Generation Of Libraries Research Articles

Molli: A General Purpose Python Toolkit for Combinatorial Small Molecule Library Generation, Manipulation, and Feature Extraction.

The construction, management, and analysis of large in silico molecular libraries is critical in many areas of modern chemistry. Herein, we introduce the MOLecular LIibrary toolkit, "molli", which is a Python 3 cheminformatics module that provides a streamlined interface for manipulating large in silico libraries. Three-dimensional, combinatorial molecule libraries can be expanded directly from two-dimensional chemical structure fragments stored in CDXML files with high stereochemical fidelity. Geometry optimization, property calculation, and conformer generation are executed by interfacing with widely used computational chemistry programs such as OpenBabel, RDKit, ORCA, NWChem, and xTB/CREST. Conformer-dependent grid-based feature calculators provide numerical representation and interface to robust three-dimensional visualization tools that provide comprehensive images to enhance human understanding of libraries with thousands of members. The package includes a command-line interface in addition to Python classes to streamline frequently used workflows. Parallel performance is benchmarked on various hardware platforms, and common workflows are demonstrated for different tasks ranging from optimized grid-based descriptor calculation on catalyst libraries to an NMR chemical shift prediction workflow from CDXML files.

Carafe enables high quality in silico spectral library generation for data-independent acquisition proteomics.

Data-independent acquisition (DIA)-based mass spectrometry is becoming an increasingly popular mass spectrometry acquisition strategy for carrying out quantitative proteomics experiments. Most of the popular DIA search engines make use of in silico generated spectral libraries. However, the generation of high-quality spectral libraries for DIA data analysis remains a challenge, particularly because most such libraries are generated directly from data-dependent acquisition (DDA) data or are from in silico prediction using models trained on DDA data. In this study, we developed Carafe, a tool that generates high-quality experiment-specific in silico spectral libraries by training deep learning models directly on DIA data. We demonstrate the performance of Carafe on a wide range of DIA datasets, where we observe improved fragment ion intensity prediction and peptide detection relative to existing pretrained DDA models.

Semisynthetic Phage Display Library Construction: Design and Synthesis of Diversified Single-Chain Variable Fragments and Generation of Primary Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the first of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library construction, and (3) secondary library construction. The first step, described here, entails design, synthesis, and cloning of four PLs. These PLs are designed with specific properties amenable to therapeutic antibody development using one universal variable heavy (VH) scaffold and four distinct variable light (VL) scaffolds. The scaffolds are diversified in positions that bind both protein and peptide targets identified in antibody-antigen complexes of known structure using the amino acid frequencies found in those positions in known human antibody sequences, avoiding residues that may lead to developability liabilities. The diversified scaffolds are combined with 90 synthetic neutral HCDR3 sequences designed with developable human diversity genes (IGHD) and joining heavy genes (IGHJ) in germline configuration, and assembled as single-chain variable fragments (scFvs) in a VL-linker-VH orientation. The four designed PLs are synthesized using trinucleotide phosphoramidites (TRIMs) and cloned independently into a phagemid vector for M13 pIII display. Quality control of the cloning of the four PLs is also described, which involves sequencing scFvs in each library.

Semisynthetic Phage Display Library Construction: Generation of Filtered Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the second of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Gold Plus Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library (FL) construction, and (3) secondary library construction. The second step, described here, involves display of the PLs as single-chain variable fragment (scFv) fusions to protein pIII of the M13 phage, as well as heat shock treatment and subsequent selection of well-folded and thermostable scFvs via protein L binding, whereas unstable and defective scFvs are removed by washing steps and centrifugation. The quality of the filtration process is assessed by sequencing clones chosen at random from the FLs. These libraries, enriched with thermostable antibodies, are then ready to be used for the third and final step of the process: generation of secondary libraries.

