Regulation of ribosomal RNA gene copy number, transcription and nucleolus organization in eukaryotes.

One of the first biological machineries to be created seems to have been the ribosome. Since then, organisms have dedicated great efforts to optimize this apparatus. The ribosomal RNA (rRNA) contained within ribosomes is crucial for protein synthesis and maintenance of cellular function in all known organisms. In eukaryotic cells, rRNA is produced from ribosomal DNA clusters of tandem rRNA genes, whose organization in the nucleolus, maintenance and transcription are strictly regulated to satisfy the substantial demand for rRNA required for ribosome biogenesis. Recent studies have elucidated mechanisms underlying the integrity of ribosomal DNA and regulation of its transcription, including epigenetic mechanisms and a unique recombination and copy-number control system to stably maintain high rRNA gene copy number. In this Review, we disucss how the crucial maintenance of rRNA gene copy number through control of gene amplification and of rRNA production by RNA polymerase I are orchestrated. We also discuss how liquid-liquid phase separation controls the architecture and function of the nucleolus and the relationship between rRNA production, cell senescence and disease.

The MBD-ACD DNA methylation reader complex recruits MICRORCHIDIA6 to regulate ribosomal RNA gene expression in Arabidopsis.

DNA methylation is an important epigenetic mark implicated in selective rRNA gene expression, but the DNA methylation readers and effectors remain largely unknown. Here, we report a protein complex that reads DNA methylation to regulate variant-specific 45S ribosomal RNA (rRNA) gene expression in Arabidopsis (Arabidopsis thaliana). The complex, consisting of METHYL-CpG-BINDING DOMAIN PROTEIN5 (MBD5), MBD6, ALPHA-CRYSTALLIN DOMAIN PROTEIN15.5 (ACD15.5), and ACD21.4, directly binds to 45S rDNA. While MBD5 and MBD6 function redundantly, ACD15.5 and ACD21.4 are indispensable for variant-specific rRNA gene expression. These 4 proteins undergo phase separation in vitro and in vivo and are interdependent for their phase separation. The α-crystallin domain of ACD15.5 and ACD21.4, which is essential for their function, enables phase separation of the complex, likely by mediating multivalent protein interactions. The effector MICRORCHIDIA6 directly interacts with ACD15.5 and ACD21.4, but not with MBD5 and MBD6, and is recruited to 45S rDNA by the MBD-ACD complex to regulate variant-specific 45S rRNA expression. Our study reveals a pathway in Arabidopsis through which certain 45S rRNA gene variants are silenced, while others are activated.

Establishment and maintenance of alternative chromatin states at a multicopy gene locus.

In eukaryotes, each of the more than 100 copies of ribosomal RNA (rRNA) genes exists in either an RNA polymerase I transcribed open chromatin state or a nucleosomal, closed chromatin state. Open rRNA genes guarantee the cell's supply with structural components of the ribosome, whereas closed rRNA genes ensure genomic integrity. We report that the observed balance between open and closed rRNA gene chromatin states in proliferating yeast cells is due to a dynamic equilibrium of transcription-dependent removal and replication-dependent assembly of nucleosomes. Pol I transcription is required for the association of the HMG box protein Hmo1 with open rRNA genes, counteracting replication-independent nucleosome deposition and maintaining the open rRNA gene chromatin state outside of S phase. The findings indicate that the opposing effects of replication and transcription lead to a de novo establishment of chromatin states for rRNA genes during each cell cycle.

RIC-seq for global in situ profiling of RNA-RNA spatial interactions.

Highly structured RNA molecules usually interact with each other, and associate with various RNA-binding proteins, to regulate critical biological processes. However, RNA structures and interactions in intact cells remain largely unknown. Here, by coupling proximity ligation mediated by RNA-binding proteins with deep sequencing, we report an RNA in situ conformation sequencing (RIC-seq) technology for the global profiling of intra- and intermolecular RNA-RNA interactions. This technique not only recapitulates known RNA secondary structures and tertiary interactions, but also facilitates the generation of three-dimensional (3D) interaction maps of RNA in human cells. Using these maps, we identify noncoding RNA targets globally, and discern RNA topological domains and trans-interacting hubs. We reveal that the functional connectivity of enhancers and promoters can be assigned using their pairwise-interacting RNAs. Furthermore, we show that CCAT1-5L-a super-enhancer hub RNA-interacts with the RNA-binding protein hnRNPK, as well as RNA derived from the MYC promoter and enhancer, to boost MYC transcription by modulating chromatin looping. Our study demonstrates the power and applicability of RIC-seq in discovering the 3D structures, interactions and regulatory roles of RNA.

