Search

Methanogens are the main biological producers of methane on Earth. Methanosarcina acetivorans is one of the best characterized methanogens that has powerful genetic tools for genome editing. To study the physiology of this methanogen in further detail as well as to effectively balance the flux of their engineered metabolic pathways in expansive project undertakings, there is the need for controlled gene expression, which then requires the availability of well-characterized promoters and ribosome-binding sites (RBS). In this study, we constructed a library of 33 promoter-RBS combinations that includes 13 wild-type and 14 hybrid combinations, as well as six combination variants in which the 5'-untranslated region (5'UTR) was rationally engineered. The expression strength for each combination was calculated by inducing the expression of the β-glucuronidase reporter gene in M. acetivorans cells in the presence of the two most used growth substrates, either methanol (MeOH) or trimethyl amine (TMA). In this study, the constructed library covers a relatively wide range (140-fold) between the weakest and strongest promoter-RBS combination as well as shows a steady increase and allows different levels of gene expression. Effects on the gene expression strength were also assessed by making measurements at three distinct growth phases for all 33 promoter-RBS combinations. Our promoter-RBS library is effective in enabling the fine-tuning of gene expression in M. acetivorans for physiological studies and the design of metabolic engineering projects that, e.g., aim for the biotechnological valorization of one-carbon compounds., Importance: Methanogenic archaea are potent producers of the greenhouse gas methane and thus contribute substantially to global warming. Under controlled conditions, these microbes can catalyze the production of biogas, which is a renewable fuel, and might help counter global warming and its effects. Engineering the primary metabolism of Methanosarcina acetivorans to render it better and more useful requires controllable gene expression, yet only a few well-characterized promoters and RBSs are presently available. Our study rectifies this situation by providing a library of 33 different promoter-RBS combinations with a 140-fold dynamic range in expression strength. Future metabolic engineering projects can take advantage of this library by using these promoter-RBS combinations as an efficient and tunable gene expression system for M. acetivorans . Furthermore, the methodologies we developed in this study could also be utilized to construct promoter libraries for other types of methanogens., Competing Interests: The authors declare no conflict of interest.

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. Any AAV genome with a 3' ITR is converted to a template suitable to adapter addition by a Bst DNA polymerase-mediated extension reaction. This extension reaction helps fix ITR heterogeneity in the AAV population and allows efficient adapter addition to even noncanonical AAV genomes. The resulting library maintains the original AAV genome configurations without introducing undesired changes. Subsequently, long-read sequencing can be performed by the Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology platform. Finally, through comprehensive data analysis, we can recover canonical, noncanonical AAV DNA, and non-AAV vector DNA sequences, along with their molecular configurations. Our method is a robust tool for profiling thorough AAV-population genomes. TMCA-AAVseq can be further extended to all parvoviruses and their derivative vectors., Competing Interests: W.X. holds equity in Ivygen Corporation and Nikegen., (© 2024 The Authors.)

The design and generation of an optimal protein expression construct is the first and essential step in the characterization of any protein of interest. However, the exchange and modification of the coding and/or noncoding elements to analyze their effect on protein function or generating the optimal result can be a tedious and time-consuming process using standard molecular biology cloning methods. To streamline the process to generate defined expression constructs or libraries of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed (Weber E, PloS One 12(5):e0176314, 2017). The system applies Golden Gate cloning as an assembly method and follows the standardized modular cloning (MoClo) principle (Weber E, PloS One 6(2):e16765, 2011). This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules effecting protein expression with a focus on minimizing the cloning burden in coding regions. The original MoPET system consists of 53 defined DNA modules divided into eight functional main classes and can be flexibly expanded dependent on the need of the experimenter and expression host. However, already with a limited set of only 53 modules, 792,000 different constructs can be rationally designed or used to generate combinatorial expression optimization libraries. We provide here a detailed protocol for the (1) design and generation of level 0 basic parts, (2) generation of defined expressions constructs, and (3) generation of combinatorial expression libraries., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Yeast surface display (YSD) is a technology that fuses the exogenous target protein gene sequence with a specific vector gene sequence, followed by introduction into yeast cells. Subsequently, the target protein is expressed and localized on the yeast cell surface by using the intracellular protein transport mechanism of yeast cells, whereas the most widely used YSD system is the α-agglutinin expression system. Yeast cells possess the eukaryotic post-translational modification mechanism, which helps the target protein fold correctly. This mechanism could be used to display various eukaryotic proteins, including antibodies, receptors, enzymes, and antigenic peptides. YSD has become a powerful protein engineering tool in biotechnology and biomedicine, and has been used to improve a broad range of protein properties including affinity, specificity, enzymatic function, and stability. This review summarized recent advances in the application of YSD technology from the aspects of library construction and screening, antibody engineering, protein engineering, enzyme engineering and vaccine development.

