Exploring taxonomic and functional microbiome of Hawaiian stream and spring irrigation water systems using Illumina and Oxford Nanopore sequencing platforms.

Irrigation water is a common source of contamination that carries plant and foodborne human pathogens and provides a niche for proliferation and survival of microbes in agricultural settings. Bacterial communities and their functions in irrigation water were investigated by analyzing samples from wetland taro farms on Oahu, Hawaii using different DNA sequencing platforms. Irrigation water samples (stream, spring, and storage tank water) were collected from North, East, and West sides of Oahu and subjected to high quality DNA isolation, library preparation and sequencing of the V3-V4 region, full length 16S rRNA, and shotgun metagenome sequencing using Illumina iSeq100, Oxford Nanopore MinION and Illumina NovaSeq, respectively. Illumina reads provided the most comprehensive taxonomic classification at the phylum level where Proteobacteria was identified as the most abundant phylum in the stream source and associated water samples from wetland taro fields. Cyanobacteria was also a dominant phylum in samples from tank and spring water, whereas Bacteroidetes were most abundant in wetland taro fields irrigated with spring water. However, over 50% of the valid short amplicon reads remained unclassified and inconclusive at the species level. In contrast, Oxford Nanopore MinION was a better choice for microbe classification at the genus and species levels as indicated by samples sequenced for full length 16S rRNA. No reliable taxonomic classification results were obtained while using shotgun metagenome data. In functional analyzes, only 12% of the genes were shared by two consortia and 95 antibiotic resistant genes (ARGs) were detected with variable relative abundance. Full descriptions of microbial communities and their functions are essential for the development of better water management strategies aimed to produce safer fresh produce and to protect plant, animal, human and environmental health. Quantitative comparisons illustrated the importance of selecting the appropriate analytical method depending on the level of taxonomic delineation sought in each microbiome.

Comparison of RT-qPCR and RT-ddPCR with Rift valley fever virus (RVFV) RNA.

Rift valley fever (RVF) is an important zoonotic disease caused by the Rift valley fever virus (RVFV) which can affect ruminants and humans. In this study, a comparison was done of the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription-droplet digital PCR (RT-ddPCR) assays with synthesized RVFV RNA, cultured viral RNA, and mock clinical RVFV RNA samples. The genomic segments (L, M, and S) of three RVFV strains (BIME01, Kenya56, and ZH548) were synthesized and used as templates for in vitro transcription (IVT). Both the RT-qPCR and RT-ddPCR assays for RVFV did not react with any of the negative reference viral genomes. Thus, both the RT-qPCR and RT-ddPCR assays are specific to RVFV. The comparison of both the RT-qPCR and RT-ddPCR assays with serially diluted templates showed that the LoD of both assays are similar, and a concordant of the results was observed. The LoD of both assays reached the practical measurable minimum concentration. Taken altogether, the sensitivity of the RT-qPCR and RT-ddPCR assays is similar, and the material measured by RT-ddPCR can be used as a reference material for RT-qPCR.

Seasonal variations of the airborne microbial assemblages of the Seoul subway, South Korea from 16S and ITS gene profiles with chemical analysis.

In this study, we determined the seasonal airborne microbial diversity profiles at SMRT stations by sequencing the 16S rRNA and ITS. Particulate matter samples were collected from air purifiers installed in the platform area of the SMRT subway stations. Three stations that included the most crowded one were selected for the sampling. The sampling was done at each season during 2019. After extracting the total DNA from all seasonal samples, PCR was performed with Illumina overhang adapter primers for the V3-V4 region of the 16S rRNA gene and ITS2 region of the ITS gene. The amplified products were further purified, and sequencing libraries were made. Sequencing was carried with the Illumina Miseq Sequencing system (Illumina, USA) followed by in-depth diversity analyses. The elemental composition of the particulate matter samples collected from the different subway stations were obtained using a WD-XRF spectrometer. The SMRT microbiome showed extensive taxonomic diversity with the most common bacterial genera at the subway stations associated with the skin. Overall, the stations included in this study harbored different phylogenetic communities based on α- and ß-diversity comparisons. Microbial assemblages also varied depending upon the season in which the samples were taken and the station. Major elements present at the subway stations were from aerosols generated between wheels and brake cushions and between the catenaries and the pantographs. This study shows that the microbial composition of the SMRT subway stations comes from a diverse combination of environmental and human sources, the season and the lifestyle of commuters.

Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2.

As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.

Comparison of Digital PCR and Quantitative PCR with Various SARS-CoV-2 Primer-Probe Sets.

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.