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Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively-both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1-B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Stojan, Trumbić, Lepen Pleić and Šantić.)

Microbial community analysis based on the 16S rRNA-gene is used to investigate both beneficial and harmful microorganisms in various fields and environments. Recently, the next-generation sequencing (NGS) technology has enabled rapid and accurate microbial community analysis. Despite these advantages of NGS based metagenomics study, sample transport, storage conditions, amplification, library preparation kits, sequencing, and bioinformatics procedures can bias microbial community analysis results. In this study, eight mock communities were pooled from genomic DNA of Lactobacillus acidophilus KCTC 3164 T , Limosilactobacillus fermentum KCTC 3112 T , Lactobacillus gasseri KCTC 3163 T , Lacticaseibacillus paracasei subsp. paracasei KCTC 3510 T , Limosilactobacillus reuteri KCTC 3594 T , Lactococcus lactis subsp. lactis KCTC 3769 T , Bifidobacterium animalis subsp. lactis KCTC 5854 T , and Bifidobacterium breve KCTC 3220 T . The genomic DNAs were quantified by droplet digital PCR (ddPCR) and were mixed as mock communities. The mock communities were amplified with various 16S rRNA gene universal primer pairs and sequenced by MiSeq, IonTorrent, MGIseq-2000, Sequel II, and MinION NGS platforms. In a comparison of primer-dependent bias, the microbial profiles of V1-V2 and V3 regions were similar to the original ratio of the mock communities, while the microbial profiles of the V1-V3 region were relatively biased. In a comparison of platform-dependent bias, the sequence read from short-read platforms (MiSeq, IonTorrent, and MGIseq-2000) showed lower bias than that of long-read platforms (Sequel II and MinION). Meanwhile, the sequences read from Sequel II and MinION platforms were relatively biased in some mock communities. In the data of all NGS platforms and regions, L. acidophilus was greatly underrepresented while Lactococcus lactis subsp. lactis was generally overrepresented. In all samples of this study, the bias index (BI) was calculated and PCA was performed for comparison. The samples with biased relative abundance showed high BI values and were separated in the PCA results. In particular, analysis of regions rich in AT and GC poses problems for genome assembly, which can lead to sequencing bias. According to this comparative analysis, the development of reference material (RM) material has been proposed to calibrate the bias in microbiome analysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Park, Kim, Choi and Kim.)

Next-generation sequencing has revolutionized our ability to investigate the microbiota composition of diverse and complex environments. However, a number of factors can affect the accuracy of microbial community assessment, such as the DNA extraction method, the hypervariable region of 16S rRNA gene targeted, or the PCR primers used for amplification. The aim of this study was to assess the influence of commercially available DNA extraction kits and different primer pairs to provide a non-biased vision of the composition of bacterial communities present in olive xylem sap. For that purpose, branches from "Picual" and "Arbequina" olive cultivars were used for xylem sap extraction using a Scholander chamber device. The DNA extraction protocol significantly affected xylem sap bacterial community assessment. That resulted in significant differences in alpha (Richness) and beta diversity (UniFrac distances) metrics among DNA extraction protocols, with the 12 DNA extraction kits evaluated being clustered in four groups behaving differently. Although the core number of taxa detected by all DNA extraction kits included four phyla, seven classes, 12 orders, 16 or 21 families, and 12 or 14 genera when using the Greengenes or Silva database for taxonomic assignment, respectively, some taxa, particularly those identified at low frequency, were detected by some DNA extraction kits only. The most accurate depiction of a bacterial mock community artificially inoculated on sap samples was generated when using the PowerPlant DNA extraction kit, the combination of 799F/1193R primers amplifying the hypervariable V5-V7 region, and the Silva 132 database for taxonomic assignment. The DESeq2 analysis displayed significant differences among genera abundance between the different PCR primer pairs tested. Thus, Enterobacter , Granulicatella , Prevotella , and Brevibacterium presented a significant higher abundance in all PCR protocols when compared with primer pair 799F/1193R, while the opposite was true for Pseudomonas and Pectobacterium . The methodological approach followed in this study can be useful to optimize plant-associated microbiome analysis, especially when exploring new plant niches. Some of the DNA extraction kits and PCR primers selected in this study will contribute to better characterize bacterial communities inhabiting the xylem sap of olives or other woody crop species., Competing Interests: MM was employed by the company Testsystems LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Haro, Anguita-Maeso, Metsis, Navas-Cortés and Landa.)