Evolving Together: Cassandra Retrotransposons Gradually Mirror Promoter Mutations of the 5S rRNA Genes.

The 5S rRNA genes are among the most conserved nucleotide sequences across all species. Similar to the 5S preservation we observe the occurrence of 5S-related nonautonomous retrotransposons, so-called Cassandras. Cassandras harbor highly conserved 5S rDNA-related sequences within their long terminal repeats, advantageously providing them with the 5S internal promoter. However, the dynamics of Cassandra retrotransposon evolution in the context of 5S rRNA gene sequence information and structural arrangement are still unclear, especially: (1) do we observe repeated or gradual domestication of the highly conserved 5S promoter by Cassandras and (2) do changes in 5S organization such as in the linked 35S-5S rDNA arrangements impact Cassandra evolution? Here, we show evidence for gradual co-evolution of Cassandra sequences with their corresponding 5S rDNAs. To follow the impact of 5S rDNA variability on Cassandra TEs, we investigate the Asteraceae family where highly variable 5S rDNAs, including 5S promoter shifts and both linked and separated 35S-5S rDNA arrangements have been reported. Cassandras within the Asteraceae mirror 5S rDNA promoter mutations of their host genome, likely as an adaptation to the host's specific 5S transcription factors and hence compensating for evolutionary changes in the 5S rDNA sequence. Changes in the 5S rDNA sequence and in Cassandras seem uncorrelated with linked/separated rDNA arrangements. We place all these observations into the context of angiosperm 5S rDNA-Cassandra evolution, discuss Cassandra's origin hypotheses (single or multiple) and Cassandra's possible impact on rDNA and plant genome organization, giving new insights into the interplay of ribosomal genes and transposable elements.

Acidic growth conditions stabilize the ribosomal RNA gene cluster and extend lifespan through noncoding transcription repression.

Blackcurrant (Ribes nigrum L.) is a classical fruit that has long been used to make juice, jam, and liqueur. Blackcurrant extract is known to relieve cells from DNA damage caused by hydrogen peroxide (H2 O2 ), methyl methane sulfonate (MMS), and ultraviolet (UV) radiation. We found that blackcurrant extract (BCE) stabilizes the ribosomal RNA gene cluster (rDNA), one of the most unstable regions in the genome, through repression of noncoding transcription in the intergenic spacer (IGS) which extended the lifespan in budding yeast. Reduced formation of extrachromosomal circles (ERCs) after exposure to fractionated BCE suggested that acidity of the growth medium impacted rDNA stability. Indeed, alteration of the acidity of the growth medium to pH ~4.5 by adding HCl increased rDNA stability and extended the lifespan. We identified RPD3 as the gene responsible for this change, which was mediated by the RPD3L histone deacetylase complex. In mammals, as inflammation sites in a tissue are acidic, DNA maintenance may be similarly regulated to prevent genome instability from causing cancer.

SCRAPT: an iterative algorithm for clustering large 16S rRNA gene data sets.

16S rRNA gene sequence clustering is an important tool in characterizing the diversity of microbial communities. As 16S rRNA gene data sets are growing in size, existing sequence clustering algorithms increasingly become an analytical bottleneck. Part of this bottleneck is due to the substantial computational cost expended on small clusters and singleton sequences. We propose an iterative sampling-based 16S rRNA gene sequence clustering approach that targets the largest clusters in the data set, allowing users to stop the clustering process when sufficient clusters are available for the specific analysis being targeted. We describe a probabilistic analysis of the iterative clustering process that supports the intuition that the clustering process identifies the larger clusters in the data set first. Using real data sets of 16S rRNA gene sequences, we show that the iterative algorithm, coupled with an adaptive sampling process and a mode-shifting strategy for identifying cluster representatives, substantially speeds up the clustering process while being effective at capturing the large clusters in the data set. The experiments also show that SCRAPT (Sample, Cluster, Recruit, AdaPt and iTerate) is able to produce operational taxonomic units that are less fragmented than popular tools: UCLUST, CD-HIT and DNACLUST. The algorithm is implemented in the open-source package SCRAPT. The source code used to generate the results presented in this paper is available at https://github.com/hsmurali/SCRAPT.

