Complete genomic and transcriptional landscape analysis using third-generation sequencing: a case study of Saccharomyces cerevisiae CEN.PK113-7D

Completion of eukaryal genomes can be difficult task with the highly repetitive sequences along the chromosomes and short read lengths of secondgeneration sequencing. Saccharomyces cerevisiae strain CEN. PK113-7D, widely used as a model organism and a cell factory, was selected for this study to demonstrate the superior capability of very long sequence reads for de novo genome assembly. We generated long reads using two common third-generation sequencing technologies (Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio)) and used short reads obtained using Illumina sequencing for error correction. Assembly of the reads derived from all three technologies resulted in complete sequences for all 16 yeast chromosomes, as well as themitochondrial chromosome, in one step. Further, we identified three types of DNA methylation (5mC, 4mC and 6mA). Comparison between the reference strain S288C and strain CEN. PK113-7D identified chromosomal rearrangements against a background of similar gene content between the two strains. We identified full-length transcripts through ONT direct RNA sequencing technology. This allows for the identification of transcriptional landscapes, including untranslated regions (UTRs) (5\u27 UTR and 3\u27 UTR) as well as differential gene expression quantification. About 91% of the predicted transcripts could be consistently detected across biological replicates grown either on glucose or ethanol. Direct RNA sequencing identified many polyadenylated non-coding RNAs, rRNAs, telomere-RNA, long non-coding RNA and antisense RNA. This work demonstrates a strategy to obtain complete genome sequences and transcriptional landscapes that can be applied to other eukaryal organisms

Genomic characterization of vancomycin-resistant Enterococcus faecium clonal complex 17 isolated from urine in tertiary hospitals in Northeastern Thailand

Vancomycin-resistant Enterococci (VREs) have increasingly become a major nosocomial pathogen worldwide, earning high-priority category from the World Health Organization (WHO) due to their antibiotic resistance. Among VREs, vancomycin-resistant Enterococcus faecium (VREfm) is particularly concerning, frequently isolated and resistant to many antibiotics used in hospital-acquired infections. This study investigated VREfm isolates from rural tertiary hospitals in Northeastern Thailand based both antibiotic susceptibility testing and whole-genome sequencing. All isolates showed resistance to vancomycin, ampicillin, erythromycin, tetracycline, ciprofloxacin, norfloxacin, and rifampin. Nitrofurantoin and tigecycline resistance were also observed in nearly all isolates. Conversely, all isolates remained susceptible to chloramphenicol, daptomycin, and linezolid. Genomic characterization revealed that all VREfm isolates belonged to clonal complex 17 (CC17), primarily consisting of sequence type (ST) 80, followed by ST17, ST761, and ST117. Additionally, all isolates harbored numerous antimicrobial-resistant genes, including vanA, tet(L), tet(M), aac(6′)-li, ant(6)-Ia, aph(3′)-III, aac(6′)-aph(2″), aph(2″)-la, ant(9)-la, erm(B), msr(C), erm(T), erm(A), fosB, dfrG, and cfr(B). Notably, all isolates contained virulence genes, for collagen adhesin (acm) and cell wall adhesin (efafm), while hylEfm (glycosyl hydrolase) was detected in VREfm ST80. This study provided important information for understanding the genomic features of VREfm isolated from urine

Ultrarapid and high-resolution HLA class I typing using transposase-based nanopore sequencing applied in pharmacogenetic testing

Nanopore sequencing has been examined as a method for rapid and high-resolution human leukocyte antigen (HLA) typing in recent years. We aimed to apply ultrarapid nanopore-based HLA typing for HLA class I alleles associated with drug hypersensitivity, including HLA-A*31:01, HLA-B*15:02, and HLA-C*08:01. Most studies have used the Oxford Nanopore Ligation Sequencing kit for HLA typing, which requires several enzymatic reactions and remains relatively expensive, even when the samples are multiplexed. Here, we used the Oxford Nanopore Rapid Barcoding kit, which is transposase-based, with library preparation taking less than 1 h of hands-on time and requiring minimal reagents. Twenty DNA samples were genotyped for HLA-A, -B, and -C; 11 samples were from individuals of different ethnicity and nine were from Thai individuals. Two primer sets, a commercial set and a published set, were used to amplify the HLA-A, -B, and -C genes. HLA-typing tools that used different algorithms were applied and compared. We found that without using several third-party reagents, the transposase-based method reduced the hands-on time from approximately 9 h to 4 h, making this a viable approach for obtaining same-day results from 2 to 24 samples. However, an imbalance in the PCR amplification of different haplotypes could affect the accuracy of typing results. This work demonstrates the ability of transposase-based sequencing to report 3-field HLA alleles and its potential for race- and population-independent testing at considerably decreased time and cost

