Benchmarking and optimization of a high-throughput sequencing based method for transgene sequence variant analysis in biotherapeutic cell line development

In recent years, High-Throughput Sequencing (HTS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ∼60% down to ∼0.3% frequencies. We subsequently tested the effect of 16 different sample and HTS library preparation protocols on the assay's ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ∼0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations.     

Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing

Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day.     

Effective use of nitrocellulose-blotted antigens for phage display monoclonal antibody selection

The combinatorial phage display library approach to antibody repertoire cloning offers a powerful tool for the isolation of specific antibodies to defined target antigens. Panning strategy is often a very critical point for selecting antibody displayed on the surface of bacteriophages. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well defined antigen source. However, when the antigen is difficult to purify by means of laborious and time-consuming chromatography procedures, panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental complications. In the present work, we successfully generated a mouse monoclonal antibody fragment from a phage display library directed against protein E7 of HPV18 avoiding antigen purification as for immunizing mice as for antibody library selection. Our work demonstrates the feasibility of phage antibody selections on antigens transferred to a nitrocellulose membrane as solid support, using one-dimensional polyacrylamide gel electrophoresis system as the only practice to separate a given antigen present in bacterial crude cell lysate.     

Modular tagging of amplicons using a single PCR for high‐throughput sequencing

High‐throughput sequencing (HTS) of PCR amplicons is becoming the method of choice to sequence one or several targeted loci for phylogenetic and DNA barcoding studies. Although the development of HTS has allowed rapid generation of massive amounts of DNA sequence data, preparing amplicons for HTS remains a rate‐limiting step. For example, HTS platforms require platform‐specific adapter sequences to be present at the 5′ and 3′ end of the DNA fragment to be sequenced. In addition, short multiplex identifier (MID) tags are typically added to allow multiple samples to be pooled in a single HTS run. Existing methods to incorporate HTS adapters and MID tags into PCR amplicons are either inefficient, requiring multiple enzymatic reactions and clean‐up steps, or costly when applied to multiple samples or loci (fusion primers). We describe a method to amplify a target locus and add HTS adapters and MID tags via a linker sequence using a single PCR. We demonstrate our approach by generating reference sequence data for two mitochondrial loci (COI and 16S) for a diverse suite of insect taxa. Our approach provides a flexible, cost‐effective and efficient method to prepare amplicons for HTS.     

From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows

Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore imperative that careful attention is given to sample preparation and library generation within workflows, especially those involving multiple PCR steps. This paper redresses this imbalance by focusing on aspects pertaining to the benchtop within typical amplicon workflows: sample screening, the target region, and library generation. Empirical data is provided to illustrate the scope of the problem. Lastly, the impact of various data analysis parameters is also investigated in the context of how the data was initially generated. It is hoped this paper may serve to highlight the importance of pre-analysis workflows in achieving meaningful, future-proof data that can be analysed appropriately. As amplicon sequencing gains traction in a variety of diagnostic applications from forensics to environmental DNA (eDNA) it is paramount workflows and analytics are both fit for purpose.     

Bacteriophage Vehicles for Phage Display: Biology,Mechanism, and Application

The phage display technique is a powerful tool for selection of various biological agents. This technique allows construction of large libraries from the antibody repertoire of different hosts and provides a fast and high-throughput selection method. Specific antibodies can be isolated based on distinctive characteristics from a library consisting of millions of members. These features made phage display technology preferred method for antibody selection and engineering. There are several phage display methods available and each has its unique merits and application. Selection of appropriate display technique requires basic knowledge of available methods and their mechanism. In this review, we describe different phage display techniques, available bacteriophage vehicles, and their mechanism.     

The analysis of clonal expansions in normal and autoimmune B cell repertoires

Clones are the fundamental building blocks of immune repertoires. The number of different clones relates to the diversity of the repertoire, whereas their size and sequence diversity are linked to selective pressures. Selective pressures act both between clones and within different sequence variants of a clone. Understanding how clonal selection shapes the immune repertoire is one of the most basic questions in all of immunology. But how are individual clones defined? Here we discuss different approaches for defining clones, starting with how antibodies are diversified during different stages of B cell development. Next, we discuss how clones are defined using different experimental methods. We focus on high-throughput sequencing datasets, and the computational challenges and opportunities that these data have for mining the antibody repertoire landscape. We discuss methods that visualize sequence variants within the same clone and allow us to consider collections of shared mutations to determine which sequences share a common ancestry. Finally, we comment on features of frequently encountered expanded B cell clones that may be of particular interest in the setting of autoimmunity and other chronic conditions.     