Full-length 16S rRNA gene sequencing by PacBio improves taxonomic resolution in human microbiome samples.

BACKGROUND: Sequencing variable regions of the 16S rRNA gene (≃300 bp) with Illumina technology is commonly used to study the composition of human microbiota. Unfortunately, short reads are unable to differentiate between highly similar species. Considering that species from the same genus can be associated with health or disease it is important to identify them at the lowest possible taxonomic rank. Third-generation sequencing platforms such as PacBio SMRT, increase read lengths allowing to sequence the whole gene with the maximum taxonomic resolution. Despite its potential, full length 16S rRNA gene sequencing is not widely used yet. The aim of the current study was to compare the sequencing output and taxonomic annotation performance of the two approaches (Illumina short read sequencing and PacBio long read sequencing of 16S rRNA gene) in different human microbiome samples. DNA from saliva, oral biofilms (subgingival plaque) and faeces of 9 volunteers was isolated. Regions V3-V4 and V1-V9 were amplified and sequenced by Illumina Miseq and by PacBio Sequel II sequencers, respectively. RESULTS: With both platforms, a similar percentage of reads was assigned to the genus level (94.79% and 95.06% respectively) but with PacBio a higher proportion of reads were further assigned to the species level (55.23% vs 74.14%). Regarding overall bacterial composition, samples clustered by niche and not by sequencing platform. In addition, all genera with > 0.1% abundance were detected in both platforms for all types of samples. Although some genera such as Streptococcus tended to be observed at higher frequency in PacBio than in Illumina (20.14% vs 14.12% in saliva, 10.63% vs 6.59% in subgingival plaque biofilm samples) none of the differences were statistically significant when correcting for multiple testing. CONCLUSIONS: The results presented in the current manuscript suggest that samples sequenced using Illumina and PacBio are mostly comparable. Considering that PacBio reads were assigned at the species level with higher accuracy than Illumina, our data support the use of PacBio technology for future microbiome studies, although a higher cost is currently required to obtain an equivalent number of reads per sample.

Using digital PCR to predict ciliate abundance from ribosomal RNA gene copy numbers.

Ciliates play a key role in most ecosystems. Their abundance in natural samples is crucial for answering many ecological questions. Traditional methods of quantifying individual species, which rely on microscopy, are often labour-intensive, time-consuming and can be highly biassed. As a result, we investigated the potential of digital polymerase chain reaction (dPCR) for quantifying ciliates. A significant challenge in this process is the high variation in the copy number of the taxonomic marker gene (ribosomal RNA [rRNA]). We first quantified the rRNA gene copy numbers (GCN) of the model ciliate, Paramecium tetraurelia, during different stages of the cell cycle and growth phases. The per-cell rRNA GCN varied between approximately 11,000 and 130,000, averaging around 50,000 copies per cell. Despite these variations in per-cell rRNA GCN, we found a highly significant correlation between GCN and cell numbers. This is likely due to the coexistence of different cellular stages in an uncontrolled (environmental) ciliate population. Thanks to the high sensitivity of dPCR, we were able to detect the target gene in a sample that contained only a single cell. The dPCR approach presented here is a valuable addition to the molecular toolbox in protistan ecology. It may guide future studies in quantifying and monitoring the abundance of targeted (even rare) ciliates in natural samples.

Widespread genetic heterogeneity of human ribosomal RNA genes.

Polymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling and used high-coverage whole-genome sequencing (WGS) data from the 1000 Genomes Project to analyze variants in 2504 individuals from 26 populations. We identified a total of 3791 variant positions. The variants positioned nonrandomly on the rRNA gene. Invariant regions included the promoter, early 5' ETS, most of 18S, 5.8S, ITS1, and large areas of the intragenic spacer. A total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants were located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. Several variants were validated based on RNA-seq analyses. Population analyses showed remarkable ancestry-linked genetic variance and the presence of both high penetrance and frequent variants in the 5' ETS, ITS2, and 28S regions segregating according to the continental populations. These findings provide a genetic view of rRNA gene array heterogeneity and raise the need to functionally assess how the 28S rRNA variants affect ribosome functions.