Activation-induced cytidine deaminase localizes to G-quadruplex motifs at mutation hotspots in lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells in vitro were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, CBFA2T3, SPIB, BCL6, HLA-DRB5 and MEF2C, along with the established BCL2 and MYC structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent BCL2 and MYC oncogene amplification and BCL2 mutations. Ninety-seven percent of the BCL2 mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified BCL2 and MYC, supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis

Decaffeinated green tea extract does not elicit hepatotoxic effects and modulates the gut microbiome in lean B6C3F\u3csub\u3e1\u3c/sub\u3e mice

© 2019 The Author(s) The aim of this study is the development of validated HPTLC method for the quantification of vitexin from Passiflora foetida commercial herbal formulations. The developed method was validated, in accordance with ICH guidelines for precision, accuracy, specificity and robustness. The plate was developed using ethyl acetate:methanol:water:formic acid 30:4:2:1(%, v/v/v/v) on 20 × 10 cm glass coated silica gel 60 F254 plates and the developed plate was scanned and quantified densitometrically at λ = 340 nm. Linear regression analysis revealed a good linear relationship between peak area and amount of vitexin in the range of 100–700 ng/spot. The amount of vitexin in nine commercial herbal formulations was successfully quantified by the developed HPTLC method. The developed and validated high performance thin layer chromatographic method offers a new sensitive and reliable tool for quantification of vitexinin in various herbal formulations containing Passiflora foetida

Genetic aberration analysis in thai colorectal adenoma and early-stage adenocarcinoma patients by whole-exome sequencing

Colorectal adenomas are precursor lesions of colorectal adenocarcinoma. The transition from adenoma to carcinoma in patients with colorectal cancer (CRC) has been associated with an accumulation of genetic aberrations. However, criteria that can screen adenoma progression to adenocarcinoma are still lacking. This present study is the first attempt to identify genetic aberrations, such as the somatic mutations, copy number variations (CNVs), and high-frequency mutated genes, found in Thai patients. In this study, we identified the genomic abnormality of two sample groups. In the first group, five cases matched normal-colorectal adenoma-colorectal adenocarcinoma. In the second group, six cases matched normal-colorectal adenomas. For both groups, whole-exome sequencing was performed. We compared the genetic aberration of the two sample groups. In both normal tissues compared with colorectal adenoma and colorectal adenocarcinoma analyses, somatic mutations were observed in the tumor suppressor gene APC (Adenomatous polyposis coli) in eight out of ten patients. In the group of normal tissue comparison with colorectal adenoma tissue, somatic mutations were also detected in Catenin Beta 1 (CTNNB1), Family With Sequence Similarity 123B (FAM123B), F-Box And WD Repeat Domain Containing 7 (FBXW7), Sex-Determining Region Y-Box 9 (SOX9), Low-Density Lipoprotein Receptor-Related Protein 5 (LRP5), Frizzled Class Receptor 10 (FZD10), and AT-Rich Interaction Domain 1A (ARID1A) genes, which are involved in the Wingless-related integration site (Wnt) signaling pathway. In the normal tissue comparison with colorectal adenocarcinoma tissue, Kirsten retrovirus-associated DNA sequences (KRAS), Tumor Protein 53 (TP53), and Ataxia-Telangiectasia Mutated (ATM) genes are found in the receptor tyrosine kinase-RAS (RTK–RAS) signaling pathway and p53 signaling pathway, respectively. These results suggest that APC and TP53 may act as a potential screening marker for colorectal adenoma and early-stage CRC. This preliminary study may help identify patients with adenoma and early-stage CRC and may aid in establishing prevention and surveillance strategies to reduce the incidence of CRC