Optimal construction of non-immune scFv phage display libraries from mouse bone marrow and spleen established to select specific scFvs efficiently binding to antigen

Monoclonal antibodies (MAbs) are widely applied in basic research, medicine, and the pharmaceutical industry. Recently, applications and generations of MAbs have been increasingly attracting attention in many research areas since MAbs could be produced in large quantities with the development of genetic technology and antibody engineering. On the other hand, in recent years, phage display system has been developed for high-throughput isolation and generation of novel MAbs that have high affinity with various antigens. This technology is capable of constructing "Library" containing billions of phage repertoires displaying various antibody fragments, and rapid selection of a specific MAb from this phage library. Additionally, this technology has a great advantage that MAbs can be generated without immunization to animals. However, there are still relatively few reports confirming that useful MAbs can be derived from non-immune antibody libraries. The latter, as undertaken by current methods, seem unable to achieve the high quality required to produce useful MAbs for any desired antigen because cloning of antibody gene from non-immune donors is inefficient. This problem is caused by the fact that their RT-PCR primer sets, PCR conditions, and efficiency of subcloning through construction of antibody gene library cannot encompass all the antibody diversity. In an attempt to overcome some of these earlier problems, here we describe an optimized method to establish a high quality, non-immune library from mouse bone-marrow and spleen, and assess its diversity in terms of content of multiple antibodies for a wide antigenic repertoire. As an example of the application of the methodology, we describe the selection of specific MAbs binding to Luciferase and identify at least 18 different clones. Using this non-immune mouse antibody library, we also obtained MAbs for VEGF, VEGF receptor 2, TNF-alpha, and Pseudomonas Exotoxin, confirming the high quality of the library and its suitability for this application.     

Functional study of 14-3-3 protein epsilon (YWHAE) in keratinocytes: microarray integrating bioinformatics approaches

Abstract

Previously, we detected that 14-3-3 protein epsilon (YWHAE) was involved in the pathogenesis of atopic dermatitis (AD) and tyrosinase-mediated pigmentation. In this study, we aimed to identify critical factors associated with YWHAE in human keratinocytes using high-throughput screening (HTS) approaches to reveal its functions in skin. We overexpressed YWHAE in human HaCaT keratinocytes and then conducted serial HTS studies, including RNA sequencing integrated with antibody arrays and the implementation of bioinformatics algorithms. Cumulatively, these approaches identified several novel genes in keratinocytes associated with the function of YWHAE including KRT9, KRT1, KRT6C, BST2, CIB2, APH1B, ACTC1, IFI27, TUBA1A, CAPN6, UTY, MX2, and MAPK15, based on RNA sequencing data, and MAPK1, MMP2, TYK2, NOS3, and CASP3, based on antibody array data. In particular, CD37 is a unique gene that was detected and validated in all the methods applied in this study. By integrating the datasets obtained from these HTS studies and utilizing the strengths of each method, we obtained new insights into the functional role of YWHAE in skin keratinocytes. The approach used here could contribute to the clinical understanding of YWHAE-associated applications in the treatment of AD disease. Abbreviations DAVID the database for annotation, visualization and integrated discovery

HTS High-throughput screening

KEGG Kyoto Encyclopedia of Genes and Genomes

PPI protein-protein interactions

Communicated by Ramaswamy H. Sarma     

Library preparation methods for next-generation sequencing: Tone down the bias

Next-generation sequencing (NGS) has caused a revolution in biology. NGS requires the preparation of libraries in which (fragments of) DNA or RNA molecules are fused with adapters followed by PCR amplification and sequencing. It is evident that robust library preparation methods that produce a representative, non-biased source of nucleic acid material from the genome under investigation are of crucial importance. Nevertheless, it has become clear that NGS libraries for all types of applications contain biases that compromise the quality of NGS datasets and can lead to their erroneous interpretation. A detailed knowledge of the nature of these biases will be essential for a careful interpretation of NGS data on the one hand and will help to find ways to improve library quality or to develop bioinformatics tools to compensate for the bias on the other hand. In this review we discuss the literature on bias in the most common NGS library preparation protocols, both for DNA sequencing (DNA-seq) as well as for RNA sequencing (RNA-seq). Strikingly, almost all steps of the various protocols have been reported to introduce bias, especially in the case of RNA-seq, which is technically more challenging than DNA-seq. For each type of bias we discuss methods for improvement with a view to providing some useful advice to the researcher who wishes to convert any kind of raw nucleic acid into an NGS library.     