Semisynthetic Phage Display Library Construction: Generation of Single-Chain Variable Fragment Secondary Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the third and final step of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library construction, (2) filtered library (FL) construction, and (3) secondary library (SL) construction. In the third step, described here, the nucleotide sequences encoding the single-chain variable fragments (scFvs) of FLs are amplified by PCR and combined with the heavy- chain CDR3 region (HCDR3) and joining fragments (H3J) obtained from a pool of donors to maximize diversity ("natural H3J fragments"). These natural H3J fragments are amplified with a set of primers designed to capture >95% of the natural H3J repertoire. The resultant fragments replace the neutral H3J fragments of the FLs, resulting in the final semisynthetic secondary libraries. The quality of these libraries is assessed by sequencing clones chosen at random from the libraries, typically 96 clones. These libraries are then ready to be used for phage selections on targets of interest, providing a robust antibody discovery platform.

Generation of Antibody Libraries for Phage Display: Preparation of Helper Phage.

The generation and selection of antibody libraries by phagemid-based phage display requires three components; namely, phagemid library, host bacterial cells, and helper phage. The use of helper phage is necessary for the selection of phagemid libraries by phage display because it provides all genes needed for production of infectious phage particles. Here, we describe the generation of high-titer helper phage preparations suitable for phagemid-based phage display. The approach is based on helper phage VCSM13, which includes a gene for kanamycin resistance and a mutated packaging signal that, in the presence of a phagemid with an unmutated packaging signal, favors the production of infectious phage particles with phagemid phenotype and genotype.

Generation of Antibody Libraries for Phage Display: Human Fab Format.

Phage display is a powerful method for the de novo generation and affinity maturation of human monoclonal antibodies from naive, immune, and synthetic antibody repertoires. The pComb3 phagemid family of phage display vectors facilitates the selection of human monoclonal antibody libraries in the monovalent Fab format, which consists of human variable domains VH and VL (Vκ or Vλ), fused to the human constant domains CH1 of IgG1 and CL (Cκ or Cλ), respectively. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of human Fab libraries with randomly combined human variable domains (VH, Vκ, and Vλ) of high sequence diversity, starting from the preparation of mononuclear cells from blood and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 108-1011 independent human Fab clones. As such, it can be used, for instance, for the generation of a large naive human Fab library from healthy individuals or for the generation of a specialized immune human Fab library from individuals with an endogenous antibody response of interest.

Generation of Antibody Libraries for Phage Display: Preparation of Electrocompetent E. coli.

The size of an antibody library, that is, the phage display-selectable diversity, is restricted mainly by its transformation into the host bacterial cells. Electroporation is the most efficient method for transforming Escherichia coli with plasmids, including phagemids. Here, we describe the preparation of electrocompetent E. coli for the generation of phagemid-encoded antibody libraries encompassing 109-1011 independent transformants. To become electrocompetent, the bacterial suspension has to have high resistance, i.e., low ionic strength, which is achieved by gradually and gently transferring bacteria grown to mid-log phase to 10% (v/v) glycerol in highly pure water. The electrocompetent E. coli must be F plasmid-harboring bacteria, referred to as F+ or male, in order to express F pili and be susceptible to infection by filamentous phage during library generation. In addition, it is necessary to apply antibiotic (e.g., tetracycline) pressure to retain the F plasmid, as it tends to segregate from bacteria. This protocol also includes assays for analyzing the prepared electrocompetent E. coli for competency, and evaluating potential contamination with helper phage, phagemid and phagemid-derived filamentous phage, and lytic phage.

Generation of Antibody Libraries for Phage Display: Chimeric Rabbit/Human Fab Format.

Rabbit monoclonal antibodies are attractive reagents for research, and have also found use in diagnostic and therapeutic applications. This is owed to their high affinity and specificity, along with their ability to recognize epitopes conserved between mouse and human antigens. Phage display is a powerful method for the de novo generation, affinity maturation, and humanization of rabbit monoclonal antibodies from naive, immune, and synthetic antibody repertoires. Using phagemid family pComb3, a preferred phage display format is chimeric rabbit/human Fab, which consists of rabbit variable domains (VH, Vκ, and Vλ) fused to human constant domains. The human constant domains, CH1 of IgG1 and CL (Cκ or Cλ), not only provide established purification and detection handles but also facilitate higher expression in Escherichia coli compared to the corresponding rabbit constant domains. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of chimeric rabbit/human Fab libraries with randomly combined rabbit variable domains of high sequence diversity, starting from the preparation of total RNA from rabbit spleen and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 108-1011 independent chimeric rabbit/human Fab clones. As such, it can be used, for instance, for the generation of either specialized immune or large naive rabbit antibody libraries.

