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We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library construction including all enzymatic steps usually required, but replaces radioactive labelling of nucleic acids with fluorescence detection. The separation and quantification steps all involve ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). This quantitative method replaces the use of autoradiography and size exclusion chromatography with combined ion-pair reversed-phase high performance liquid chromatography and in line fluorescence detection. The result of this approach is combination of speed with the generation of reproducible, high quality cDNA libraries.

Background Cholesterol gallstone (CG) is the most common gallstone disease, which is induced by biliary cholesterol supersaturation. The purpose of this study is to investigate the pathogenesis of CG. Methods Sixteen mice were equally and randomly divided into model group and normal control group. The model group was fed with lithogenic diets to induce CG, and then gallbladder bile lipid analysis was performed. After RNA-seq library was constructed, differentially expressed mRNAs (DE-mRNAs) and differentially expressed lncRNAs (DE-lncRNAs) between model group and normal control group were analyzed by DESeq2 package. Using the cluster Profiler package, enrichment analysis for the DE-mRNAs was carried out. Based on Cytoscape software, the protein-protein interaction (PPI) network and competing endogenous RNA (ceRNA) network were built. Using quantitative real-time reverse transcription-PCR (qRT-PCR) analysis, the key RNAs were validated. Results The mouse model of CG was suc cessfully established, and then 181 DE-mRNAs and 33 DE-lncRNAs between model and normal groups were obtained. Moreover, KDM4A was selected as a hub node in the PPI network, and lncRNA MEG3 was considered as a key lncRNA in the regulatory network. Additionally, the miR-107-5p/miR-149-3p/miR-346-3-MEG3 regulatory pairs and MEG3-PABPC4/CEP131/NUMB1 co-expression pairs existed in the regulatory network. The qRT-PCR analysis showed that KDM4A expression was increased, and the expressions of MEG3, PABPC4, CEP131, and NUMB1 were downregulated. Conclusion These RNAs might be related to the pathogenesis of CG.

The analysis of cell-free circulating tumor DNA (ctDNA) is potentially a less invasive, more dynamic assessment of cancer progression and treatment response than characterizing solid tumor biopsies. Standard isolation methods require separation of plasma by centrifugation, a time-consuming step that complicates automation. To address these limitations, we present an automatable magnetic bead-based ctDNA isolation method that eliminates centrifugation to purify ctDNA directly from peripheral blood (PB). To develop and test our method, ctDNA from cancer patients was purified from PB and plasma. We found that allelic fractions of somatic single-nucleotide variants from target gene capture libraries were comparable, indicating that the PB ctDNA purification method may be a suitable replacement for the plasma-based protocols currently in use.

Background: Candida auris is characterized for having a high genetic variability among species. MALDI-TOF MS library contains spectra from only three strains of C. auris, which makes difficult the identification process and gives low scores at the species level. Our aim was to construct and validate an internal library to improve C. auris identification with Colombian clinical strains. Methods: From 30 clinical strains, 770 mass spectra were obtained for the construction of the database. The validation was performed with 300 strains to compare the identification results in the BDAL and C. auris Colombia libraries. Results: Our library allowed a complete, 100% identification of the evaluated strains and a significant improvement in the scores obtained, showing a better performance compared to the Bruker BDAL library. Conclusions: The strengthening of the database is a great opportunity to improve the scoring and C. auris identification. Library data are available via ProteomeXchange with identifier PXD016387.

A transposon-based approach for the construction of sequencing libraries is an efficient way of preparing samples for processing on both Illumina and Ion Torrent platforms. However, PCR-mediated incorporation of adaptors in tagged DNA fragments leaves behind self-complementary regions flanking the DNA fragment. These regions are capable of forming hairpin structures and, together with adaptors, create conditions for the potential formation of template heteroduplexes. These negatively affect the sequencing process on the Ion Torrent platform and can lead to a more than 3-fold decline in output data compared with sequencing of conventional libraries. To address this problem, we have developed MuPlus, a transposon-based protocol for barcoded library preparation for Ion Torrent, in which one adaptor is integrated by PCR and the second is integrated by ligation as a single-stranded oligonucleotide after enzymatic cleavage of a complementary part on one strand of the tag. The resulting library does not contain self-complementary, hairpin-forming regions, is free of heteroduplexes, and can be analyzed on the Ion Torrent platform with the same efficiency as a library created with a ligation-based protocol.

Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel “small-intelligent” strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.

Metagenomic techniques, notably the cloning of environmental DNA (eDNA) into surrogate hosts, have given access to the genome of uncultured bacteria. However, the determination of gene functions based on DNA sequences alone remains a significant challenge. The functional screening of metagenomic libraries represents an interesting approach in the discovery of microbial metabolites. We describe here an optimized screening approach that facilitates the identification of new antimicrobials among large metagenomic libraries. Notably, we report a detailed genomic library construction protocol using Escherichia coli DH10B as a surrogate host, and demonstrate how vector/genomic DNA dephosphorylation, ligase inactivation, dialysis of the ligation product and vector/genomic DNA ratio greatly influence clone recovery. Furthermore, we describe the use of an airbrush device to screen E. coli metagenomic libraries for their antibacterial activity against Staphylococcus aureus, a method we called bacteriospray. This bacterial spraying tool greatly facilitates and improves the functional screening of large genomic libraries, as it conveniently allows the production of a thinner and more uniform layer of target bacteria compared to the commonly used overlay method, resulting in the screening of 5–10 times more clones per agar plate. Using the Burkholderia thailandensis E264 genomic DNA as a proof of concept, four clones out of 70,000 inhibited the growth of S. aureus and were found to each contain a DNA insert. Analysis of these chromosomic fragments revealed genomic regions never previously reported to be responsible for the production of antimicrobials, nor predicted by bioinformatics tools.