噬菌体抗体库技术及其应用研究进展

噬菌体呈现抗体库是近年发展的一项分子生物学新技术,它的建立是抗体技术领域中的一次革命性进展。它以其独特的构建和筛选系统,彻底改变了抗体制备的传统途径,使抗体工程技术进入了一个新的发展阶段,并对生物学领域中许多技术的发展起到了巨大的推动作用。该技术是迄今发展最成熟、应用最广泛的制备抗体技术。我们简要综述此项技术的研究应用进展。     

An anti-leukemic single chain Fv antibody selected from a synthetic human phage antibody library

The display of human antibody repertoire on the cell surface of the filamentous bacteriophage has offered a novel strategy for selecting antibodies to a diverse range of purified targets. However, the selection of antibodies with biological functions has not yet been fully investigated. To select phage antibodies with therapeutic potential, a synthetic human single chain Fv (scFv) phage antibody library was panned on whole premyelocytic leukemia cell line (HL60). Phages binding to common receptors and undesirable phages were subtracted by incubating the library with human glioma cells. High affinity binding phages to HL60 cells were enriched by fluorescence-activated cell sorting. After the 6th round of selection, 50% of the selected phage antibodies showed significant binding to HL60 cells, whereas none of the analyzed phage antibodies bound to human pre-B cells (Nalm-6). In addition to binding, one scFv antibody inhibited HL60 cell proliferation by 90% compared to irrelevant scFv antibodies. Taken together the data demonstrate that specific scFv antibodies with biological functions can be isolated by using whole cells as affinity matrix.     

Environmental DNA metabarcoding: Transforming how we survey animal and plant communities

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High‐throughput sequencing (“HTS”) platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed “environmental DNA” or “eDNA”). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called “eDNA metabarcoding” and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.     

从噬菌体文库中筛选潜在的GMCSF的拮抗剂(英文)

以粒细胞巨噬细胞集落刺激因子（ＧＭＣＳＦ） 为筛选文库的靶分子, 通过高效筛选（Ｈｉｇｈ ｔｈｒｏｕｇｈｐｕｔｓｃｒｅｅｎｉｎｇ, ＨＴＳ） 方法来筛选多种多肽噬菌体文库, 在一个以噬菌体主要蛋白质为载体的多肽噬菌体文库中筛选到了一些与ＧＭＣＳＦ结合的多肽, 并通过了ＥＬＩＳＡ和微淘选（ｍｉｃｒｏｐａｎｎｉｎｇ） 实验的证实。这些多肽先导化合物经过进一步的优化, 可能成为ＧＭＣＳＦ细胞因子的拮抗剂     

Systematic error detection in experimental high-throughput screening

Background  

High-throughput screening (HTS) is a key part of the drug discovery process during which thousands of chemical compounds are screened and their activity levels measured in order to identify potential drug candidates (i.e., hits). Many technical, procedural or environmental factors can cause systematic measurement error or inequalities in the conditions in which the measurements are taken. Such systematic error has the potential to critically affect the hit selection process. Several error correction methods and software have been developed to address this issue in the context of experimental HTS [1–7]. Despite their power to reduce the impact of systematic error when applied to error perturbed datasets, those methods also have one disadvantage - they introduce a bias when applied to data not containing any systematic error [6]. Hence, we need first to assess the presence of systematic error in a given HTS assay and then carry out systematic error correction method if and only if the presence of systematic error has been confirmed by statistical tests.     

High-throughput assays for lipases and esterases

In the past few years a considerable number of high-throughput screening (HTS) systems have been developed, especially for lipases and esterases. In this review, a range of HTS methods for the directed evolution of these hydrolases are covered. This includes spectrophotometric and fluorimetric formats as well as other approaches to allow for fast, efficient and reliable identification of desired enzyme variants within large mutant libraries. In addition, methods for library creation and application of lipases and esterases are briefly covered.     

Cell-free immunology: construction and in vitro expression of a PCR-based library encoding a single-chain antibody repertoire

A novel cloning-independent strategy has been developed to generate a combinatorial library of PCR fragments encoding a murine single-chain antibody repertoire and express it directly in a cell-free system. The new approach provides an effective alternative to the techniques involving in vivo procedures of preparation and handling large libraries of antibodies. The possible use of the described strategy in the ribosome display is discussed.