Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.     

Looking for therapeutic antibodies in next-generation sequencing repositories

ABSTRACT

Recently it has become possible to query the great diversity of natural antibody repertoires using next-generation sequencing (NGS). These methods are capable of producing millions of sequences in a single experiment. Here we compare clinical-stage therapeutic antibodies to the ~1b sequences from 60 independent sequencing studies in the Observed Antibody Space database, which includes antibody sequences from NGS analysis of immunoglobulin gene repertoires. Of 242 post-Phase 1 antibodies, we found 16 with sequence identity matches of 95% or better for both heavy and light chains. There are also 54 perfect matches to therapeutic CDR-H3 regions in the NGS outputs, suggesting a nontrivial amount of convergence between naturally observed sequences and those developed artificially. This has potential implications for both the legal protection of commercial antibodies and the discovery of antibody therapeutics.     

Analysis of T-cell receptor repertoire in peripheral blood of patients with pancreatic cancer and other pancreatic diseases

Pancreatic cancer (PC) has been the fourth cancer-related death worldwide, diagnosed at an unresectable stage due to its rapid progression and few symptoms of this disease at early stages. The aim of this study was to determine the association between the diversity of T-cell receptor (TCR) repertoire and clinicopathological characteristics of patients with PC and other benign pancreatic diseases. In order to make a comprehensive analysis the TCR repertoire, high-throughput sequencing was used to differentiate complementarity determining region 3 (CDR3) of the TCR β chain in peripheral blood samples from 3 PC, 3 chronic pancreatitis, 3 pancreatic cystic lesions and 3 pancreatic neuroendocrine tumour patients. We found that there were significant differences related to TCR repertoire between PC and other pancreatic diseases, and PC is a relatively immunosuppressive tumour. Changes of peripheral TCR repertoire may be used to predict the progression of PC and the response to immunotherapy. And there may exist novel-specific antigens in PC patients which could be used to design targeting immunotherapy in the nearly future.     

Complexity of the microRNA repertoire revealed by next-generation sequencing

MicroRNAs (miRNAs) have been implicated to play key roles in normal physiological functions, and altered expression of specific miRNAs has been associated with a number of diseases. It is of great interest to understand their roles and a prerequisite for such study is the ability to comprehensively and accurately assess the levels of the entire repertoire of miRNAs in a given sample. It has been shown that some miRNAs frequently have sequence variations termed isomirs. To better understand the extent of miRNA sequence heterogeneity and its potential implications for miRNA function and measurement, we conducted a comprehensive survey of miRNA sequence variations from human and mouse samples using next generation sequencing platforms. Our results suggest that the process of generating this isomir spectrum might not be random and that heterogeneity at the ends of miRNA affects the consistency and accuracy of miRNA level measurement. In addition, we have constructed a database from our sequencing data that catalogs the entire repertoire of miRNA sequences (http://galas.systemsbiology.net/cgi-bin/isomir/find.pl). This enables users to determine the most abundant sequence and the degree of heterogeneity for each individual miRNA species. This information will be useful both to better understand the functions of isomirs and to improve probe or primer design for miRNA detection and measurement.     

The functional repertoire of rabbit antibodies and antibody discovery via next-generation sequencing

To gain insight into the functional antibody repertoire of rabbits, the VH and VL repertoires of bone marrow (BM) and spleen (SP) of a naïve New Zealand White rabbit (NZW; Oryctolagus cuniculus) and that of lymphocytes collected from a NZW rabbit immunized (IM) with a 16-mer peptide were deep-sequenced. Two closely related genes, IGHV1S40 (VH1a3) and IGHV1S45 (VH4), were found to dominate (~90%) the VH repertoire of BM and SP, whereas, IGHV1S69 (VH1a1) contributed significantly (~40%) to IM. BM and SP antibodies recombined predominantly with IGHJ4. A significant proportion (~30%) of IM sequences recombined with IGHJ2. The VK repertoire was encoded by nine IGKV genes recombined with one IGKJ gene, IGKJ1. No significant bias in the VK repertoire of the BM, SP and IM samples was observed. The complementarity-determining region (CDR)-H3 and -L3 length distributions were similar in the three samples following a Gaussian curve with average length of 12.2 ± 2.4 and 11.1 ± 1.1 amino acids, respectively. The amino acid composition of the predominant CDR-H3 and -L3 loop lengths was similar to that of humans and mice, rich in Tyr, Gly, Ser and, in some specific positions, Asp. The average number of mutations along the IGHV/KV genes was similar in BM, SP and IM; close to 12 and 15 mutations for VH and VL, respectively. A monoclonal antibody specific for the peptide used as immunogen was obtained from the IM rabbit. The CDR-H3 sequence was found in 1,559 of 61,728 (2.5%) sequences, at position 10, in the rank order of the CDR-H3 frequencies. The CDR-L3 was found in 24 of 11,215 (0.2%) sequences, ranking 102. No match was found in the BM and SP samples, indicating positive selection for the hybridoma sequence. Altogether, these findings lay foundations for engineering of rabbit V regions to enhance their potential as therapeutics, i.e., design of strategies for selection of specific rabbit V regions from NGS data mining, humanization and design of libraries for affinity maturation campaigns.     