As central players in cellular structure and function, proteins have long been central themes in life science research. Analyzing the impact of protein sequence variation on its structure and function is one of the important means to study proteins. In recent years, a technology called deep mutational scanning (DMS) has been widely used in the field of protein research. It introduces thousands of mutations in parallel in specific regions of proteins through high-abundance DNA libraries. After screening, high-throughput sequencing is employed to score each mutation, revealing sequence-function correlations. Due to its high-throughput, fast and easy, and labor-saving features, DMS has become an important method for protein function research and protein engineering. This review briefly summarizes the principle of DMS technology, highlighting its applications in mammalian cells. Moreover, this review analyzes the current technical bottlenecks, aiming to facilitate relevant research.

Currently, bioorthogonal coupling reactions have garnered considerable interest due to their high substrate selectivity and less restrictive reaction conditions. During recent decades, bioorthogonal coupling reactions have emerged as powerful tools in drug development. This review describes the current applications of bioorthogonal coupling reactions in compound library building mediated by the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and in situ click chemistry or conjunction with other techniques; druggability optimization with 1,2,3-triazole groups; and intracellular self-assembly platforms with ring tension reactions, which are presented from the viewpoint of drug development. There is a reasonable prospect that bioorthogonal coupling reactions will accelerate the screening of lead compounds, the designing strategies of small molecules and expand the variety of designed compounds, which will be a new trend in drug development in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

The emergence of antibiotic-resistant-bacteria is a serious public health threat, which prompts us to speed up the discovery of novel antibacterial agents. Phage display technology has great potential to screen peptides or antibodies with high binding capacities for a wide range of targets. This property is significant in the rapid search for new antibacterial agents for the control of bacterial resistance. In this paper, we not only summarized the recent progress of phage display for the discovery of novel therapeutic agents, identification of action sites of bacterial target proteins, and rapid detection of different pathogens, but also discussed several problems of this technology that must be solved. Breakthrough in these problems may further promote the development and application of phage display technology in the biomedical field in the future.

The human microbiome plays a crucial role in human health. In the past decade, advances in high-throughput sequencing technologies and analytical software have significantly improved our knowledge of the human microbiome. However, most studies concerning the human microbiome did not provide repeatable protocols to guide the sample collection, handling, and processing procedures, which impedes obtaining valid and timely microbial taxonomic and functional results. This protocol provides detailed operation methods of human microbial sample collection, DNA extraction, and library construction for both the amplicon sequencing-based measurements of the microbial samples from the human nasal cavity, oral cavity, and skin, as well as the shotgun metagenomic sequencing-based measurements of the human stool samples among adult participants. This study intends to develop practical procedure standards to improve the reproducibility of microbiota profiling of human samples., Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00097-y., (© International Human Phenome Institutes (Shanghai) 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Naturally occurring heavy chain antibodies (HCAbs) in Camelidae species were a surprise discovery in 1993 by Hamers et al. Since that time, antibody fragments derived from HCAbs have garnered considerable attention by researchers and biotechnology companies. Due to their biophysico-chemical advantages over conventional antibody fragments, camelid single-domain antibodies (sdAbs, V H Hs, nanobodies) are being increasingly utilized as viable immunotherapeutic modalities. Currently there are multiple V H H-based therapeutic agents in different phases of clinical trials in various formats such as bi- and multivalent, bi- and multi-specific, CAR-T, and antibody-drug conjugates. The first approved V H H, a bivalent humanized V H H (caplacizumab), was approved for treating rare blood clotting disorders in 2018 by the EMA and the FDA in 2019. This was followed by the approval of an anti-BCMA V H H-based CAR-T cell product in 2022 (ciltacabtagene autoleucel; CARVYKTI™) and more recently a trivalent antitumor necrosis factor alpha-based V H H drug (ozoralizumab; Nanozora ® ) in Japan for the treatment of rheumatoid arthritis. In this chapter we provide protocols describing the latest developments in isolating antigen-specific V H Hs including llama immunization, construction of phage-displayed libraries, phage panning and screening of the soluble V H Hs by ELISA, affinity measurements by surface plasmon resonance, functional cell binding by flow cytometry, and additional validation by immunoprecipitation. We present and discuss comprehensive, step-by-step methods for isolating and characterization of antigen-specific V H Hs. This includes protocols for expression, biotinylation, purification, and characterization of the isolated V H Hs. To demonstrate the feasibility of the entire strategy, we present examples of V H Hs previously isolated and characterized in our laboratory., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Next-generation sequencing (NGS) of small RNA (sRNA) cDNA libraries permits the identification and characterization of sRNA species de novo. However, the method through which these libraries are constructed can often introduce artifacts such as over- or underrepresentation of specific sequences or adapter oligonucleotides due to sequence biases held by the enzymes used. In this chapter we describe a protocol for sRNA library construction making use of high-definition (HD) adapters for the Illumina sequencing platform, which reduce ligation bias. This protocol leads to drastically reduced direct 5'/3' adapter ligation products and can be used for the synthesis of sRNA libraries from total RNA or sRNA of various plant, animal, and fungal samples. This protocol also includes a method for total RNA extraction from plant leaf and cultured cells or body fluids., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