In food safety the detection of food contaminations with pathogenic microorganisms is a race against time and often outpaced by error-prone epidemiological approaches. For evidence-based outbreak investigations fast and reliable techniques and procedures are required to identify the source of infection. Metagenomics has the potential to become a powerful tool in the field of modern food safety, since it allows the detection, identification and characterization of a broad range of pathogens in a single experiment without pre-cultivation within a couple of days. Nevertheless, sample handling, sequencing and data analysis are challenging and can introduce errors and biases into the analysis. In order to evaluate the potential of metagenomics in food safety, we generated a mock community containing DNA of foodborne bacteria. Herewith, we compare the aptitude of the two prevalent approaches - 16S rDNA amplicon sequencing and whole genome shotgun sequencing - for the detection of foodborne bacteria using different parameters during sample preparation, sequencing and data analysis. 16S rDNA sequencing did not only result in high deviations from the expected sample composition on genus and species level, but more importantly lacked the detection of several pathogenic species. While shotgun sequencing is more suitable for species detection, abundance estimation, genome assembly and species characterization, the performance can vary depending on the library preparation kit, which was confirmed for a naturally Francisella tularensis contaminated game meat sample. The application of the Nextera XT DNA Library Preparation Kit for shotgun sequencing did not only result in lower reference genome recovery and coverage, but also in distortions of the mock community composition. For data analysis, we propose a publicly available workflow for pathogen detection and characterization and demonstrate its benefits on the usability of metagenomic sequencing in food safety by analyzing an authentic metagenomic sample.

The amount of host DNA poses a major challenge to metagenome analysis. However, there is no guidance on the levels of host DNA, nor on the depth of sequencing needed to acquire meaningful information from whole metagenome sequencing (WMS). Here, we evaluated the impact of a wide range of amounts of host DNA and sequencing depths on microbiome taxonomic profiling using WMS. Synthetic samples with increasing levels of host DNA were created by spiking DNA of a mock bacterial community, with DNA from a mouse-derived cell line. Taxonomic analysis revealed that increasing proportions of host DNA led to decreased sensitivity in detecting very low and low abundant species. Reduction of sequencing depth had major impact on the sensitivity of WMS for profiling samples with 90% host DNA, increasing the number of undetected species. Finally, analysis of simulated datasets with fixed depth of 10 million reads confirmed that microbiome profiling becomes more inaccurate as the level of host DNA increases in a sample. In conclusion, samples with high amounts of host DNA coupled with reduced sequencing depths, decrease WMS coverage for characterization of the microbiome. This study highlights the importance of carefully considering these aspects in the design of WMS experiments to maximize microbiome analyses.

Invasive fungal infections are an increasingly important cause of human morbidity and mortality. We generated a next-generation sequencing (NGS)-based method designed to detect a wide range of fungi and applied it to analysis of the fungal microbiome (mycobiome) of the lung during fungal infection. Internal transcribed spacer 1 (ITS1) amplicon sequencing and a custom analysis pipeline detected 96% of species from three mock communities comprised of potential fungal lung pathogens with good recapitulation of the expected species distributions (Pearson correlation coefficients r = 0.63, p = 0.004; r = 0.71, p < 0.001; r = 0.62, p = 0.002). We used this pipeline to analyze mycobiomes of bronchoalveolar lavage (BAL) specimens classified as culture-negative ( n = 50) or culture-positive ( n = 39) for Blastomyces dermatitidis/gilchristii , the causative agent of North America blastomycosis. Detected in 91.4% of the culture-positive samples, Blastomyces dominated (>50% relative abundance) the mycobiome in 68.6% of these culture-positive samples but was absent in culture-negative samples. To overcome any bias in relative abundance due to between-sample variation in fungal biomass, an abundance-weighting calculation was used to normalize the data by accounting for sample-specific PCR cycle number and PCR product concentration data utilized during sample preparation. After normalization, there was a statistically significant greater overall abundance of ITS1 amplicon in the Blastomyces -culture-positive samples versus culture-negative samples. Moreover, the normalization revealed a greater biomass of yeast and environmental fungi in several Blastomyces -culture-positive samples than in the culture-negative samples. Successful detection of Coccidioides , Scedosporium , Phaeoacremonium , and Aspergillus in 6 additional culture-positive BALs by ITS1 amplicon sequencing demonstrates the ability of this method to detect a broad range of fungi from clinical specimens, suggesting that it may be a potentially useful adjunct to traditional fungal microbiological testing for the diagnosis of respiratory mycoses.

Metaproteomics, the study of protein expression in microbial communities, is a versatile tool for environmental microbiology. Achieving sufficiently high metaproteome coverage to obtain a comprehensive picture of the activities and interactions in microbial communities is one of the current challenges in metaproteomics. An essential step to maximize the number of identified proteins is peptide separation via liquid chromatography (LC) prior to mass spectrometry (MS). Thorough optimization and comparison of LC methods for metaproteomics are, however, currently lacking. Here, we present an extensive development and test of different 1D and 2D-LC approaches for metaproteomic peptide separations. We used fully characterized mock community samples to evaluate metaproteomic approaches with very long analytical columns (50 and 75 cm) and long gradients (up to 12 h). We assessed a total of over 20 different 1D and 2D-LC approaches in terms of number of protein groups and unique peptides identified, peptide spectrum matches (PSMs) generated, the ability to detect proteins of low-abundance species, the effect of technical replicate runs on protein identifications and method reproducibility. We show here that, while 1D-LC approaches are faster and easier to set up and lead to more identifications per minute of runtime, 2D-LC approaches allow for a higher overall number of identifications with up to >10,000 protein groups identified. We also compared the 1D and 2D-LC approaches to a standard GeLC workflow, in which proteins are pre-fractionated via gel electrophoresis. This method yielded results comparable to the 2D-LC approaches, however with the drawback of a much increased sample preparation time. Based on our results, we provide recommendations on how to choose the best LC approach for metaproteomics experiments, depending on the study aims.