The functional repertoire of rabbit antibodies and antibody discovery via next-generation sequencing

To gain insight into the functional antibody repertoire of rabbits, the VH and VL repertoires of bone marrow (BM) and spleen (SP) of a naïve New Zealand White rabbit (NZW; Oryctolagus cuniculus) and that of lymphocytes collected from a NZW rabbit immunized (IM) with a 16-mer peptide were deep-sequenced. Two closely related genes, IGHV1S40 (VH1a3) and IGHV1S45 (VH4), were found to dominate (~90%) the VH repertoire of BM and SP, whereas, IGHV1S69 (VH1a1) contributed significantly (~40%) to IM. BM and SP antibodies recombined predominantly with IGHJ4. A significant proportion (~30%) of IM sequences recombined with IGHJ2. The VK repertoire was encoded by nine IGKV genes recombined with one IGKJ gene, IGKJ1. No significant bias in the VK repertoire of the BM, SP and IM samples was observed. The complementarity-determining region (CDR)-H3 and -L3 length distributions were similar in the three samples following a Gaussian curve with average length of 12.2 ± 2.4 and 11.1 ± 1.1 amino acids, respectively. The amino acid composition of the predominant CDR-H3 and -L3 loop lengths was similar to that of humans and mice, rich in Tyr, Gly, Ser and, in some specific positions, Asp. The average number of mutations along the IGHV/KV genes was similar in BM, SP and IM; close to 12 and 15 mutations for VH and VL, respectively. A monoclonal antibody specific for the peptide used as immunogen was obtained from the IM rabbit. The CDR-H3 sequence was found in 1,559 of 61,728 (2.5%) sequences, at position 10, in the rank order of the CDR-H3 frequencies. The CDR-L3 was found in 24 of 11,215 (0.2%) sequences, ranking 102. No match was found in the BM and SP samples, indicating positive selection for the hybridoma sequence. Altogether, these findings lay foundations for engineering of rabbit V regions to enhance their potential as therapeutics, i.e., design of strategies for selection of specific rabbit V regions from NGS data mining, humanization and design of libraries for affinity maturation campaigns.     

Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals

Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.     

Comparing CDRH3 diversity captured from secondary lymphoid organs for the generation of recombinant human antibodies

The plasticity of natural immunoglobulin repertoires can be exploited for the generation of phage display libraries. Secondary lymphoid organs, such as the spleen and the lymph nodes, constitute interesting sources of diversity because they are rich in B cells, part of which can be affinity matured. These organs, however, differ in their anatomical structure, reflecting the different fluids they drain, which affects the B cell repertoires. The CDRH3 repertoires from these organs, extracted from naïve or immunized mice, were compared in the context of phage display libraries using human antibody framework families. Deep sequencing analysis revealed that all libraries displayed different CDRH3 repertoires, but the one derived from lymph nodes of naïve mice was the most diverse. Library performance was assessed by in vitro selection. For both organs, immunization increased substantially the frequency of molecules able to bind to the immunogen. The library derived from lymph nodes from naïve mice, however, was the most effective in generating diverse and high affinity candidates. These results illustrate that the use of a biased CDRH3 repertoire increases the performance of libraries, but reduces the clonal diversity, which may be detrimental for certain strategies.     

