Sequence library construction

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of the Burkholderia cepacia complex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants in B. cenocepacia K56-2, focusing on cell envelope structural and functional processes. We identified structures mechanistically important for resistance to singular and multiple antibiotic classes...

The microbial community structure plays an important role in the internal environment of brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae), which is an indispensable part to reflect the internal environment of BPH. Wing dimorphism is a strategy for balancing flight and reproduction of insects. Here, quantitative fluorescence PCR was used to analyse the number and changes of the symbionts in the fat body of long- and short-winged BPHs at different developmental stages. A metagenomic library was constructed based on the 16 S rRNA sequence and internal transcribed spacer sequence for high-throughput sequencing, to analyze the community structure and population number of the symbionts of long- and short-winged BPHs, and to make functional prediction...

The Chinese white wax scale insect (CWWSI), Ericerus pela, can secret an amount of wax equivalent to their body weight. Previous studies demonstrated the fatty acyl-CoA reductase (far3) plays a pivotal role in wax secretion of CWWSI. The high expression of far3 is crucial for the massive wax secretion. However, the transcription regulation of far3 was not clear. To identify regulatory factors that control the expression of far3, the assay for transposase-accessible chromatin (ATAC) and yeast one-hybrid (Y1H) were carried out in this study...

BACKGROUND: Heterologous expression of active, native-folded protein in Escherichia coli is critical in both academic research and biotechnology settings. When expressing non-native recombinant proteins in E. coli , obtaining soluble and active protein can be challenging. Numerous techniques can be used to enhance a proteins solubility, and largely focus on either altering the expression strain, plasmid vector features, growth conditions, or the protein coding sequence itself. However, there is no one-size-fits-all approach for addressing issues with protein solubility, and it can be both time and labor intensive to find a solution...

Transposable elements (TEs) are a major component of eukaryotic genomes and are present in almost all eukaryotic organisms. TEs are highly dynamic between and within species, which significantly affects the general applicability of the TE databases. Orthoptera is the only known group in the class Insecta with a significantly enlarged genome (0.93-21.48 Gb). When analyzing the large genome using the existing TE public database, the efficiency of TE annotation is not satisfactory. To address this limitation, it becomes imperative to continually update the available TE resource library and the need for an Orthoptera-specific library as more insect genomes are publicly available...

Coronaviruses (CoVs), including severe acute respiratory syndrome coronavirus 2, can infect a variety of mammalian and avian hosts with significant medical and economic consequences. During the life cycle of CoV, a coordinated series of subgenomic RNAs, including canonical subgenomic messenger RNA and non-canonical defective viral genomes (DVGs), are generated with different biological implications. Studies that adopted the Nanopore sequencer (ONT) to investigate the landscape and dynamics of viral RNA subgenomic transcriptomes applied arbitrary bioinformatics parameters without justification or experimental validation...

Asians have a higher carrier rate of pulmonary arterial hypertension (PAH)-related genetic variants than Caucasians do. This study aimed to identify PAH-related genetic variants using whole exome sequencing (WES) in Asian idiopathic and heritable PAH cohorts. A WES library was constructed, and candidate variants were further validated by polymerase chain reaction and Sanger sequencing in the PAH cohort. In a total of 69 patients, the highest incidence of variants was found in the BMPR2, ATP13A3 , and GDF2 genes...

Metabolite biosensors, through which the intracellular metabolite concentrations could be converted to changes in gene expression, are widely used in a variety of applications according to the different output signals. However, it remains challenging to fine-tune the dose-response relationships of biosensors to meet the needs of various scenarios. On the other hand, the short read length of next-generation sequencing (NGS) has greatly limited the design capability of sequence libraries. To address these issues, we describe a DNA trackable assembly method, coupled with fluorescence-activated cell sorting and NGS (Sort-Seq), to achieve the characterization of dose-response curves in a massively parallel manner...

The unique palindromic inverted terminal repeats (ITRs) and single-stranded nature of adeno-associated virus (AAV) DNA are major hurdles to current sequencing technologies. Due to these characteristics, sequencing noncanonical AAV genomes present in AAV vector preparations remains challenging. To address this limitation, we developed thorough molecule configuration analysis of noncanonical AAV genomes (TMCA-AAV-seq). TMCA-AAV-seq takes advantage of the documented AAV packaging mechanism in which encapsidation initiates from its 3' ITR, for AAV-seq library construction...

Next-generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR-based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG-seq primer set (MIG-seq being a PCR method enabling library construction with low-quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops...