Optimized bacterial community characterization through full-length 16S rRNA gene sequencing utilizing MinION nanopore technology.

BACKGROUND: Accurate identification of bacterial communities is crucial for research applications, diagnostics, and clinical interventions. Although 16S ribosomal RNA (rRNA) gene sequencing is a widely employed technique for bacterial taxonomic classification, it often results in misclassified or unclassified bacterial taxa. This study sought to refine the full-length 16S rRNA gene sequencing protocol using the MinION sequencer, focusing on the V1-V9 regions. Our methodological enquiry examined several factors, including the number of PCR amplification cycles, choice of primers and Taq polymerase, and specific sequence databases and workflows employed. We used a microbial standard comprising eight bacterial strains (five gram-positive and three gram-negative) in known proportions as a validation control. RESULTS: Based on the MinION protocol, we employed the microbial standard as the DNA template for the 16S rRNA gene amplicon sequencing procedure. Our analysis showed that an elevated number of PCR amplification cycles introduced PCR bias, and the selection of Taq polymerase and primer sets significantly affected the subsequent analysis. Bacterial identification at genus level demonstrated Pearson correlation coefficients ranging from 0.73 to 0.79 when assessed using BugSeq, Kraken-Silva and EPI2ME-16S workflows. Notably, the EPI2ME-16S workflow exhibited the highest Pearson correlation with the microbial standard, minimised misclassification, and increased alignment accuracy. At the species taxonomic level, the BugSeq workflow was superior, with a Pearson correlation coefficient of 0.92. CONCLUSIONS: These findings emphasise the importance of careful selection of PCR settings and a well-structured analytical framework for 16S rRNA full-length gene sequencing. The results showed a robust correlation between the predicted and observed bacterial abundances at both the genus and species taxonomic levels, making these findings applicable across diverse research contexts and with clinical utility for reliable pathogen identification.

Comparison of 16S rRNA gene sequencing microbiota among children with serological IgE-mediated food hypersensitivity.

BACKGROUND: We hypothesized that specific food hypersensitivity (FH) in children is linked to specific gut microbiota. The aim of our study was to quantify and evaluate differences in gut microbial composition among children with different IgE-mediated FH. METHODS: Children (n = 81) aged 18 to 36 months were enrolled, fecal samples of 57 children with FH and 24 healthy children were evaluated using next-generation sequencing. Individual microbial diversity and composition were analyzed via targeting the 16 S rRNA gene hypervariable V3-V5 regions. RESULTS: Children with IgE-mediated FH (in milk, egg white, soy) had significantly lower gut microbiota diversity and richness than healthy children. Children with IgE-mediated FH exhibited relatively high abundances of Firmicutes and relative underrepresentation of the phylum Bacteroidetes. We observed significant increases in relative abundances of Ruminococcaceae, Clostridiaceae, and Erysipelotrichaceae (p < 0.01, compared to control) in children with milk hypersensitivity and of Clostridiaceae and Erysipelotrichaceae (p < 0.01) in children with peanut hypersensitivity. We also found significant increases in the numbers of Clostridiaceae, Lachnospiraceae and Pasteurellaceae (p < 0.01) in children with egg white hypersensitivity. CONCLUSIONS: These findings identify early evidence of different gut microbiota development/ differentiation in children with food hypersensitivity. Specific food hypersensitivities may be associated with compositional changes in intestinal microbiota. IMPACT: These findings identify early evidence of different gut microbiota development/differentiation in children with food hypersensitivity. We built a gut microbial profile that could identify toddlers at risk for food hypersensitivity. Children with enriched Firmicutes (phylum) with partial different families may be associated with food hypersensitivity. Enriched family Clostridiaceae, Ruminococcaceae, Lachnospiraceae, or Erysipelotrichaceae in gut microbiota may be associated with specific food hypersensitivities (such as milk, egg white, peanut) in children.

Highly divergent morphology but a close molecular phylogenetic relationship between two little-known ciliate genera Actinobolina and Papillorhabdos (Protozoa: Ciliophora: Litostomatea) with description of two new species.