An important contributor to the successful generation of recombinant affinity reagents via phage display is a large and diverse library. We describe, herein, the application of Kunkel mutagenesis and rolling circle amplification (RCA) to the construction of a 1.1 × 10 11 member library, with only 26 electroporations, and isolation of low- to sub-nanomolar monobodies to a number of protein targets, including human COP9 signalosome subunit 5 (COPS5), HIV-1 Rev. binding protein-like protein (HRBL), X-ray repair cross-complementing 5/6 (Ku70/80) heterodimer, the receptor-binding domain (RBD) of SARS-CoV-2, and transforming growth factor beta 1 (TGF-β1)., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction., Competing Interests: ZS, JM, and DD are co-inventors of a patent, filed on March 12 by the University of Pittsburgh, related to MSLN specific antibodies described in this paper. Binders against CD22 and PD-L1 are in invention disclosures in the University of Pittsburgh. Sequences of binders will be provided upon request and collaboration. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sun, Li, Mellors, Orentas and Dimitrov.)

Cardiac fibrosis (CF) and heart failure (HF) are common heart diseases, and severe CF can lead to HF. In this study, we tried to find their common potential molecular markers, which may help the diagnosis and treatment of CF and HF. RNA library construction and high-throughput sequencing were performed. The DESeq2 package in R was used to screen differentially expressed mRNAs (DEmRNAs), differentially expressed lncRNA (DElncRNAs) and differentially expressed miRNA (DEmiRNAs) between different samples. The common DEmRNAs, DElncRNAs and DEmiRNAs for the two diseases were obtained. The ConsensusPathDB (CPDB) was used to perform biological function enrichment for common DEmRNAs. Gene interaction network was constructed to screen out key genes. Subsequently, real-time polymerase chain reaction (RT-PCR) verification was performed. Lastly, GSE104150 and GSE21125 data sets were utilized for expression validation and diagnostic analysis. There were 1477 DEmRNAs, 502 DElncRNAs and 36 DEmiRNAs between CF and healthy control group. There were 607 DEmRNAs, 379DElncRNAs,s and 42 DEmiRNAs between HF and healthy control group. CH and FH shared 146 DEmRNAs, 80 DElncRNAs, and 6 DEmiRNAs. Hsa-miR-144-3p, CCNE2, C9orf72, MAP3K20-AS1, LEF1-AS1, AC243772.2, FLJ46284, and AC239798.2 were key molecules in lncRNA-miRNA-mRNA network. In addition, hsa-miR-144-3p and CCNE2 may be considered as potential diagnostic gene biomarkers in HF. In this study, the identification of common biomarkers of CF and HF may help prevent CF to HF transition as early as possible.

