Post-replicative DNA methylation is essential for diverse biological processes in both eukaryotes and prokaryotes. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, ...remains one of the most formidable threats worldwide. Although DNA methylation of M. tuberculosis has been documented, little information is available for clinical drug-resistant M. tuberculosis. Single-molecule real-time (SMRT) sequencing was used to profile the core methylome of three clinical isolates, namely multidrug-resistant (MDR), extensively drug-resistant (XDR) and extremely drug-resistant (XXDR) strains. 3812, 6808 and 6041 DNA methylated sites were identified in MDR-MTB, XDR-MTB and XXDR-MTB genome, respectively. There are two types of methylated motifs, namely N
6
-methyladenine (m6A) and N
4
-methylcytosine (m4C). A novel widespread 6 mA methylation motif 5′-CACGCAG-3′ was found in XDR-MTB and XXDR-MTB. The methylated genes are involved in multiple cellular processes, especially metabolic enzymes engaged in glucose metabolism, fatty acid and TCA cycle. Many methylated genes are involved in mycobacterial virulence, antibiotic resistance and tolerance. This provided a comprehensive list of methylated genes in drug-resistant clinical isolates and the basis for further functional elucidation.
The modular Snow Microwave Radiative Transfer (SMRT) model simulates microwave scattering behavior in snow via different selectable theories and snow microstructure representations, which is well ...suited to intercomparisons analyses. Here, five microstructure models were parameterized from X-ray tomography and thin-section images of snow samples and evaluated with SMRT. Three field experiments provided observations of scattering and absorption coefficients, brightness temperature, and/or backscatter with the increasing complexity of snowpack. These took place in Sodankylä, Finland, and Weissfluhjoch, Switzerland. Simulations of scattering and absorption coefficients agreed well with observations, with higher errors for snow with predominantly vertical structures. For simulation of brightness temperature, difficulty in retrieving stickiness with the Sticky Hard Sphere microstructure model resulted in relatively poor performance for two experiments, but good agreement for the third. Exponential microstructure gave generally good results, near to the best performing models for two field experiments. The Independent Sphere model gave intermediate results. New Teubner-Strey and Gaussian Random Field models demonstrated the advantages of SMRT over microwave models with restricted microstructural geometry. Relative model performance is assessed by the quality of the microstructure model fit to micro-computed tomography (CT) data and further improvements may be possible with different fitting techniques. Careful consideration of simulation stratigraphy is required in this new era of high-resolution microstructure measurement as layers thinner than the wavelength introduce artificial scattering boundaries not seen by the instrument.
This study examined the storage of smoked bacon. Gas chromatography-mass spectrometry (GC–MS) and single-molecule real-time DNA sequencing technology (SMRT) were used to quantitate volatile ...constituents and microbial communities. The results indicated that the quality of bacon and its volatile constituents at room temperature are higher than those stored at 4 °C (P < 0.05). The bacterial abundance of smoked bacon in the H group that were hung from a beam at room temperature was higher than those of the L group, which were packaged under vacuum conditions and stored at 4 °C. The fungal diversity was lower in the H group than in the L group. Staphylococcus equorum was the most prominent bacterial species in the two groups, while both the fungal species Candida zeylanoides and Debaryomyces prosopidis were in all groups of smoked bacon. Pearson correlation coefficients indicated that Staphylococcus equorum positively correlated with heptanal in both groups. Lactobacillus sakei was positively (P < 0.01) related to heptanal in refrigeration, and Debaryomyces prosopidis positively correlated with hexanal in both groups. This study should help to improve the quality of meat by mediating microbial communities in Sichuan smoked bacon.
•Volatile constituents of smoked bacon at 25 °C were significantly higher than those at 4 °C.•Analysis of microbial diversity in Sichuan bacon using single-molecule real-time DNA sequencing.•Storage at room temperature facilitated maintaining the diversity of the microbiota.•Pearson correlation coefficient analysis was performed on the microbiology and quality of bacon.•Microbial diversity and quality of the bacon were significantly affected by different storage methods.
The mite
is distributed worldwide and parasitism the ear canals of cats and dogs, causing otitis externa. Molecular biology of
is poorly understood, with only a few genes being deposited in public ...databases. In the present study, we aimed to perform transcriptome analysis of
using SMRT and Illumina sequencing of RNA from different development stages. SMRT-Seq of
demonstrated 5,431 final transcripts, including 406 long non-coding RNAs and 2,698 differentially expressed genes (DEGs), including 1,357 up-regulated genes and 1,341 down-regulated genes between adult mites and nymph/larva. A total of 397 putative allergen genes were detected, 231 of which were DEGs. Among them, 77 were homologous of known mite allergens. The expression level of allergen genes hints at the pathogenicity of mites in different life stages, and the protein interaction network analysis could identify possible key genes in the pathogenic mechanism. Intriguingly, Gene Ontology analysis showed that most of the (DEGs) were associated with the terms hydrolase activity and proteolysis. Kyoto Encyclopedia of genes and genomes (KEGG) analysis identified drug metabolism-cytochrome P450 signal pathway as one of the top pathways. SMRT-Seq of the full-length transcriptome of
was performed first, and a valuable resource was acquired through the combination analysis with the Illumina sequencing data. The results of our analyses provide new information for further research into
.