Free-living litostomatean ciliates, prominent microeukaryote predators commonly encountered in freshwater and marine habitats, play vital roles in maintaining energy flow and nutrient cycles. Nevertheless, understanding their biodiversity and phylogenetic relationships remains challenging due to insufficient morphological information and molecular data. As a new contribution to this group, three haptorian ciliates, including two new species (Actinobolina bivacuolata sp. nov. and Papillorhabdos foissneri sp. nov.) and the insufficiently described type species, Actinobolina radians, were isolated from wetlands around Lake Weishan, China and investigated by a combination of living morphology, stained preparations, and 18S rRNA gene sequence data. An illustrated key of the valid species within the two genera is provided. In addition, we reveal the phylogenetic positions of these two genera for the first time. Although they differ in all key morphologic characters such as general appearance (ellipsoidal with numerous tentacles vs. cylindrical), extrusomes (stored in tentacles vs. anchored to pellicle), circumoral kinety (present vs. absent), composition of somatic kineties (kinetosome clusters vs. monokinetids), and number of dorsal brush rows (1 vs. 4), they both cluster in a fully supported clade in the phylogenetic tree, which indicates that the biodiversity and additional molecular markers of this group need further exploration.

Investigation of the effect of oligo-xylulose on the intestinal flora under pressure overload in mice based on 16S rRNA gene sequencing.

The relationship between gut microbiota dysbiosis and heart failure has been drawing increasing attention. This study aimed to investigate the effects of oligo-xylulose (XOS) on the gut microbiota of mice with heart failure induced by pressure overload. A chronic heart failure mouse model was constructed by pressure overload, and XOS were administered in their diet. The gut microbiota was analyzed using 16S rRNA gene sequencing, and the effects of XOS on the microbiota composition were evaluated. . XOS supplementation improved the balance of intestinal microbiota in mice under pressure overload, increasing the abundance of beneficial bacteria, such as Bifidobacterium and Lactobacillus, while decreasing the abundance of harmful bacteria, such as Desulfovibrio and Enterococcus. XOS has potential as a dietary supplement to improve the balance of intestinal microbiota and benefit individuals with heart failure. The findings of this study suggest that modulating the gut microbiota could be a novel strategy for treating heart failure.

Hemimastigophora is a novel supra-kingdom-level lineage of eukaryotes.

Almost all eukaryote life forms have now been placed within one of five to eight supra-kingdom-level groups using molecular phylogenetics1-4. The 'phylum' Hemimastigophora is probably the most distinctive morphologically defined lineage that still awaits such a phylogenetic assignment. First observed in the nineteenth century, hemimastigotes are free-living predatory protists with two rows of flagella and a unique cell architecture5-7; to our knowledge, no molecular sequence data or cultures are currently available for this group. Here we report phylogenomic analyses based on high-coverage, cultivation-independent transcriptomics that place Hemimastigophora outside of all established eukaryote supergroups. They instead comprise an independent supra-kingdom-level lineage that most likely forms a sister clade to the 'Diaphoretickes' half of eukaryote diversity (that is, the 'stramenopiles, alveolates and Rhizaria' supergroup (Sar), Archaeplastida and Cryptista, as well as other major groups). The previous ranking of Hemimastigophora as a phylum understates the evolutionary distinctiveness of this group, which has considerable importance for investigations into the deep-level evolutionary history of eukaryotic life-ranging from understanding the origins of fundamental cell systems to placing the root of the tree. We have also established the first culture of a hemimastigote (Hemimastix kukwesjijk sp. nov.), which will facilitate future genomic and cell-biological investigations into eukaryote evolution and the last eukaryotic common ancestor.

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast.

The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell size in polyploid cells. In haploid mutant cells with larger cell sizes, the rDNA repeat copy number rises. By combining mathematical modeling and experimental work with the large-size cln3 strain, we observed that the increasing repeat copy number is based on a feedback mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of rDNA repeats in a volume-dependent manner. This amplification is paralleled with an increase in rRNA nTR, which indicates a control of the RNA pol I synthesis rate by cell volume.

Enhanced phylogenetic insights into the microbiome of chronic rhinosinusitis through the novel application of long read 16S rRNA gene amplicon sequencing.