Over the last two decades, yeast display methodology has served as a popular tool for discovery, humanization, stability improvement, and affinity maturation of antibodies and antibody fragments, but also for development of diverse non-antibody protein scaffolds towards the ability of antigen recognition. Yeast display is particularly well suited for multiparametric analysis of properties of derivatized proteins, allowing the evolution of most diverse protein structures into antigen binding entities with favorable expression, stability, and folding properties. Here we present the methodological basics of a novel yeast display-based approach for the functionalization of the large extracellular loop of CD81 into a de novo antigen binding unit. CD81 is intrinsically overrepresented on the surface of extracellular vesicles (EVs), naturally occurring nanoparticle units that act as cell-to-cell messengers by delivering their intracellular cargo from the source cell into a recipient cell. This amazing feature makes them of highest biotechnological interest, yet methods for their targeted delivery are still in their infancy. As a novel approach for introducing EV surface modifications enabling specific target cell recognition and internalization, we have prepared yeast display libraries of CD81 large extracellular loop mutants, which are selected towards specific antigen binding and resulting mutants conveniently clicked into the full-length EV surface protein. Resulting EVs display wild-type-like characteristics regarding the expression level and distribution of recombinant proteins and are hence promising therapeutic tools., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Abbreviations: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; k a : association rate; k d : dissociation rate; K D : dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.

Protein engineering through directed evolutison is facilitated by the screening and characterization of protein libraries. Efficient and effective methods for multiple site-saturation mutagenesis, such as Darwin Assembly, can accelerate the sampling of relevant sequence space and the identification of variants with desired functionalities. Here, we present the automation of the Darwin Assembly method, using a Gilson PIPETMAX™ liquid handling platform under the control of the Antha software platform, which resulted in the accelerated construction of complex, multiplexed gene libraries error-free and with minimal hands-on time, while maintaining flexibility over experimental parameters through a graphical user interface rather than requiring user-driven library-dependent programming of the liquid handling platform. We also present an approach for barcoding libraries that overcomes amplicon length limitations in next generation sequencing and enables fast reconstruction of library reads., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Standardisation of genetic parts has become a topic of increasing interest over the last decades. The promise of simplifying molecular cloning procedures, while at the same time making them more predictable and reproducible has led to the design of several biological standards, one of which is modular cloning (MoClo). The Yeast MoClo toolkit provides a large library of characterised genetic parts combined with a comprehensive and flexible assembly strategy. Here we aimed to (1) simplify the adoption of the standard by providing a simple design tool for including new parts in the MoClo library, (2) characterise the toolkit further by demonstrating the impact of a BglII site in promoter parts on protein expression, and (3) expand the toolkit to enable efficient construction of gRNA arrays, marker-less integration cassettes and combinatorial libraries. These additions make the toolkit more applicable for common engineering tasks and will further promote its adoption in the yeast biological engineering community.

Microbes employ sophisticated cellular networks encoded by complex genomes to rapidly adapt to changing environments. High-throughput genome engineering methods are valuable tools for functionally profiling genotype-phenotype relationships and understanding the complexity of cellular networks. However, current methods either rely on special homologous recombination systems and are thus applicable in only limited bacterial species or can generate only nonspecific mutations and thus require extensive subsequent screening. Here, we report a site-specific transposon-assisted genome engineering (STAGE) method that allows high-throughput Cas12k-guided mutagenesis in various microorganisms, such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Exploiting the powerful STAGE technique, we construct a site-specific transposon mutant library that focuses on all possible transcription factors (TFs) in P. aeruginosa, enabling the comprehensive identification of essential genes and antibiotic-resistance-related factors. Given its broad host range activity and easy programmability, this method can be widely adapted to diverse microbial species for rapid genome engineering and strain evolution., Competing Interests: Declaration of interests A patent application has been submitted for the Cas12k-guided transposition system., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Discovering therapeutic agents: New bioactive agents, either as sole or combinational agents, have been constructed through the synthetic manipulation of the intermediates within the total synthesis of the uvaretin class of natural products. It was found that increasing the hydrophobic character of the phenolic core correlates to a decrease in sole agent cytotoxicity. The synthesis of new, small chemical screening libraries (CSL) constructed from the intermediates of our total synthesis route of the uvaretin class of natural products is demonstrated herein. Numerous chalcone-based CSLs with various substitution on the phenolic groups within the chalcone core were assembled. Through cytotoxicity investigations, it was found that the level of hydrophobicity of the phenolic core of the chalcones gives biases: less cytotoxicity with more hydrophobic cores. In addition, it was observed that the potentiation, evaluated with 6-thiopurine in the pancreatic cancer cell line MIA PaCa-2, is tunable by the inclusion of less-hydrophobic character on the phenolic core. The role of the o-hydroxybenzyl group, present within the uvaretin family, was revealed to be cytotoxic in character. Merging all of the structure-activity relationship studies performed on the CSLs constructed in this effort led to the construction of a new chalcone hybrid possessing both a cytotoxic enone group and a small-molecule-potentiating, reduced enone group., (© 2021 Wiley-VCH GmbH.)

