments. The rational for this approach was to allow the metabarcoding of multiple genes without extra costs associated with additional single amplicon dual indexing and library preparations. Our results showed that the technique effectively recovered very similar proportions of sequence reads and dominant Symbiodiniaceae clades among the three pooled gene amplicons investigated per sample, and captured varying levels of phylogenetic resolution enabling a more comprehensive assessment of the diversity present. The pooled Symbiodiniaceae multi-gene metabarcoding approach decribed here is readily scalable, offering considerable analytical cost savings while providing sufficient phylogenetic information and sequence coverage.

Towards an in-depth characterization of Symbiodiniaceae in tropical giant clams via metabarcoding of pooled multi-gene amplicons

High-throughput sequencing is revolutionizing our ability to comprehensively characterize free-living and symbiotic Symbiodiniaceae, a diverse dinoflagellate group that plays a critical role in coral reef ecosystems. Most studies however, focus on a single marker for metabarcoding Symbiodiniaceae, potentially missing important ecological traits that a combination of markers may capture. In this proof-of-concept study, we used a small set of symbiotic giant clam (Tridacna maxima) samples obtained from nine French Polynesian locations and tested a dual-index sequence library preparation method that pools and simultaneously sequences multiple Symbiodiniaceae gene amplicons per sample for in-depth biodiversity assessments. The rational for this approach was to allow the metabarcoding of multiple genes without extra costs associated with additional single amplicon dual indexing and library preparations. Our results showed that the technique effectively recovered very similar proportions of sequence reads and dominant Symbiodiniaceae clades among the three pooled gene amplicons investigated per sample, and captured varying levels of phylogenetic resolution enabling a more comprehensive assessment of the diversity present. The pooled Symbiodiniaceae multi-gene metabarcoding approach decribed here is readily scalable, offering considerable analytical cost savings while providing sufficient phylogenetic information and sequence coverage.