Generation of Antibody Libraries for Phage Display: Library Reamplification.

Phage display of Fab libraries enables the de novo discovery and in vitro evolution of monoclonal antibodies. Fab libraries are collections of millions to billions of different antibodies that collectively cover a large antigen or epitope binding space. To preserve the diversity of the Fab library for repeated selection campaigns, it is recommended to use the original phage from the Fab library generation rather than reamplified phage, if practically possible. This is because reamplification will bias the Fab library for clones that are expressed at higher rates. Fab-phage, however, should only be used if they have been prepared on the same day, to avoid proteolytic cleavage of the physical linkage of phenotype (phage-displayed Fab protein) and genotype (phage-encapsulated Fab DNA). Thus, in practice, reamplification of a Fab-phage library cannot usually be avoided. Here, we describe the steps for the reamplification of an original Fab-phage library prior to its selection. The protocol can also be used to reamplify Fab-phage from the third or later panning rounds when enriched clones are unlikely to be lost by reamplification biases.

Cloning, Expression, and Purification of Phage Display-Selected Fab for Biophysical and Biological Studies.

The antigen-binding fragment (Fab) is the ∼50-kDa monovalent arm of an antibody molecule. In the laboratory, the Fab can be produced via either enzymatic digestion or recombinant expression, and its use facilitates the accurate assessment of affinity and specificity of monoclonal antibodies. The high melting temperature of the Fab, together with its low tendency to aggregate and ready conversion to natural and nonnatural immunoglobulin (Ig) formats (without affecting antigen binding properties), have made it a preferred format for phage display, as well as a tool for accurate assessment of affinity, specificity, and developability of monoclonal antibodies. Here, we outline a strategy to clone, express, and purify human or chimeric nonhuman/human Fabs that have previously been selected by phage display. Fabs purified using this approach, which results in milligram amounts, enable a variety of downstream biophysical and biological assays that ultimately inform the success of phage display library generation and selection.

IDEIS: a tool to identify PTPRC/CD45 isoforms from single-cell transcriptomic data.

Single-cell RNA sequencing (scRNA-seq) methods are widely used in life sciences, including immunology. Typical scRNA-seq analysis pipelines quantify the abundance of particular transcripts without accounting for alternative splicing. However, a well-established pan-leukocyte surface marker, CD45, encoded by the PTPRC gene, presents alternatively spliced variants that define different immune cell subsets. Information about some of the splicing patterns in particular cells in the scRNA-seq data can be obtained using isotype-specific DNA oligo-tagged anti-CD45 antibodies. However, this requires generation of an additional sequencing DNA library. Here, we present IDEIS, an easy-to-use software for CD45 isoform quantification that uses single-cell transcriptomic data as the input. We showed that IDEIS accurately identifies canonical human CD45 isoforms in datasets generated by 10× Genomics 5' sequencing assays. Moreover, we used IDEIS to determine the specificity of the Ptprc splicing pattern in mouse leukocyte subsets.

FastAd: A versatile toolkit for rapid generation of single adenoviruses or diverse adenoviral vector libraries

Adenoviruses (Ads) are potent gene delivery vectors for in vitro and in vivo applications. However, current methods for their construction are time-consuming and inefficient, limiting their rapid production and utility in generating complex genetic libraries. Here, we introduce FastAd, a rapid and easy-to-use technology for inserting recombinant “donor” DNA directly into infectious “receiver” Ads in mammalian cells by the concerted action of two efficient recombinases: Cre and Bxb1. Subsequently, the resulting mixed recombinant Ad population is subjected to negative selections by flippase (FLP) recombinase to remove viruses that missed the initial recombination. With this approach, recombinant Ad production time is reduced from two months to 10 days or less. FastAd can be applied for inserting complex genetic DNA libraries into Ad genomes, as evidenced by the generation of barcode libraries with over 3 million unique clones from a T25 flask-scale of transfection. Furthermore, we leveraged FastAd to construct an Ad library containing a comprehensive genome-wide CRISPR/Cas9 guide RNA (gRNA) library and demonstrated its effectiveness in uncovering novel virus-host interactions. In summary, FastAd enables the rapid generation of single Ad vectors or complex genetic libraries, facilitating not only novel applications of Ad vectors but also research in foundational Ad biology.