The European ash ( Fraxinus excelsior ) is threatened by the introduced ascomycete Hymenoscyphus fraxineus , the causal agent of ash dieback. Endophytic fungi are known to modulate their host's resistance against pathogens. To understand possible consequences of ash dieback on the endophytic mycobiome, F. excelsior leaves were collected in naturally regenerated forests and the fungal communities analyzed by classic culture and Illumina amplicon sequencing using a newly developed and validated fungal-specific primer. Collections were done in the area infested by ash dieback north of the Alps, and in the disease free area on the south side. Sycamore maple ( Acer pseudoplatanus ) was additionally collected, as well as the flowering ash ( F. ornus ), which occurs naturally in the south and shows tolerance to ash dieback. Both cultivation and amplicon sequencing revealed characteristic endophytic fungal communities dominated by several strictly host specific Venturia species. On A. pseudoplatanus , a hitherto undescribed Venturia species was identified. Due to its dominance on F. excelsior , V. fraxini is unlikely to go extinct in case of reduced host densities. A majority of species was not strictly host specific and is therefore likely less affected by ash dieback in the future. Still, shifts in community structure and loss of genetic diversity cannot be excluded. The potentially endangered endophyte Hymenoscyphus albidus was rarely found. In addition to host specificity, species with preferences for leaf laminae or petioles were found. We also detected considerable geographical variation between sampling sites and clear differences between the two sides of the Alps for endophytes of F. excelsior , but not A. pseudoplatanus . Since sycamore maple is not affected by an epidemic, this could point toward an influence of ash dieback on ash communities, although firm conclusions are not possible because of host preferences and climatic differences. Furthermore, the mycobiota of F. excelsior trees with or without dieback symptoms were compared, but no clear differences were detected. Besides methodical refinement, our study provides comprehensive data on the ash mycobiome that we expect to be subject to changes caused by an emerging disease of the host tree.

The rumen microbiome scientific community has utilized amplicon sequencing as an aid in identifying potential community compositional trends that could be used as an estimation of various production and performance traits including methane emission, animal protein production efficiency, and ruminant health status. In order to translate rumen microbiome studies into executable application, there is a need for experimental and analytical concordance within the community. The objective of this study was to assess these factors in relation to selected currently established methods for 16S phylogenetic community analysis on a microbial community standard (MC) and a DNA standard (DS; ZymoBIOMICS TM ). DNA was extracted from MC using the RBBC method commonly used for microbial DNA extraction from rumen digesta samples. 16S rRNA amplicon libraries were generated for the MC and DS using primers routinely used for rumen bacterial and archaeal community analysis. The primers targeted the V4 and V3-V4 region of the 16S rRNA gene and samples were subjected to both 20 and 28 polymerase chain reaction (PCR) cycles under identical cycle conditions. Sequencing was conducted using the Illumina MiSeq platform. As the bacteria contained in the microbial mock community were well-classified species, and for ease of explanation, we used the results of the Basic Local Alignment Search Tool classification to assess the DNA, PCR cycle number, and primer type. Sequence classification methodology was assessed independently. Spearman's correlation analysis indicated that utilizing the repeated bead beating and column method for DNA extraction in combination with primers targeting the 16S rRNA gene using 20 first-round PCR cycles was sufficient for amplicon sequencing to generate a relatively accurate depiction of the bacterial communities present in rumen samples. These results also emphasize the requirement to develop and utilize positive mock community controls for all rumen microbiomic studies in order to discern errors which may arise at any step during a next-generation sequencing protocol.

Profiling of microbial community composition is frequently performed by partial 16S rRNA gene sequencing on benchtop platforms following PCR amplification of specific hypervariable regions within this gene. Accuracy and reproducibility of this strategy are two key parameters to consider, which may be influenced during all processes from sample collection and storage, through DNA extraction and PCR based library preparation to the final sequencing. In order to evaluate both the reproducibility and accuracy of 16S rRNA gene based microbial profiling using the Ion Torrent PGM platform, we prepared libraries and performed sequencing of a well-defined and validated 20-member bacterial DNA mock community on five separate occasions and compared results with the expected even distribution. In general the applied method had a median coefficient of variance of 11.8% (range 5.5-73.7%) for all 20 included strains in the mock community across five separate sequencing runs, with underrepresented strains generally showing the largest degree of variation. In terms of accuracy, mock community species belonging to Proteobacteria were underestimated, whereas those belonging to Firmicutes were mostly overestimated. This could be explained partly by premature read truncation, but to larger degree their genomic GC-content, which correlated negatively with the observed relative abundances, suggesting a PCR bias against GC-rich species during library preparation. Increasing the initial denaturation time during the PCR amplification from 30 to 120 s resulted in an increased average relative abundance of the three mock community members with the highest genomic GC%, but did not significantly change the overall evenness of the community distribution. Therefore, efforts should be made to optimize the PCR conditions prior to sequencing in order to maximize accuracy.