Rett syndrome (RTT) is an X-linked neurological disorder caused by mutations in the methyl-CpG–binding protein 2 (MeCP2) gene. The majority of RTT missense mutations disrupt the interaction of the ...MeCP2 with DNA or the nuclear receptor corepressor (NCoR)/silencing mediator of retinoic acid and thyroid receptors (SMRT) corepressor complex. Here, we show that the “NCoR/SMRT interaction domain” (NID) of MeCP2 directly contacts transducin beta-like 1 (TBL1) and TBL1 related (TBLR1), two paralogs that are core components of NCoR/SMRT. We determine the cocrystal structure of the MeCP2 NID in complex with the WD40 domain of TBLR1 and confirm by in vitro and ex vivo assays that mutation of interacting residues of TBLR1 and TBL1 disrupts binding to MeCP2. Strikingly, the four MeCP2-NID residues mutated in RTT are those residues that make the most extensive contacts with TBLR1. Moreover, missense mutations in the gene for TBLR1 that are associated with intellectual disability also prevent MeCP2 binding. Our study therefore reveals the molecular basis of an interaction that is crucial for optimal brain function.
A circular consensus sequencing (CCS) strategy involving single molecule, real‐time (SMRT) DNA sequencing technology was applied to de novo assembly and single nucleotide polymorphism (SNP) detection ...of chloroplast genomes. Chloroplast DNA was purified from enriched chloroplasts of pooled individuals to construct a shotgun library for each species. The sequencing reactions were performed on a PacBio RS platform. CCS sub‐reads were generated from polymerase reads that passed the native dumbbell‐shaped DNA templates multiple times. The complete chloroplast genome sequence was generated by mapping all reads to the draft sequence constructed in a step‐by‐step manner. The full‐chain, PCR‐free approach eliminates the possible context‐specific biases in library construction and sequencing reaction. The chloroplast genome was easily and completely assembled using the data generated from one SMRT Cell without requiring a reference genome. Comparisons of the three assembled Fritillaria genomes to 34.1 kb of validation Sanger sequences revealed 100% concordance, and the detected intraspecies SNPs at a minimum variant frequency of 15% were all confirmed. This simple approach with potential for parallel sequencing yields high‐quality chloroplast genomes for sensitive SNP detection and comparative analyses. We recommend this approach for its powerful applicability for evolutionary genetics and genomics studies in plants based on the sequences of chloroplast genomes.
The underlying mechanism of sex differentiation and development remains mysterious concerning the economic crustaceans, hindering the development of mono-sex breeding technology of those species. ...Gene discovery by next generation sequencing (NGS) has been widely applied in those species without reference genome. Nevertheless, the pivotal molecular differences driving sex dimorphisms before gonad differentiation are largely unknown. Besides, accurate assembly and downstream analysis remain a challenge due to the short-read nature of NGS. Here, we reported a comprehensive long read transcriptome by single-molecule real-time (SMRT) sequencing in an economic crustacean species, mud crab (Scylla paramamosain). Combined with NGS, differentially expressed genes (DEGs) were further analyzed and validated between sexes with different gonadal differentiation status. It was demonstrated that the quality of the long-read transcriptome (N50 of unigenes, annotation rate, species-hit accuracy, etc.) was significantly higher than previously assembled transcriptomes. Furthermore, it was founded that narrow transcriptomic differences existed between sexes before gonad differentiation in the mud crab. Estradiol 17-beta-dehydrogenase 8 (17β-HSD8) gene involved in steroid biosynthesis was significantly up-regulated in genetic male juvenile crab, suggesting estradiol-17β (E2) may promote female differentiation in the mud crab. Moreover, novel enriched pathways including Hippo signaling pathway were firstly identified to be involved in gonadal development in crustaceans. Our data will not only provide valuable genetic resource for further diverse genetic research, but also help to understand the mechanism underlying sex differentiation and gonadal development of this species.
•DEGs analysis combining long read transcriptome and next generation sequencing was reported in crustaceans firstly;•Novel DEGs between sexes at first crablet stage (C1) before gonadal differentiation were identified in the mud crab;•17β-HSD8 was expressed with sexual difference at C1 stage, suggesting E2 may promote female differentiation in mud crab;•KEGG pathway enrichment and FISH analysis revealed that Hippo pathway may play important role in gonadal development.
Second-generation, high-throughput sequencing methods have greatly improved our understanding of the ecology of soil microorganisms, yet the short barcodes (< 500 bp) provide limited taxonomic and ...phylogenetic information for species discrimination and taxonomic assignment.