Large-scale investigation of zoonotic viruses in the era of high-throughput sequencing

Zoonotic diseases considerably impact public health and socioeconomics. RNA viruses reportedly caused approximately 94% of zoonotic diseases documented from 1990 to 2010, emphasizing the importance of investigating RNA viruses in animals. Furthermore, it has been estimated that hundreds of thousands of animal viruses capable of infecting humans are yet to be discovered, warning against the inadequacy of our understanding of viral diversity. High-throughput sequencing (HTS) has enabled the identification of viral infections with relatively little bias. Viral searches using both symptomatic and asymptomatic animal samples by HTS have revealed hidden viral infections. This review introduces the history of viral searches using HTS, current analytical limitations, and future potentials. We primarily summarize recent research on large-scale investigations on viral infections reusing HTS data from public databases. Furthermore, considering the accumulation of uncultivated viruses, we discuss current studies and challenges for connecting viral sequences to their phenotypes using various approaches: performing data analysis, developing predictive modeling, or implementing high-throughput platforms of virological experiments. We believe that this article provides a future direction in large-scale investigations of potential zoonotic viruses using the HTS technology.     

High-throughput sequencing exclusively identified a novel Torque teno virus genotype in serum of a patient with fatal fever

Torque teno virus (TTV) has been found to be prevalent world-wide in healthy populations and in patients with various diseases, but its etiological role has not yet been determined. Using high-throughput unbiased sequencing to screen for viruses in the serum of a patient with persistent high fever who died of suspected viral infection and prolonged weakness, we identified the complete genome sequence of a TTV (isolate Hebei-1). The genome of TTV-Hebei-1 is 3649 bp in length, encoding four putative open reading frames, and it has a G+C content of 49%. Genomic comparison and a BLASTN search revealed that the assembled genome of TTV-Hebei-1 represented a novel isolate, with a genome sequence that was highly heterologous to the sequences of other reported TTV strains. A phylogenetic tree constructed using the complete genome sequence showed that TTV-Hebei-1 and an uncharacterized Taiwanese strain, TW53A37, constitute a new TTV genotype. The patient was strongly suspected of carrying a viral infection and died eventually without any other possible causes being apparent. No virus other than the novel TTV was identified in his serum sample. Although a direct causal link between the novel TTV genotype infection and the patient’s disease could not be confirmed, the findings suggest that surveillance of this novel TTV genotype is necessary and that its role in disease deserves to be explored.     

马属动物全基因组高通量测序研究进展

马属(Equus)动物的祖先大约在5500万年前出现,经过持续分化形成现今的马、驴和斑马,统称马属动物。马作为家畜中的重要一员,是推动人类文明发展的载体,在人类的饮食、战争、农耕、运输和娱乐等领域做出了巨大贡献。然而,人类为了满足需求,或多或少影响着马的进化方向,从而在长时间自然和人工选择过程中形成了多种独具特色性状的不同马种。驴和骡在全球的存栏量也较多,在人类的生产和生活中起到的作用同样不可忽视,不但为人类提供了生产力而且还提供了食物和营养保健品。可见,马属动物对人类的重要性。近年来,高通量测序技术和生物信息学分析方法被广泛运用于家畜的遗传学研究。人们利用高通量测序手段探索马属动物在进化过程中的种群变化历史,解析形成独特性状的分子机制,为其育种工作提供有效的数据支持。本文综述了马属动物全基因组高通量测序的研究进展,以及利用该技术在马属动物的进化历史和功能基因挖掘研究领域所取得的成就,以期今后对马属动物的深入研究、产业开发和利用等方面提供参考信息。     

