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.
•A full-length transcriptome of Urechis unicinctus was generated by using SMRT Sequencing technology.•A total of 59,371 unigenes with a 2,779 bp average length were obtained.•2,613 lncRNAs, 59,190 ...CDSs, 35,166 SSRs, and 1,733 TFs were identified in the transcriptome.•211 PRRs were identified, including TLRs, CLTs, SRs, PGRPs, and GNBPs.•Numerous transcript variations of immune molecules provide new insights into the innate immune system of Urechis unicinctus.
The Echiura worm Urechis unicinctus refers to a common benthic invertebrate found in the intertidal zone of Huanghai as well as Bohai Bay. U. unicinctus is known to contain various physiologically active substances, making it highly valuable in terms of its edibility, medicinal properties, and economic potential. Nonetheless, the limited study on the immune system of U. unicinctus poses difficulties for its aquaculture and artificial reproduction. Marine invertebrates, including shellfish and U. unicinctus, are thought to primarily depend on their innate immune system for disease protection, owing to the severalinnate immune molecules they possess. Herein, we employed PacBio single-molecule real-time (SMRT) sequencing technology to perform the full-length transcriptome analysis of U. unicinctus individuals under five different conditions (room temperature (RT), low temperature (LT), high temperature (HT), without water (DRY), ultraviolet irradiation (UV)). Concequently, we identified 59,371 unigenes that had a 2,779 bp average length, 2,613 long non-coding RNAs (lncRNAs), 59,190 coding sequences (CDSs), 35,166 simple sequence repeats (SSRs), and 1,733 transcription factors (TFs), successfully annotating 90.58 % (53,778) of the unigenes. Subsequently, key factors associated with immune-related processes, such as non-self-recognition, cellular immune defenses, and humoral immune defenses, were searched. Our study also identified pattern recognition receptors (PRRs) that included 17 peptidoglycan recognition proteins (PGRPs), 13 Gram-negative binding proteins (GNBPs), 18 scavenger receptors (SRs), 74 toll-like receptors (TLRs), and 89 C-type lectins (CLTs). Altogether, the high-quality transcriptome obtained data will offer valuable insights for further investigations into U. unicinctus innate immune response, laying the foundation for subsequent molecular biology studies and aquaculture.
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
GenomeBiology.com,
01/2013, Volume:
14, Issue:
7
Journal Article
Peer reviewed
Open access
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.
Over 160 RNA modifications have been identified, including N7-methylguanine (m7G), N6-methyladenosine (m6A), and 5-methylcytosine (m5C). These modifications play key roles in regulating the fate of ...RNA. In eukaryotes, m6A is the most abundant mRNA modification, accounting for over 80% of all RNA methylation modifications. Highly dynamic m6A modification may exert important effects on organismal reproduction and development. Significant advances in understanding the mechanism of m6A modification have been made using immunoprecipitation, chemical labeling, and site-directed mutagenesis, combined with next-generation sequencing. Single-molecule real-time and nanopore direct RNA sequencing (DRS) approaches provide additional ways to study RNA modifications at the cellular level. In this review, we explore the technical history of identifying m6A RNA modifications, emphasizing technological advances in detecting m6A modification. In particular, we discuss the challenge of generating accurate dynamic single-base resolution m6A maps and also strategies for improving detection specificity. Finally, we outline a roadmap for future research in this area, focusing on the application of RNA epigenetic modification, represented by m6A modification.
SUMMARY
Australian pine (Casuarina spp.) is extensively planted in tropical and subtropical regions for wood production, shelterbelts, environmental protection, and ecological restoration due to ...their superior biological characteristics, such as rapid growth, wind and salt tolerance, and nitrogen fixation. To analyze the genomic diversity of Casuarina, we sequenced the genomes and constructed de novo genome assemblies of the three most widely planted Casuarina species: C. equisetifolia, C. glauca, and C. cunninghamiana. We generated chromosome‐scale genome sequences using both Pacific Biosciences (PacBio) Sequel sequencing and chromosome conformation capture technology (Hi‐C). The total genome sizes for C. equisetifolia, C. glauca, and C. cunninghamiana are 268 942 579 bp, 296 631 783 bp, and 293 483 606 bp, respectively, of which 25.91, 27.15, and 27.74% were annotated as repetitive sequences. We annotated 23 162, 24 673, and 24 674 protein‐coding genes in C. equisetifolia, C. glauca, and C. cunninghamiana, respectively. We then collected branchlets from male and female individuals for whole‐genome bisulfite sequencing (BS‐seq) to explore the epigenetic regulation of sex determination in these three species. Transcriptome sequencing (RNA‐seq) revealed differential expression of phytohormone‐related genes between male and female plants. In summary, we generated three chromosome‐level genome assemblies and comprehensive DNA methylation and transcriptome datasets from both male and female material for three Casuarina species, providing a basis for the comprehensive investigation of genomic diversity and functional gene discovery of Casuarina in the future.
Significance Statement
In this study we generated three chromosome‐level genome assemblies and comprehensive DNA methylation and transcriptome datasets from both male and female material for three Casuarina species, providing a basis for the comprehensive investigation of genomic diversity and functional gene discovery of Casuarina in the future.
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