Genome Sequence of Halobacterium sp. Strain BOL4-2, Isolated and Cultured from Salar de Uyuni, Bolivia

Halobacterium sp. strain BOL4-2 was isolated from an Andean salt flat, Salar de Uyuni, in Bolivia. Single-molecule real-time (SMRT) sequencing revealed a 2.4-Mbp genome with a 2.0-Mbp chromosome and four plasmids (2 to 299 kb). Its isolation from an environment experiencing multiple extremes makes the strain interesting for astrobiology.

Integration of the Salmonella Typhimurium methylome and transcriptome following environmental or metabolic perturbation reveals DNA methylation and transcriptional regulation are largely decoupled

Despite being in a golden age of prokaryotic epigenomics, little work has systematically examined the plasticity and functional impacts of the bacterial DNA methylome. Here, we leveraged SMRT sequencing to examine the m6A DNA methylome of two Salmonella enterica ser. Typhimurium strains: 14028s and a ∆metJ mutant with derepressed methionine metabolism, grown in Luria Broth or a media that simulates the intracellular environment. We find that the methylome is remarkably static-over 95% of adenosine bases retain their methylation status across conditions. Integration of methylation with transcriptomic data revealed no correlation between methylation and gene expression. Further, examining the transcriptome in ∆yhdJ bacteria, lacking the m6A methylase with the most dynamic methylation pattern in our dataset, revealed little evidence of YhdJ-mediated gene regulation. Curiously, despite G(m6A)TC motifs being particularly resistant to change across conditions, we found that the Dam methylase is required for the ∆metJ motility defect. This ∆;metJ motility defect may be partially driven by hypermethylation of the chemotaxis gene tsr. Together, these data redefine the S. Typhimurium epigenome as a highly stable system that has rare, but important, roles in transcriptional regulation. Incorporating these lessons into future studies will be critical as we progress through the epigenomic era.

SMRT Sequencing of the Full-Length Transcriptome of the Coelomactra antiquata

Coelomactra antiquata is an important aquatic economic shellfish with high medicinal value. However, because C. antiquata has no reference genome, a lot of molecular biology research cannot be carried out, so the analysis of its transcripts is an important step to study the regulatory genes of various substances in C. antiquata. In the present study, we conducted the first full-length transcriptome analysis of C. antiquata by using PacBio single-molecule real-time (SMRT) sequencing technology. The results identified a total of 39,209 unigenes with an average length of 2,732 bp, 23,338 CDSs, 251 AS events, 9,881 lncRNAs, 20,106 SSRs, and 2,316 TFs. Subsequently, 59.22% (23,220) of the unigenes were successfully annotated, of which 23,164, 18,711, 15,840, 13,534, and 13,474 unigenes could be annotated using NR, Swiss-prot, KOG, GO, and KEGG databases, respectively. This study lays the foundation for the follow-up research of molecular biology and provides a reference for studying the more medicinal value of C. antiquata.

Tandem repeats structure of gel-forming mucin domains could be revealed by SMRT sequencing data

Mucins are large glycoproteins that cover and protect epithelial surface of the body. Gel-forming mucin domains of mucin genes are rich in proline, threonine, and serine that are heavily glycosylate. These domains show great complexity with tandem repeats (TRs), thus make it difficult to study the sequences. With the coming of single molecule real-time (SMRT) sequencing technologies, we manage to present sequence structure of mucin domains via SMRT long reads for gel-forming mucins MUC2, MUC5AC, MUC5B and MUC6. Our study shows that for different individuals, single nucleotide polymorphisms (SNPs) could be found in mucin domains of MUC2, MUC5AC, MUC5B and MUC6, while different number of tandem repeats could be found in mucin domains of MUC2 and MUC6. Furthermore, we get the sequence of MUC2, MUC5AC, and MUC5B mucin domain in a Chinese individual at accuracy of possibly maximum 99.98%, 99.93%, and 99.76%, respectively. We report a new method to obtain DNA sequence of gel-forming mucin domains. This method will provided new insights on getting the sequence for Tandem Repeat parts which locate in coding region. With the sequences we obtained with this method, we can give more information for people to study the sequences of gel-forming mucin domains.

Genome Resource of a Hypervirulent Strain C9-3 of Xanthomonas oryzae pv. oryzae Causing Bacterial Blight of Rice

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight, one of the most devastating diseases of rice. Here, a hypervirulent strain, C9-3, defeating Xa1、Xa10、xa13 and Xa23 resistance genes, was used to extract genomic DNA for single molecule real-time (SMRT) sequencing. After assembly, the genome consists of a single-circular chromosome with the size of 4,924,298 bp with G+C content of 63.7%, and contains 4715 genes. Annotation and analysis of the TALE genes using a suite of application named AnnoTALE suggested that 17 transcription activator-like effectors, including 15 typical TALEs and 2 iTALEs/truncTALEs, were encoded in the genome. The approach and genome resource will contribute to the discovery of new virulence effectors and understanding on rice-X. oryzae pv. oryzae interactions.

Licorice Germplasm Resources Identification Using DNA Barcodes Inner-Variants

Based on the gradual transformation from wild growth to artificial cultivation, the accurate authentication of licorice seeds contributes to the first committed step of its quality control and is pivotal to ensure the clinical efficacy of licorice. However, it is still challenging to obtain genetically stable licorice germplasm resources due to the multi-source, multi-heterozygous, polyploid, and hybrid characteristics of licorice seeds. Here, a new method for determining the heterozygosity of licorice seed mixture, based on the various sites, and finding the composition characteristics of licorice seed is preliminarily designed and proposed. Namely, high-throughput full-length multiple DNA barcodes(HFMD), based on ITS multi-copy variation exist, the full-length amplicons of ITS2, psbA-trnH and ITS are directly sequenced by rDNA through the next-generation sequence(NGS) and single-molecule real-time (SMRT) technologies. By comparing the three sequencing methods, our results proved that SMRT sequencing successfully identified the complete gradients of complex mixed samples with the best performance. Meanwhile, HFMD is a brilliant and feasible method for evaluating the heterozygosity of licorice seeds. It shows a perfect interpretation of DNA barcoding and can be applied in multi-base multi-heterozygous and polyploid species.

Full-length transcriptome analysis and identification of transcript structures in Eimeria necatrix from different developmental stages by single-molecule real-time sequencing

Background Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix. Methods Second-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied. Results SMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2. Conclusions We obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control. Graphical Abstract