Integrative analysis of the human cis-antisense gene pairs, miRNAs and their transcription regulation patterns

Cis-antisense gene pairs (CASGPs) can transcribe mRNAs from an opposite strand of a given locus. To classify and understand diverse CASGP phenomena in the human we compiled a genome-wide catalog of CASGPs and integrated these sequences with microarray, SAGE and miRNA data. Using the concept of overlapping regions and clustering of SA transcripts by chromosome coordinates, we identified up to 9000 overlapping antisense loci. Four thousand three hundred and seventy-four of these CASGPs form 1759 complex gene architectures. We found that ∼35% (6347/18160) of RefSeq genes are overlapped with the antisense transcripts. About 30% of Affymetrix U133 microarray initial sequences map transcripts of ∼35% CASGPs and reveal mostly concordant expression in CASGPs. We found strong significant overrepresentation of human miRNA genes in loci of CASGPs. We developed a data-driven model of cross-talk between co-expressed CASGPs and DICER1-mediated miRNA pathway in normal spermatogenesis and in severe teratozoospermia. Specifically, we revealed complex SA structural–functional gene module composing the protein-coding genes, WDR6, DALRD3, NDUFAF3 and ncRNA precursors, mir-425 and mir-191, which could provide downregulation of ncRNA pathway via direct targeting DICER1 and basonuclin 2 transcripts by mir-425 and mir-191 in normal spermatogenesis, but this mechanism is switched off in severe teratozoospermia. The database is available from http://globalisland.bii.a-star.edu.sg/∼jiangtao/sas/index3.php?link =abou

Statistics of protein-DNA binding and the total number of binding sites for a transcription factor in the mammalian genome

<p>Abstract</p> <p>Background</p> <p>Transcription factor (TF)-DNA binding loci are explored by analyzing massive datasets generated with application of Chromatin Immuno-Precipitation (ChIP)-based high-throughput sequencing technologies. These datasets suffer from a bias in the information about binding loci availability, sample incompleteness and diverse sources of technical and biological noises. Therefore adequate mathematical models of ChIP-based high-throughput assay(s) and statistical tools are required for a robust identification of specific and reliable TF binding sites (TFBS), a precise characterization of TFBS avidity distribution and a plausible estimation the total number of specific TFBS for a given TF in the genome for a given cell type.</p> <p>Results</p> <p>We developed an exploratory mixture probabilistic model for a specific and non-specific transcription factor-DNA (TF-DNA) binding. Within ChiP-seq data sets, the statistics of specific and non-specific DNA-protein binding is defined by a mixture of sample size-dependent skewed functions described by Kolmogorov-Waring (K-W) function (Kuznetsov, 2003) and exponential function, respectively. Using available Chip-seq data for eleven TFs, essential for self-maintenance and differentiation of mouse embryonic stem cells (SC) (Nanog, Oct4, sox2, KLf4, STAT3, E2F1, Tcfcp211, ZFX, n-Myc, c-Myc and Essrb) reported in Chen et al (2008), we estimated (i) the specificity and the sensitivity of the ChiP-seq binding assays and (ii) the number of specific but not identified in the current experiments binding sites (BSs) in the genome of mouse embryonic stem cells. Motif finding analysis applied to the identified c-Myc TFBSs supports our results and allowed us to predict many novel c-Myc target genes.</p> <p>Conclusion</p> <p>We provide a novel methodology of estimating the specificity and the sensitivity of TF-DNA binding in massively paralleled ChIP sequencing (ChIP-seq) binding assay. Goodness-of fit analysis of K-W functions suggests that a large fraction of low- and moderate- avidity TFBSs cannot be identified by the ChIP-based methods. Thus the task to identify the binding sensitivity of a TF cannot be technically resolved yet by current ChIP-seq, compared to former experimental techniques. Considering our improvement in measuring the sensitivity and the specificity of the TFs obtained from the ChIP-seq data, the models of transcriptional regulatory networks in embryonic cells and other cell types derived from the given ChIp-seq data should be carefully revised.</p