Background: Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples., Results: In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories., Conclusions: The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. Video Abstract.

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., Competing Interests: The authors declare there are no competing interests., (©2021 Qian et al.)

Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica , where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB , VioC , and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica , and high deoxyviolacein production was found strongly associated with the weak expression of VioD . By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica . This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.

Due to crucial roles in gene regulation, noncoding small RNAs (sRNAs) of 20-30 nucleotides (nt) have been intensively studied in mammals and plants and are implicated in significant diseases and metabolic disorders. Elucidation of biogenesis mechanisms and functional characterization of sRNAs is often achieved using tools such as separation of small-sized RNA and deep sequencing. Although RNA interference pathways, such as quelling and meiotic silencing, have been well-described in Neurospora crassa, knowledge of sRNAs in other filamentous fungi is still limited compared to other eukaryotes. As a prerequisite for study, isolation and sequence analysis of sRNAs is necessary. We developed a protocol for isolation and library construction of sRNAs of 20-30 nt for deep sequencing in two filamentous fungi, N. crassa and Fusarium oxysporum f.sp. lycopersici. Using 200-300 μg total RNA, sRNA was isolated by size-fractionation and ligated with adapters and amplified by RT-PCR for deep sequencing. Sequence analysis of several cDNA clones showed that the cloned sRNAs were not tRNAs and rRNAs and were fungal genome-specific. In order to validate fungal miRNAs that were imported into the host cell, we developed a straightforward method to isolate protoplasts from tomato roots infected by Fusarium oxysporum f.sp. lycopersici using enzymatic digestion.

RNA silencing plays a critical role in diverse biological processes in plants including growth, development, and responses to abiotic and biotic stresses. RNA silencing is guided by small non-coding RNAs (sRNAs) with the length of 21-24 nucleotides (nt) that are loaded into Argonaute (AGO) to repress expression of target loci and transcripts through transcriptional or posttranscriptional gene silencing mechanisms. Identification and quantitative characterization of sRNAs are crucial steps toward appreciation of their functions in biology. Here, we developed a step-by-step protocol to precisely illustrate the process of cloning of sRNA libraries and correspondingly computational analysis of the recovered sRNAs. This protocol can be used in all kinds of organisms, including Arabidopsis, and is compatible with various high-throughput sequence technologies such as Illumina Hiseq. Thus, we wish that this protocol represents an accurate way to identify and quantify sRNAs in vivo.

Circular RNA (circRNA) is an important class of noncoding RNA. Current, protocols to detected circRNAs have utilized nonpolyadenylated RNAs, ribosomal RNA-depleted RNA samples (rRNA- library), and rRNA-depleted RNA that has been treated with RNase R to digest linear RNA (rRNA- RNase R+ library). Accumulating evidence suggests the participation of circRNAs in Alzheimer's disease (AD)-associated pathophysiology, but the details remain largely unknown. Here, we elucidated the brain circRNAs profiles of AD-model and WT mice using two methods of library construction (rRNA- RNase R+ libraries and rRNA- libraries). We focused on the construction of libraries that best allow the identification of circRNAs from next-generation RNA sequencing data. We obtained a significantly higher abundance of circRNAs in the rRNA- RNase R+ libraries than in the rRNA- libraries. Additionally, the rRNA- RNase R+ libraries more clearly revealed differentially expressed circRNAs. We performed a correlation analysis between differentially expressed circRNAs and their parental genes and performed KEGG analysis of the parental genes to explore the role of circRNA in AD. Our results identified significantly dysregulated circRNAs and KEGG analysis revealed that the identified circRNAs are involved in regulating AD development from distinct origins, including cAMP signaling, MAPK signaling, insulin secretion, Axon guidance, Long-term potentiation, dopaminergic synapse, and ErBb signaling pathways. Following rigorous selection, we identified several important circRNAs and mRNAs and propose the circRNAs regulate parental gene transcription or affect variable splicing, which were discovered to be mainly involved in Aβ production and cognitive performance (TRPC6) and neuronal differentiation and development (NME7). These newly identified circRNAs should serve as a valuable resource for the future development of potential biomarkers and therapeutic targets for AD., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

: 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., Competing Interests: The authors declare no conflict of interest.