Here, we utilized the third-generation Pacific Biosciences (PacBio) RSII and Sequel instruments to evaluate the suitability of full-length internal transcribed spacer (ITS) barcodes and longer rRNA gene amplicons for metabarcoding Fungi, Oomycetes and other eukaryotes in soil samples.
Metabarcoding revealed multiple errors and biases: Taq polymerase substitution errors and mis-incorporating indels in sequencing homopolymers constitute major errors; sequence length biases occur during PCR, library preparation, loading to the sequencing instrument and quality filtering; primer–template mismatches bias the taxonomic profile when using regular and highly degenerate primers.
The RSII and Sequel platforms enable the sequencing of amplicons up to 3000 bp, but the sequence quality remains slightly inferior to Illumina sequencing especially in longer amplicons. The full ITS barcode and flanking rRNA small subunit gene greatly improve taxonomic identification at the species and phylum levels, respectively. We conclude that PacBio sequencing provides a viable alternative for metabarcoding of organisms that are of relatively low diversity, require > 500-bp barcode for reliable identification or when phylogenetic approaches are intended.
•In this study, we utilized the publicly available RNA-seq data generated from Illumina-based short-reads and PacBio single-molecule real-time (SMRT) long-reads to assess the zebra finch brain ...transcriptome.•To analyze the high-throughput RNA-seq data, we adopted a hybrid bioinformatic approach combining short and long-read pipelines.•We added 220 novel genes and 8134 transcript variants to the Ensembl annotation, and predicted a new proteome based on the refined annotation.•Using mass-spectrometry of zebra finch caudal telencephalon, we validated 18 different novel proteins.•Our results provide additional resources for future studies of zebra finches utilizing this improved bird genome annotation and proteome.
The zebra finch (Taeniopygia guttata), a representative oscine songbird species, has been widely studied to investigate behavioral neuroscience, most notably the neurobiological basis of vocal learning, a rare trait shared in only a few animal groups including humans. In 2019, an updated zebra finch genome annotation (bTaeGut1_v1.p) was released from the Ensembl database and is substantially more comprehensive than the first version published in 2010. In this study, we utilized the publicly available RNA-seq data generated from Illumina-based short-reads and PacBio single-molecule real-time (SMRT) long-reads to assess the bird transcriptome. To analyze the high-throughput RNA-seq data, we adopted a hybrid bioinformatic approach combining short and long-read pipelines. From our analysis, we added 220 novel genes and 8,134 transcript variants to the Ensembl annotation, and predicted a new proteome based on the refined annotation. We further validated 18 different novel proteins by using mass-spectrometry data generated from zebra finch caudal telencephalon tissue. Our results provide additional resources for future studies of zebra finches utilizing this improved bird genome annotation and proteome.
Tripsacum dactyloides (2n = 4x = 72) and Zea perennis (2n = 4x = 40) are tertiary gene pools of Zea mays L. and exhibit many abiotic adaptations absent in modern maize, especially salt tolerance. A ...previously reported allopolyploid (hereafter referred to as MTP, 2n = 74) synthesized using Zea mays, Tripsacum dactyloides, and Zea perennis has even stronger salt tolerance than Z. perennis and T. dactyloides. This allopolyploid will be a powerful genetic bridge for the genetic improvement of maize. However, the molecular mechanisms underlying its salt tolerance, as well as the key genes involved in regulating its salt tolerance, remain unclear.
Single-molecule real-time sequencing and RNA sequencing were used to identify the genes involved in salt tolerance and reveal the underlying molecular mechanisms. Based on the SMRT-seq results, we obtained 227,375 reference unigenes with an average length of 2300 bp; most of the unigenes were annotated to Z. mays sequences (76.5%) in the NR database. Moreover, a total of 484 and 1053 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Functional enrichment analysis of DEGs revealed that multiple pathways responded to salt stress, including "Flavonoid biosynthesis," "Oxidoreductase activity," and "Plant hormone signal transduction" in the leaves and roots, and "Iron ion binding," "Acetyl-CoA carboxylase activity," and "Serine-type carboxypeptidase activity" in the roots. Transcription factors, such as those in the WRKY, B3-ARF, and bHLH families, and cytokinin negatively regulators negatively regulated the salt stress response. According to the results of the short time series-expression miner analysis, proteins involved in "Spliceosome" and "MAPK signal pathway" dynamically responded to salt stress as salinity changed. Protein-protein interaction analysis revealed that heat shock proteins play a role in the large interaction network regulating salt tolerance.
Our results reveal the molecular mechanism underlying the regulation of MTP in the response to salt stress and abundant salt-tolerance-related unigenes. These findings will aid the retrieval of lost alleles in modern maize and provide a new approach for using T. dactyloides and Z. perennis to improve maize.
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