Genetic screening of pools of mutants can reveal genetic determinants involved in complex biological interactions, processes, and systems. We previously constructed two single-gene deletion resources for Salmonella enterica serovar Typhimurium 14028s in which kanamycin (KanR) and chloramphenicol (CamR) cassettes were used to replace non-essential genes. We have now used lambda-red recombination to convert the antibiotic cassettes in these resources into a tetracycline-resistant (TetR) version where each mutant contains a different 21-base barcode flanked by Illumina Read1 and Read2 primer sequences...

In this report, we present OLAF-Seq, a novel strategy to construct a long-read sequencing library such that adjacent fragments are linked with end-terminal duplications. We use the CRISPR-Cas9 nickase enzyme and a pool of multiple sgRNAs to perform non-random fragmentation of targeted long DNA molecules (> 300kb) into smaller library-sized fragments (about 20 kbp) in a manner so as to retain physical linkage information (up to 1000 bp) between adjacent fragments. DNA molecules targeted for fragmentation are preferentially ligated with adaptors for sequencing, so this method can enrich targeted regions while taking advantage of the long-read sequencing platforms...

Whole-genome sequencing (WGS) of microbial pathogens recovered from patients with infectious disease facilitates high-resolution strain characterization and molecular epidemiology. However, increasing reliance on culture-independent methods to diagnose infectious diseases has resulted in few isolates available for WGS. Here, we report a novel culture-independent approach to genome characterization of Bordetella pertussis , the causative agent of pertussis and a paradigm for insufficient genomic surveillance due to limited culture of clinical isolates...

The binding process of insulin to its transmembrane receptor entails a sophisticated interplay between two proteins, each possessing two binding sites. Given the difficulties associated with the use of insulin in the treatment of diabetes, despite its remarkable efficacy, there is interest in smaller and more stable compounds than the native hormone that would effectively activate the receptor. Our study adopts a strategy focused on synthesizing extensive combinatorial libraries of bipodal compounds consisting of two distinct peptides linked to a molecular scaffold...

BACKGROUND: We aimed to identify some potential biomarkers for elderly osteoporosis (OP) by integral analysis of lncRNA and mRNA expression data. METHODS: A total of 8 OP cases and 5 healthy participants were included in the study. Fasting peripheral venous blood samples were collected from individuals, and total RNA was extracted. RNA-seq library was prepared and sequenced on the Illumina HiSeq platform. Differential gene expression analysis was performed using "DESeq2" package in R language...

Genomes from yeast to humans are subject to pervasive transcription. A single round of pervasive transcription is sufficient to alter local chromatin conformation, nucleosome dynamics and gene expression, but is hard to distinguish from background signals. Size fractionated native elongating transcript sequencing (sfNET-Seq) was developed to precisely map nascent transcripts independent of expression levels. RNAPII-associated nascent transcripts are fractionation into different size ranges before library construction...

Genomics can be used to study the complex relationships between hosts and their microbiota. Many bacteria cannot be cultured in the laboratory, making it difficult to obtain adequate amounts of bacterial DNA and to limit host DNA contamination for the construction of metagenome-assembled genomes (MAGs). For example, Wolbachia is a genus of exclusively obligate intracellular bacteria that live in a wide range of arthropods and some nematodes. While Wolbachia endosymbionts are frequently described as facultative reproductive parasites in arthropods, the bacteria are obligate mutualistic endosymbionts of filarial worms...

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on plasmid replication proteins (Rep) in cyanobacteria is limited. Synechocystis sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown...

The major drawbacks of RNA sequencing (RNA-seq), a remarkably accurate transcriptome profiling method, is its high cost and poor scalability. Here, we report a highly scalable and cost-effective method for transcriptomics profiling called Bulk transcriptOme profiling of cell Lysate in a single poT (BOLT-seq), which is performed using unpurified bulk 3'-end mRNA in crude cell lysates. During BOLT-seq, RNA/DNA hybrids are directly subjected to tagmentation, and second-strand cDNA synthesis and RNA purification are omitted, allowing libraries to be constructed in 2 h of hands-on time...

INTRODUCTION: Several studies have reported a significant correlation between noise-induced hearing loss and cognitive decline. However, comprehensive analyses of this relationship are rare. This study aimed to assess the influence of hearing impairment on cognitive functions by analyzing organ samples in the afferent auditory pathway of deafened mice using mRNA sequencing. METHODS: We prepared 10 female 12-week-old C57BL/6N mice as the experimental and control groups in equal numbers...