INTRODUCTION: 16S rRNA next generation sequencing (NGS) has been the de facto standard of microbiome profiling. A limitation of this technology is the inability to accurately assign taxonomy to a species order. Long read 16S sequencing platforms, including Oxford Nanopore Technologies (ONT), have the potential to overcome this limitation. The paranasal sinuses are an ideal niche to apply this technology, being a low biomass environment where bacteria are implicated in disease propagation. Characterising the microbiome to a species order may offer new pathophysiological insights. METHODOLOGY: Cohort series comparing ONT and NGS biological conclusions. Swabs obtained endoscopically from the middle meatus of 61 CRSwNP patients underwent DNA extraction, amplification and dual sequencing (Illumina Miseq (NGS) and ONT GridION). Agreement, relative abundance, prevalence, and culture correlations were compared. RESULTS: Mean microbiome agreement between sequencers was 61.4%. Mean abundance correlations were strongest at a familial/genus order and declined at a species order where NGS lacked resolution. The most significant discrepancies applied to Corynebacterium and Cutibacterium, which were estimated in lower abundance by ONT. ONT accurately identified 84.2% of cultured species, which was significantly higher than NGS. CONCLUSIONS: ONT demonstrated superior resolution and culture correlations to NGS, but underestimated core sinonasal taxa. Future application and optimisation of this technology can advance our understanding of the sinonasal microenvironment.

Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis.

In eukaryotes, scores of excess ribosomal RNA (rRNA) genes are silenced by repressive chromatin modifications. Given the near sequence identity of rRNA genes within a species, it is unclear how specific rRNA genes are reproducibly chosen for silencing. Using Arabidopsis thaliana ecotype (strain) Col-0, a systematic search identified sequence polymorphisms that differ between active and developmentally silenced rRNA gene subtypes. Recombinant inbred mapping populations derived from three different ecotype crosses were then used to map the chromosomal locations of silenced and active RNA gene subtypes. Importantly, silenced and active rRNA gene subtypes are not intermingled. All silenced rRNA gene subtypes mapped to the nucleolus organizer region (NOR) on chromosome 2 (NOR2). All active rRNA gene subtypes mapped to NOR4. Using an engineered A. thaliana line in which a portion of Col-0 chromosome 4 was replaced by sequences of another ecotype, we show that a major rRNA gene subtype silenced at NOR2 is active when introgressed into the genome at NOR4. Collectively, these results reveal that selective rRNA gene silencing is not regulated gene by gene based on mechanisms dependent on subtle gene sequence variation. Instead, we propose that a subchromosomal silencing mechanism operates on a multimegabase scale to inactivate NOR2.

Fine-Tuning of DADA2 Parameters for Multiregional Metabarcoding Analysis of 16S rRNA Genes from Activated Sludge and Comparison of Taxonomy Classification Power and Taxonomy Databases.

Taxonomic classification using metabarcoding is a commonly used method in microbiological studies of environmental samples and during monitoring of biotechnological processes. However, it is difficult to compare results from different laboratories, due to the variety of bioinformatics tools that have been developed and used for data analysis. This problem is compounded by different choices regarding which variable region of the 16S rRNA gene and which database is used for taxonomic identification. Therefore, this study employed the DADA2 algorithm to optimize the preprocessing of raw data obtained from the sequencing of activated sludge samples, using simultaneous analysis of three frequently used regions of 16S rRNA (V1-V3, V3-V4, V4-V5). Additionally, the study evaluated which variable region and which of the frequently used microbial databases for taxonomic classification (Greengenes2, Silva, RefSeq) more accurately classify OTUs into taxa. Adjusting the values of selected parameters of the DADA2 algorithm, we obtained the highest possible numbers of OTUs for each region. Regarding biodiversity within regions, the V3-V4 region had the highest Simpson and Shannon indexes, and the Chao1 index was similar to that of the V1-V3 region. Beta-biodiversity analysis revealed statistically significant differences between regions. When comparing databases for each of the regions studied, the highest numbers of taxonomic groups were obtained using the SILVA database. These results suggest that standardization of metabarcoding of short amplicons may be possible.

Improved DNA Extraction and Amplification Strategy for 16S rRNA Gene Amplicon-Based Microbiome Studies.

Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent ('Rapid' protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel 'Rapid' method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the 'Rapid' protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel 'Rapid' DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel 'Rapid' DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing.