The human microbiome includes trillions of bacteria, many of which play a vital role in host physiology. Numerous studies have now detected bacterial DNA in first-pass meconium and amniotic fluid ...samples, suggesting that the human microbiome may commence
. However, these data have remained contentious due to underlying contamination issues. Here, we have used a previously described method for reducing contamination in microbiome workflows to determine if there is a fetal bacterial microbiome beyond the level of background contamination. We recruited 50 women undergoing non-emergency cesarean section deliveries with no evidence of intra-uterine infection and collected first-pass meconium and amniotic fluid samples. Full-length 16S rRNA gene sequencing was performed using PacBio SMRT cell technology, to allow high resolution profiling of the fetal gut and amniotic fluid bacterial microbiomes. Levels of inflammatory cytokines were measured in amniotic fluid, and levels of immunomodulatory short chain fatty acids (SCFAs) were quantified in meconium. All meconium samples and most amniotic fluid samples (36/43) contained bacterial DNA. The meconium microbiome was dominated by reads that mapped to
. Aside from this species, the meconium microbiome was remarkably heterogeneous between patients. The amniotic fluid microbiome was more diverse and contained mainly reads that mapped to typical skin commensals, including
and
spp. All meconium samples contained acetate and propionate, at ratios similar to those previously reported in infants.
reads were inversely correlated with meconium propionate levels. Amniotic fluid cytokine levels were associated with the amniotic fluid microbiome. Our results demonstrate that bacterial DNA and SCFAs are present
, and have the potential to influence the developing fetal immune system.
Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich ...repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes.
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•Species-wide NLR diversity is high but not unlimited•A large fraction of NLR diversity is recovered with 40–50 accessions•Presence/absence variation in NLRs is widespread, resulting in a mosaic population•A high diversity of NLR-integrated domains favor known virulence targets
In plants, NLR proteins are important intracellular receptors with roles in innate immunity and disease resistance. This work provides a panoramic view of this diverse and complicated gene family in the model species A. thaliana and provides a foundation for the identification and functional study of disease-resistance genes in agronomically important species with complex genomes.
•This is the first report on metabolic gene clusters in paper mulberry silage.•Ensiling causes a shift in dominant bacteria from Gram-negative to Gram-positive.•Enterobacter and Lactobacillus species ...determine silage fermentation quality.•PacBio sequencing reveals microbial dynamics during silage fermentation.•Paper mulberry can be used to prepare high-quality nutrient-rich silage.
To develop a new high-protein woody forage resource for livestock to alleviate feed shortages in the tropics, we applied PacBio single-molecule, real-time (SMRT) sequencing to explore the community structure, species diversity and metabolic gene clusters of natural microorganisms associated with paper mulberry (PM) silage fermentation. High levels of microbial diversity and abundance were observed in PM raw material, and these levels decreased with the progression of silage fermentation. During woody ensiling, the dominant bacteria shifted from pathogenic Gram-negative Proteobacteria to beneficial Gram-positive Firmicutes. Lactic acid bacteria became the most dominant bacteria that affected fermentation quality in terminal silages. Global and overview maps, carbohydrate metabolism and amino-acid metabolism were the important microbial metabolic pathways that impacted the final fermentation product of silage. PacBio SMRT sequencing revealed specific microbial-related information concerning silage. PM is rich in nutrients and macro mineral contents, which are preserved well during ensiling, indicating that PM silage can serve as a new woody resource suitable for ruminants.
Histone deacetylases (HDACs) are key epigenetic regulators, and transcriptional complexes with deacetylase function are among the epigenetic corepressor complexes in the nucleus that target the ...epigenome. HDAC-bearing corepressor complexes such as the Sin3 complex, NuRD complex, CoREST complex, and SMRT/NCoR complex are common in biological systems. These complexes activate the otherwise inactive HDACs in a solitary state. HDAC complexes play vital roles in the regulation of key biological processes such as transcription, replication, and DNA repair. Moreover, deregulated HDAC complex function is implicated in human diseases including cancer. Therapeutic strategies targeting HDAC complexes are being sought actively. Thus, illustration of the nature and composition of HDAC complexes is vital to understanding the molecular basis of their functions under physiologic and pathologic conditions, and for designing targeted therapies. This review presents key aspects of large multiprotein HDAC-bearing complexes including their structure, function, regulatory mechanisms, implication in disease development, and role in therapeutics.
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Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon ...sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system.
By examining templates from more than 5000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL can reduce greatly reduce sequencing costs in comparison to first (Sanger) and second generation platforms (Illumina, Ion).
SMRT analysis generates high-fidelity sequences from amplicons with varying GC content and is resilient to homopolymer tracts. Analytical costs are low, substantially less than those for first or second generation sequencers. When implemented on the SEQUEL platform, SMRT analysis enables massive amplicon characterization because each instrument can recover sequences from more than 5 million DNA extracts a year.
The advantages of SMRT sequencing Roberts, Richard J; Carneiro, Mauricio O; Schatz, Michael C
Genome biology,
01/2013, Letnik:
14, Številka:
7
Journal Article
Recenzirano
Odprti dostop
Of the current next-generation sequencing technologies, SMRT sequencing is sometimes overlooked. However, attributes such as long reads, modified base detection and high accuracy make SMRT a useful ...technology and an ideal approach to the complete sequencing of small genomes.
To determine the phase of NUDT15 sequence variants for more comprehensive star (*) allele diplotyping, we developed a novel long‐read single‐molecule real‐time HiFi amplicon sequencing method. A 10.5 ...kb NUDT15 amplicon assay was validated using reference material positive controls and additional samples for specimen type and blinded accuracy assessment. Triplicate NUDT15 HiFi sequencing of two reference material samples had nonreference genotype concordances of >99.9%, indicating that the assay is robust. Notably, short‐read genome sequencing of a subset of samples was unable to determine the phase of star (*) allele‐defining NUDT15 variants, resulting in ambiguous diplotype results. In contrast, long‐read HiFi sequencing phased all variants across the NUDT15 amplicons, including a *2/*9 diplotype that previously was characterized as *1/*2 in the 1000 Genomes Project v3 data set. Assay throughput was also tested using 8.5 kb amplicons from 100 Ashkenazi Jewish individuals, which identified a novel NUDT15 *1 suballele (c.−121G>A) and a rare likely deleterious coding variant (p.Pro129Arg). Both novel alleles were Sanger confirmed and assigned as *1.007 and *20, respectively, by the PharmVar Consortium. Taken together, NUDT15 HiFi amplicon sequencing is an innovative method for phased full‐gene characterization and novel allele discovery, which could improve NUDT15 pharmacogenomic testing and subsequent phenotype prediction.
Sichuan paocai, a traditional Chinese fermented vegetable, is rife with lactic acid bacteria (LAB). However, the precise bacterial profiles of home-made Sichuan paocai brine (HSPB) remain unclear. In ...this study, the bacterial compositions of 38 aged HSPB samples with varying titratable acidity (TA) were determined by SMRT sequencing of the full-length 16S rRNA gene. The lactic and acetic acids of HSPBs were also measured to determine any relevance with the bacterial profiles. The SMRT sequencing results reveal that the HSPB bacterial communities were comprised of numerous phylogenetic taxa, including 35 phyla, 371 genera, and 593 species; the bacterial diversity decreased as HSPB acidity increased. Lactobacillus acetotolerans, which was positively correlated to HSPB acidity, was the most dominant species followed by Lactobacillus brevis, which was positively related to acetic acid in the samples. A few opportunistic pathogens (e.g. Serratia marcescens and Stenotrophomonas maltophilia) were also detected. Sample groups with lower acidity had higher bacterial diversity and more Lactobacillus species with relative abundance >1% and opportunistics than higher-acidity samples. The results presented here report the comprehensive bacterial profiles of home-made Sichuan paocai for the first time via SMRT sequencing technology and the correlation between TA and bacterial compositions. It is necessary to further investigate the opportunistics detected in this work as they relate to the safety and quality of paocai.
•Bacterial profiles in Sichuan paocai were revealed by SMRT sequencing technology.•Lactobacillus acetotolerans was the most dominant species.•New species and some opportunistic pathogens were also detected.•Bacterial diversity decreased as the acidity of home-made Sichuan paocai increased.•Low acidity group had more Lactobacilli and opportunistic pathogens.
DNA methylation serves as the primary mode of epigenetic regulation in prokaryotes, particularly through transcriptional regulation. With the rapid implementation of third‐generation sequencing ...technology, we are currently experiencing a golden age of bacterial epigenomics. However, there has been a lack of comprehensive research exploring the versatility and consequential impact of bacterial DNA methylome on cellular and physiological functions. There is a critical need for a user‐friendly bioinformatics tool that can effectively characterize DNA methylation modification features and predict the regulation patterns. To address this gap, the current study introduces Bacmethy, an innovative tool that utilizes SMRT‐seq data and offers a range of analytical modules. First, the tool classifies methylation sites in the genome, highlighting the distinct regulations present under varying modification fractions and location enrichment. Furthermore, this tool enables us to identify regulatory region methylation and potential cis and trans interactions between methylation sites and regulatory effectors. Using benchmark data sets and our data, we show that our tool facilitates the understanding of the distinctive traits of DNA methylation modifications and predicts transcriptional regulation effects on important physiological and pathological functions. Bacmethy code is freely available, and the Docker image is downloadable. Bacmethy has been made available as a user‐friendly web server interface at https://bacmethy.med.sustech.edu.cn.
Bacmethy tool provides a one‐stop analysis and visualization pipeline for effectively characterizing bacterial DNA methylation modification features and predicting the regulation patterns. Bacmethy offers both a local run function and an online interface analysis service, providing significant convenience for researchers without coding abilities. Bacmethy provides useful information for decoding the underlying molecular mechanisms of how DNA methylation regulates bacterial cellular and physiological functions.
Highlights
Bacmethy tool provides a one‐stop analysis and visualization pipeline for effectively characterizing bacterial DNA methylation modification features and predicting the regulation patterns.
Bacmethy offers both a local run function and an online interface analysis service, providing significant convenience for researchers without coding abilities.
Bacmethy provides useful information for decoding the underlying molecular mechanisms of how DNA methylation regulates bacterial cellular and physiological functions.
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