Phage display is a commonly used technology for the screening of large clonal libraries of proteins and peptides. The construction of peptide libraries containing very short sequences, however, poses certain problems for conventional restriction-based cloning procedures, which are rooted in the necessity to purify restricted library oligos. Herein, we present an alternative cloning method especially suitable for such very short sequences of about only 21 base pairs resulting in a 60 bp insert. The employed restriction-free hot fusion cloning strategy allows for facile library construction bypassing the need for purification of the small oligo. The library includes one well-defined disulfide bridge rendering the displayed macrocyclic peptide sequences as attractive scaffolds for novel active principles.

The status of T cell receptors (TCRs) repertoire is associated with the occurrence and progress of various diseases and can be used in monitoring the immune responses, predicting the prognosis of disease and other medical fields. High-throughput sequencing promotes the studying in TCR repertoire. The chapter focuses on the whole process of TCR profiling, including DNA extraction, library construction, high-throughput sequencing, and how to analyze data.

Since two decades, yeast display methodology is a popular tool for discovery, stability improvement, and affinity maturation of diverse protein scaffolds, intended for antigen recognition. Yeast display is particularly well suited for the selection of heterodimeric proteins, such as antibodies and T-cell receptors (TCRs), as it allows rapid library creation via gap-repair-driven homologous recombination and subsequent construction of a combinatorial library after mating of yeast of opposite mating types. Certain properties of the TCR scaffold, such as its stability, inferior to most antibody fragments, require modifications of traditional antigen selection strategies. Their selection can be monitored and guided upon staining with the soluble versions of their original antigen, peptide-major histocompatibility complex (MHC), or clonotypic antibodies, whose binding is critically dependent on the TCR structural integrity. Overall, this chapter underlines the importance of the versatile yeast display technique for the diversification of the TCR scaffold for antigen recognition and optimization of its stability.

Suppression analysis is used for the identification of new genes and genetic interactions when there is a notable phenotype available for genetic selection or screening. A random genomic DNA library constructed on a multi-copy plasmid is a useful tool for suppression analysis when one expects that an overdose of a few genes will suppress the phenotype. These libraries have been successfully used to determine the function of a gene by revealing genes whose functions are related to the gene of interest. They have also been used to identify the targets of chemical or biological agents by increasing the number of unaffected target gene products in a cell. In this article, I will discuss important considerations for constructing multicopy genomic DNA libraries. The protocol provided in this paper should be a useful guide for constructing genomic DNA libraries in many bacterial species for which multi-copy plasmids are available.

At present, approaches to hybrid capture sequencing have many limitations, from their significant complexity and labor requirements, to their low enrichment efficiency, resulting in their limited utilization. In an effort to overcome these drawbacks, we have developed a novel method that relied upon direct genomic DNA hybridization and single-stranded DNA library preparation. Using this novel protocol, we were able to achieve a targeting efficiency as high as 75%, and we found this approach to overall be an efficient and simple approach to DNA library construction., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Identification of genomic alterations from formalin-fixed paraffin-embedded (FFPE) samples using next-generation sequencing (NGS) is very important for cancer-targeted therapy today. To achieve a higher efficiency and shorter turn-around time for NGS library preparation, here, we compared NGS library preparation processes and outcomes with three commercial library construction methods and two hybridization capture methods thus, developed an improved NGS library construction approach. This improved approach took advantage of both methods and resulted in a higher output from the same input DNA, including higher library construction success rate, higher probe capture rate, and shorter turn-around time. Using this approach, targeted region libraries could be constructed within only 1 day for FFPE samples; therefore, this approach has potential applications of NGS in routine clinical tests. Anat Rec, 302:941-946, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Target enrichment is increasingly used for genotyping of plant and animal species or to better understand the evolutionary history of important lineages through the inference of statistically robust phylogenies. Limitations to routine target enrichment are both the complexity of current protocols and low input DNA quantity. Thus, working with tiny organisms such as microarthropods can be challenging. Here, we propose easy to set up optimizations for DNA extraction and library preparation prior to target enrichment. Prepared libraries were used to capture 1,432 ultraconserved elements (UCEs) from microhymenoptera (Chalcidoidea), which are among the tiniest insects on Earth and the most commercialized worldwide for biological control purposes. Results show no correlation between input DNA quantities (1.8-250 ng, 0.4 ng with an extra whole genome amplification step) and the number of sequenced UCEs on an Illumina MiSeq. Phylogenetic inferences highlight the potential of UCEs to solve relationships within the families of chalcid wasps, which has not been achieved so far. The protocol (library preparation + target enrichment) allows processing 96 specimens in five working days, by a single person, without requiring the use of expensive robotic molecular biology platforms, which could help to generalize the use of target enrichment for minute specimens., (© 2019 John Wiley & Sons Ltd.)