基于高通量测序的不同年龄恒河猴肠道菌群结构差异分析

目的观察不同年龄恒河猴肠道菌群结构和丰度的变化。方法 50只恒河猴,分为成年(5～10岁)、老年(10岁以上)2组,其中成年组33只,老年组17只。采集动物的新鲜直肠粪便,提取DNA后使用Illumina高通量测序平台对样本中细菌16S rDNA-V3区进行测序,定量分析肠道菌群的结构和丰度。结果老年组和成年组获得的优化序列数差异无显著性(P> 0.05)。α-多样性分析,老年组恒河猴肠道菌群的Chao1指数(P=0.0174)、Simpson指数(P=0.0258)、ACE指数(P=0.0121)与成年组比较降低,Shannon指数与成年组比较升高(P=0.0132)。老年组与成年组相比,在门水平,拟杆菌门(Bacteroidetes)相对丰度升高(P=0.0013),厚壁菌门(Firmicutes)、螺旋体门(Spirochaetes)、疣微菌门(Verrucomicrobia)、粘胶球形菌门(Lentisphaerae)相对丰度降低(P=0.0283,P=0.0002,P=0.0482,P=0.0242)。在科水平,Prevotellaceae相对丰度升高(P=0.0001),Ruminococcaceae、Clostridiales、Spirochaetaceae、Christensenellaceae相对丰度降低(P=0.0039,P=0.0080,P=0.0002,P=0.0021)。在属水平,老年组unidentified_Prevotellaceae相对丰度升高(P=0.0001),乳杆菌属(Lactobacillus)、八迭球菌(Sarcina)、unidentified_Spirochaetaceae相对丰度降低(P=0.0114,P=0.0227,P=0.0028)。β-多样性分析,老年组肠道菌群与成年组分布在不同区域,差异有显著性(P=0.003)。LEfSe分析,在成年组,链球菌属(Streptococcus)、布赫纳氏菌属(buchnera)、乳杆菌属(lactobacillus)是具有统计学意义的生物标记物。结论恒河猴肠道菌群的结构随年龄增长而改变。丰度降低,多样性增高。     

基于高通量测序技术分析使它隆对玉米土壤细菌多样性的影响

【目的】研究使它隆对玉米土壤细菌多样性的影响。【方法】利用Illumina Miseq高通量测序技术,分别测定了玉米土壤细菌的16S rRNA基因的V4-V5可变区序列,进而对不同时期的不喷施除草剂和喷施除草剂的玉米土壤中细菌群落组成和多样性进行分析。【结果】研究共获得260940条有效序列,167191条优质序列,12656个OTUs。多样性分析结果表明,使它隆处理10 d后的土壤细菌多样性和丰度降低;使它隆处理60 d后的土壤细菌多样性和丰度提高。对土壤细菌群落组成分析发现,5个土壤样品中的优势菌门均为酸酐菌门、变形菌门、放线菌门、绿弯菌门和芽单胞菌门。使它隆处理10 d后的样品酸杆菌门的比例增加,放线菌门和绿弯菌门的比例降低;使它隆处理60 d后样品变形菌门的比例降低,绿弯菌门的比例明显增加。【结论】使它隆对玉米土壤细菌多样性产生一定影响,其影响随施药时间而异。     

基于高通量测序技术研究水貂远端肠道细菌群落组成

【目的】研究水貂远端肠道微生物群落组成及其多样性。【方法】通过高通量测序技术研究水貂远端肠内容物细菌的组成和多样性。【结果】从10只健康水貂远端肠道内容物样品中得到146 287高质量序列代表17个菌门、167个细菌属。水貂肠道细菌以厚壁菌门(59.99%)、拟杆菌门(16.2%)、梭杆菌门(11.5%)、放线菌门(5.9%)和变形菌门(5.3%)为主,其中厚壁菌门最为丰富。厚壁菌门中的梭菌目是最丰富的目,而梭菌目中的链球菌占有50%以上的OTUs,是最大的细菌属。【结论】水貂肠道内存在复杂的微生物区系,这对进一步研究水貂对营养物质吸收利用提供了理论基础。     

PRGmatic: an efficient pipeline for collating genome-enriched second-generation sequencing data using a 'provisional-reference genome'

Second-generation sequencing is increasingly being used in combination with genome-enrichment techniques to amplify a large number of loci in many individuals for the purpose of population genetic and phylogeographic analysis. Compiling all the necessary tools to analyse these data is complex and time-consuming. Here, we assemble a set of programs and pipe them together with Perl, enabling research laboratories without a dedicated bioinformatician to utilize second-generation sequencing. User input is a folder of the second-generation sequencing reads sorted by individual (in FASTA format) and pipeline output is a folder of multi-FASTA files that correspond to loci (with 2 alleles called per individual). Additional output includes a summary file of the number of individuals per locus, observed and expected heterozygosity for each locus, distribution of multiple hits and summary statistics (θ, Tajima's D, etc.). This user-friendly, open source pipeline, which requires no a priori reference genome because it constructs its own, allows the user to set various parameters (e.g. minimum coverage) in the dependent programs (CAP3, BWA, SAMtools and VarScan) and facilitates evaluation of the nature and quality of data collected prior to analysis in software packages.