Computational Analysis of T-Cell Receptor Repertoire Workflow: From T-Cell Isolation to Bioinformatics Analysis.

The T-cell receptor (TCR) is the key molecule involved in the adaptive immune response. It is generated by the V(D)J recombination, responsible of the enormous diversity of the TCR repertoire, a crucial feature determining the individual capability to response to antigens and to build immunological memory. A pivotal role in the recognition of antigen is played by the hypervariable complementarity-determining region 3 (CDR3) of the V-beta chain of TCR. Investigating the CDR3 supports the understanding of the adaptive immune system dynamics in physiological processes, such as immune aging, and in disease, especially autoimmune disorders in which T cells are main actors. High-throughput sequencing (HTS) paved the way for a great progress in the investigation of TCR repertoire, enhancing the read depth in the process of library generation of sequencing and the number of samples that can be analyzed simultaneously. Therefore, the leverage of big datasets stressed the need to develop computational approach, by bioinformatics, to unravel the characteristics of the TCR repertoire.

Barcoding of Italian mosquitoes (BITMO): generation and validation of DNA barcoding reference libraries for native and alien species of Culicidae

BackgroundMosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2).MethodsA total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed.ResultsNovel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI.ConclusionsThis study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches.Graphical abstract

Introducing synthetic thermostable RNase inhibitors to single-cell RNA-seq

Single-cell RNA-sequencing (scRNAseq) is revolutionizing biomedicine, propelled by advances in methodology, ease of use, and cost reduction of library preparation. Over the past decade, there have been remarkable technical improvements in most aspects of single-cell transcriptomics. Yet, little to no progress has been made in advancing RNase inhibition despite maintained RNA integrity being critical during cell collection, storage, and cDNA library generation. Here, we demonstrate that a synthetic thermostable RNase inhibitor (SEQURNA) yields single-cell libraries of equal or superior quality compared to ubiquitously used protein-based recombinant RNase inhibitors (RRIs). Importantly, the synthetic RNase inhibitor provides additional unique improvements in reproducibility and throughput, enables new experimental workflows including retained RNase inhibition throughout heat cycles, and can reduce the need for dry-ice transports. In summary, replacing RRIs represents a substantial advancement in the field of single-cell transcriptomics.

Generic FPGA Pre-Processing Image Library for Industrial Vision Systems.

Currently, there is a demand for an increase in the diversity and quality of new products reaching the consumer market. This fact imposes new challenges for different industrial sectors, including processes that integrate machine vision. Hardware acceleration and improvements in processing efficiency are becoming crucial for vision-based algorithms to follow the complexity growth of future industrial systems. This article presents a generic library of pre-processing filters for execution in field-programmable gate arrays (FPGAs) to reduce the overall image processing time in vision systems. An experimental setup based on the Zybo Z7 Pcam 5C Demo project was developed and used to validate the filters described in VHDL (VHSIC hardware description language). Finally, a comparison of the execution times using GPU and CPU platforms was performed as well as an evaluation of the integration of the current work in an industrial application. The results showed a decrease in the pre-processing time from milliseconds to nanoseconds when using FPGAs.

FADO: Floorplan-Aware Directive Optimization Based on Synthesis and Analytical Models for High-Level Synthesis Designs on Multi-Die FPGAs

Multi-die FPGAs are widely adopted for large-scale accelerators, but optimizing high-level synthesis designs on these FPGAs faces two challenges. First, the delay caused by die-crossing nets creates an NP-hard floorplanning problem. Second, traditional directive optimization cannot consider resource constraints on each die or the timing issue incurred by the die-crossings. Furthermore, the high algorithmic complexity and the large scale lead to extended runtime for legalizing the floorplan of HLS designs under different directive configurations. To co-optimize the directives and floorplan of HLS designs on multi-die FPGAs, we formulate the co-search based on bin-packing variants and present two iterative optimization flows. The first (FADO 1.0) relies on a pre-built QoR library. It involves a greedy, latency-bottleneck-guided directive search, and an incremental floorplan legalization. Compared with a global floorplanning solution, it takes 693X~4925X shorter search time and achieves 1.16X~8.78X better design performance, measured in workload execution time. To remove the time-consuming QoR library generation, the second flow (FADO 2.0) integrates an analytical QoR model and redesigns the directive search to accelerate convergence. Through experiments on mixed dataflow and non-dataflow designs, compared with 1.0, FADO 2.0 further yields a 1.40X better design performance on average after implementation on the Alveo U250 FPGA.

MetaExpertPro: A Computational Workflow for Metaproteomics Spectral Library Construction and Data-Independent Acquisition Mass Spectrometry Data Analysis

Analysis of large-scale data-independent acquisition mass spectrometry metaproteomics data remains a computational challenge. Here, we present a computational pipeline called metaExpertPro for metaproteomics data analysis. This pipeline encompasses spectral library generation using data-dependent acquisition MS, protein identification and quantification using data-independent acquisition mass spectrometry, functional and taxonomic annotation, as well as quantitative matrix generation for both microbiota and hosts. By integrating FragPipe and DIA-NN, metaExpertPro offers compatibility with both Orbitrap and timsTOF MS instruments. To evaluate the depth and accuracy of identification and quantification, we conducted extensive assessments using human fecal samples and benchmark tests. Performance tests conducted on human fecal samples indicated that metaExpertPro quantified an average of 45,000 peptides in a 60-min diaPASEF injection. Notably, metaExpertPro outperformed three existing software tools by characterizing a higher number of peptides and proteins. Importantly, metaExpertPro maintained a low factual false discovery rate of approximately 5% for protein groups across four benchmark tests. Applying a filter of five peptides per genus, metaExpertPro achieved relatively high accuracy (F-score=0.67-0.90) in genus diversity and showed a high correlation (rSpearman=0.73-0.82) between the measured and true genus relative abundance in benchmark tests. Additionally, the quantitative results at the protein, taxonomy, and function levels exhibited high reproducibility and consistency across the commonly adopted public human gut microbial protein databases IGC and UHGP. In a metaproteomic analysis of dyslipidemia patients, metaExpertPro revealed characteristic alterations in microbial functions and potential interactions between the microbiota and the host.

A Versatile Method for Site-Specific Chemical Installation of Aromatic Posttranslational Modification Analogs into Proteins.

Posttranslational modifications (PTMs) of proteins play central roles in regulating the protein structure, interactome, and functions. A notable modification site is the aromatic side chain of Tyr, which undergoes modifications such as phosphorylation and nitration. Despite the biological and physiological importance of Tyr-PTMs, our current understanding of the mechanisms by which these modifications contribute to human health and disease remains incomplete. This knowledge gap arises from the absence of natural amino acids that can mimic these PTMs and the lack of synthetic tools for the site-specific introduction of aromatic PTMs into proteins. Herein, we describe a facile method for the site-specific chemical installation of aromatic PTMs into proteins through palladium-mediated S-C(sp2) bond formation under ambient conditions. We demonstrate the incorporation of novel PTMs such as Tyr-nitration and phosphorylation analogs to synthetic and recombinantly expressed Cys-containing peptides and proteins within minutes and in good yields. To demonstrate the versatility of our approach, we employed it to prepare 10 site-specifically modified proteins, including nitrated and phosphorylated analogs of Myc and Max proteins. Furthermore, we prepared a focused library of site-specifically nitrated and phosphorylated α-synuclein (α-Syn) protein, which enabled, for the first time, deciphering the role of these competing modifications in regulating α-Syn conformation aggregation in vitro. Our strategy offers advantages over synthetic or semisynthetic approaches, as it enables rapid and selective transfer of rarely explored aromatic PTMs into recombinant proteins, thus facilitating the generation of novel libraries of homogeneous posttranslationally modified proteins for biomarker discovery, mechanistic studies, and drug discovery.