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.

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.

The Enterobacteriaceae, and in particular, Escherichia coli including foodborne pathotypes are particularly amenable to transposon mutagenesis. Here we describe the use of mini-Tn5 and Mu d1(Ap lac) to generate transposon inserts for analysis of enterohemorrhagic Escherichia coli EDL933. We also discuss how to array the library in 96-well plates and sequence individual clones for further analysis.

RNA sequencing (RNA-seq) has become the preferred method for global quantification of bacterial gene expression. With the continued improvements in sequencing technology and data analysis tools, the most labor-intensive and expensive part of an RNA-seq experiment is the preparation of sequencing libraries, which is also essential for the quality of the data obtained. Here, we present a straightforward and inexpensive basic protocol for preparation of strand-specific RNA-seq libraries from bacterial RNA as well as a computational pipeline for the data analysis of sequencing reads. The protocol is based on the Illumina platform and allows easy multiplexing of samples and the removal of sequencing reads that are PCR duplicates. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

The emergence and widespread use of high-throughput sequencing technologies have promoted metagenomic studies on environmental or animal samples. Library construction for metagenome sequencing and annotation of the produced sequence reads are important steps in such studies and influence the quality of metagenomic data. In this study, we collected some marine mollusk samples, such as Crassostrea hongkongensis, Chlamys farreri, and Ruditapes philippinarum, from coastal areas in South China. These samples were divided into two batches to compare two library construction methods for shellfish viral metagenome. Our analysis showed that reverse-transcribing RNA into cDNA and then amplifying it simultaneously with DNA by whole genome amplification (WGA) yielded a larger amount of DNA compared to using only WGA or WTA (whole transcriptome amplification). Moreover, higher quality libraries were obtained by agarose gel extraction rather than with AMPure bead size selection. However, the latter can also provide good results if combined with the adjustment of the filter parameters. This, together with its simplicity, makes it a viable alternative. Finally, we compared three annotation tools (BLAST, DIAMOND, and Taxonomer) and two reference databases (NCBI's NR and Uniprot's Uniref). Considering the limitations of computing resources and data transfer speed, we propose the use of DIAMOND with Uniref for annotating metagenomic short reads as its running speed can guarantee a good annotation rate. This study may serve as a useful reference for selecting methods for Shellfish viral metagenome library construction and read annotation.

Background: Next-generation sequencing (NGS) is fundamental to the current biological and biomedical research. Construction of sequencing library is a key step of NGS. Therefore, various library construction methods have been explored. However, the current methods are still limited by some shortcomings., Results: This study developed a new NGS library construction method, Single strand Adaptor Library Preparation (SALP), by using a novel single strand adaptor (SSA). SSA is a double-stranded oligonucleotide with a 3' overhang of 3 random nucleotides, which can be efficiently ligated to the 3' end of single strand DNA by T4 DNA ligase. SALP can be started with any denatured DNA fragments such as those sheared by Tn5 tagmentation, enzyme digestion and sonication. When started with Tn5-tagmented chromatin, SALP can overcome a key limitation of ATAC-seq and become a high-throughput NGS library construction method, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells unbiasly. In this way, this study successfully characterized the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, with SALP-seq. Similarly, this study also successfully characterized the chromatin openness states of HepG2 cells with SALP-seq by using 10 5 to 500 cells., Conclusions: This study developed a new NGS library construction method, SALP, by using a novel kind of single strand adaptor (SSA), which should has wide applications in the future due to its unique performance.

RNA sequencing (RNA-seq) can not only be used to identify the expression of common or rare transcripts but also in the identification of other abnormal events, such as alternative splicing, novel transcripts, and fusion genes. In principle, RNA-seq can be carried out by almost all of the next-generation sequencing (NGS) platforms, but the libraries of different platforms are not exactly the same; each platform has its own kit to meet the special requirements of the instrument design.

Metabolomics based on mass spectrometry can provide quantitative and qualitative information of the pool of metabolites (metabolome) present intracellularly or extracellularly in a given biological system. A typical metabolomics workflow requires several key steps such as quick and robust sample preparations with quenching of metabolism, chemical derivatization if needed, instrumental measurement, data-processing with/without database information and further statistical analysis and interpretation. Here, we introduce general metabolomics workflows for global and targeted analyses using gas chromatography or liquid chromatography coupled with mass spectrometers.

Affilin ® molecules represent a new class of so-called scaffold proteins. The concept of scaffold proteins is to use stable and versatile protein structures which can be endowed with de novo binding properties and specificities by introducing mutations in surface exposed amino acid residues. Complex variations and combinations are generated by genetic methods of randomization resulting in large cDNA libraries. The selection for candidates binding to a desired target can be executed by display methods, especially the very robust and flexible phage display. Here, we describe the construction of ubiquitin based Affilin ® phage display libraries and their use in biopanning experiments for the identification of novel protein ligands.

Background: RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. METHODS: Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. RESULTS: This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments., Conclusions: These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.

Small RNAs (sRNAs) as key regulators of gene expression play fundamental roles in many biological processes. Next-generation sequencing (NGS) has become an important tool for sRNA discovery and profiling. However, NGS data often show bias for or against certain sequences which is mainly caused by adapter oligonucleotides that are ligated to sRNAs more or less efficiently by RNA ligases. In order to reduce ligation bias, High-definition (HD) adapters for the Illumina sequencing platform were developed. However, a large amount of direct 5' and 3' adapter ligation products are often produced when the current commercially available kits are used for cloning with HD adapters. In this chapter we describe a protocol for sRNA library construction using HD adapters with drastically reduced direct 5' adapter-3' adapter ligation product. The protocol can be used for sRNA library preparation from total RNA or sRNA of various plant, animal, insect, or fungal samples. The protocol includes total RNA extraction from plant leaf tissue and cultured mammalian cells and sRNA library construction using HD adapters.

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.

Next-generation sequencing has been evaluated at Genzyme as a means of identifying bioreactor contaminants due to its capability for detection of known and novel microbial species. In this approach, data obtained from next-generation sequencing is used to interrogate databases containing genomic sequences and identities of potential adventitious agents. We describe here the use of this approach to help identify the causative agent of a bioreactor contamination. We also present the results of spiking experiments to establish the limits of detection for DNA viruses, RNA viruses, and bacteria, in a background of Chinese hamster ovary cells, a cell line used for production of many human therapeutics. Using Illumina sequencing-based detection, all of the viruses included in this study were detected at less than 1 copy per cell, and bacteria were detected at 0.001 copy per cell. Thus, next-generation sequencing-based detection of adventitious agents is a valuable approach that can fill a critical unmet need in the detection of known and novel microorganisms in biopharmaceutical manufacturing., Lay Abstract: Because biological products are manufactured in cells, the living environment must be kept sterile. Any introduction of microorganisms into the culture vessel may affect the growth and other biological properties of the cells or contaminate the product. It is therefore important to monitor the culture for such contaminants, but many methods can only detect a specific microorganism. In this study, we show that next-generation sequencing-based detection is a sensitive and complementary approach that can potentially detect a wide range of organisms., (© PDA, Inc. 2014.)

The need for dynamic, elastomeric polymeric biomaterials remains high, with few options with tunable control of mechanical properties, and environmental responses. Yet the diversity of these types of protein polymers pursued for biomaterials-related needs remains limited. Robust high-throughput synthesis and characterization methods will address the need to expand options for protein-polymers for a range of applications. To address this need, a combinatorial library approach with high throughput screening is used to select specific examples of dynamic protein silk-elastin-like polypeptides (SELPs) with unique stimuli responsive features, including tensile strength, and adhesion. Using this approach 64 different SELPs with different sequences and molecular weights are selected out of over 2,000 recombinant E. coli colonies. New understanding of sequence-function relationships with this family of proteins is gained through this combinatorial-screening approach and can provide a guide to future library designs. Further, this approach yields new families of SELPs to match specific material functions.

This unit presents protocols for construction of next-generation sequencing (NGS) directional RNA sequencing libraries for the Illumina HiSeq and MiSeq from a wide variety of input RNA sources. The protocols are based on the New England Biolabs (NEB) small RNA library preparation set for Illumina, although similar kits exist from different vendors. The protocol preserves the orientation of the original RNA in the final sequencing library, enabling strand-specific analysis of the resulting data. These libraries have been used for differential gene expression analysis and small RNA discovery and are currently being tested for de novo transcriptome assembly. The protocol is robust and applicable to a broad range of RNA input types and RNA quality, making it ideal for high-throughput laboratories., (Copyright © 2014 John Wiley & Sons, Inc.)

In the last two decades, several phage display-selected monoclonal antibodies (mAbs) have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV) Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses.