Dataset of NGS-Phage display library of nanobodies derived from Indian desert camel (Camelus dromedarius L.)

We subjected to NGS methodology a fraction of our previously constructed phage display library (PDL) of nanobody (Nb) clones (Cl) for obtaining the dataset of Nbs NGS reads. This is the first phage display library of Nbs derived from the Indian desert camel and its initial preliminary characterization revealed functional Nbs against diverse Ags from infectious agents. Several Nb clones were isolated by panning against E. coli endotoxin and S. aureus β-hemolysin (an exotoxin), and were deposited, along with the library, in ICAR-Veterinary Type Culture Collection at Hisar (Haryana) (Accession no. VTCCMBA22 to VTCCMBA48). Some of these Nb clones were characterized for biochemical and functional features, and Sanger sequenced (GenBank accession no.: EU861212; KF990215; KF990216; KF990217; GU014816). For NGS, the cryo-preserved transformants library was revived to extract the Nb-encoding VHH (inserts)-pHEN4 (vector) DNA pool. The DNA sample was used for amplifying VHH pool by PCR. The VHH amplicons band was gel-purified and subjected to NGS using Illumina MiSeqTM platform. ‘Nextra XT micro V2 Index’ kit was used for the Nb library DNA sample sequencing, with the adaptors: ‘i7’ (N706: TAGGCATG) and ‘i5’ (S517: GCGTAAGA). The raw data of NGS reads was submitted to NCBI ‘Sequence Reads Archive’ repository. Raw NGS data ‘Read 1’ [Phage lib NGS seqs-ABT-28_S24_L001_R1_001.fastq (1)] and ‘Read 2’ [Phage lib NGS seqs-ABT-28_S24_L001_R2_001.fastq (2)] files were generated by Illumina® MiSeq™ system. Preliminary examination using CLC Genomics Workbench 12.0 revealed 91,073 sequence reads in each file. Quality score ‘Q30’ (1 in 1000 bases incorrect) at 2x150 bp was >80%, i.e., acceptable. A total read count was 182146 (matched= 179591; unmatched=2555), with average read length of 130.33 bases and a total of 23.74 Mb. Of 179591 matched reads, 142004 were paired reads and 37587 broken paired reads. These data can be accessed at SRA RunSelector: https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA516512 Three consensus contigs were present in the NGS reads. Contig 1 and -2 have CDR1, -2 and -3, whereas contig 3 being of shorter length, has only CDR 1 and -2. The contig 1 CDR1, -2 and -3 are identical to the T. evansi VSG binder Nb clones isolated from the library and sequenced by Sanger’s technique. The contig 2 CDR 1 and -2 are identical and CDR3 highly similar to the LPS-binder Nb clones previously isolated from the library. VHH hallmark amino acids are present in all the contigs. Blastn of contig 1-3 consensus sequences revealed homologies to sequences of Nbs specific to E. coli LPS, Trypanosoma evansi variable surface glycoprotein (VSG), and several other important antigens of veterinary and human pathogens. Anti-LPS Nb clones are highly similar to NGS sequence reads. NGS reads guided the isolation of anti-T. evansi RoTat1.2 VSG Nb clones. This approach could be used for the search of other Nbs and templates for Nb reformatting and Nb-based drug designing.本研究采用下一代测序（Next-Generation Sequencing, NGS）技术，对本课题组前期构建的纳米抗体（nanobody, Nb）克隆（clone, Cl）噬菌体展示库（phage display library, PDL）的部分样本进行测序，以获取纳米抗体NGS读段数据集。该纳米抗体噬菌体展示库是首个源自印度沙漠骆驼的纳米抗体库，初步表征结果显示，库内存在可识别多种传染性病原体抗原（antigen, Ag）的功能性纳米抗体。本研究通过针对大肠杆菌（Escherichia coli, E. coli）内毒素及金黄色葡萄球菌（Staphylococcus aureus, S. aureus）β-溶血素（一种外毒素）的淘筛实验，分离得到多株纳米抗体克隆，并与该噬菌体展示库一同保藏于印度希萨尔（哈里亚纳邦）的ICAR兽医模式菌种保藏中心，保藏编号为VTCCMBA22至VTCCMBA48。其中部分纳米抗体克隆已完成生化与功能特性表征，并通过桑格测序（Sanger sequencing）获得序列，其GenBank登录号为EU861212、KF990215、KF990216、KF990217及GU014816。本次NGS实验中，我们复苏了低温保藏的转化子库，提取编码纳米抗体的重链可变区（variable heavy chain of heavy-chain antibodies, VHH，插入片段）-pHEN4载体DNA混合样本。以该DNA样本为模板，通过聚合酶链式反应（polymerase chain reaction, PCR）扩增VHH混合片段。扩增得到的VHH产物经凝胶纯化后，采用Illumina MiSeq™平台进行NGS测序。本次测序使用Nextra XT micro V2 Index试剂盒，采用的接头为i7（N706：TAGGCATG）与i5（S517：GCGTAAGA）。本研究将NGS读段的原始数据提交至美国国家生物技术信息中心（National Center for Biotechnology Information, NCBI）的序列读段档案库（Sequence Reads Archive）。原始NGS数据包含Read 1与Read 2两个文件，文件名分别为Phage lib NGS seqs-ABT-28_S24_L001_R1_001.fastq (1)与Phage lib NGS seqs-ABT-28_S24_L001_R2_001.fastq (2)，由Illumina® MiSeq™测序系统生成。使用CLC Genomics Workbench 12.0进行初步质控分析显示，每个文件包含91073条序列读段。在2×150 bp的测序模式下，Q30质量值（即每1000个碱基中错误碱基不超过1个）占比超过80%，符合测序质量要求。本次实验总读段数为182146条（匹配读段179591条，未匹配读段2555条），平均读长为130.33 bp，总数据量为23.74 Mb。在179591条匹配读段中，142004条为成对读段，37587条为断裂成对读段。上述数据可通过SRA RunSelector平台访问：https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA516512。NGS读段中存在3条一致性重叠群（contig）：重叠群1与2包含互补决定区1、2、3（complementarity-determining region, CDR），而长度较短的重叠群3仅包含CDR1与CDR2。重叠群1的CDR1、CDR2与CDR3序列，与本库中通过桑格测序分离得到的伊氏锥虫（Trypanosoma evansi, T. evansi）可变表面糖蛋白（variable surface glycoprotein, VSG）结合纳米抗体克隆序列完全一致。重叠群2的CDR1与CDR2序列与此前从本库中分离得到的脂多糖（lipopolysaccharide, LPS）结合纳米抗体克隆完全一致，其CDR3序列与该类克隆高度相似。所有重叠群均包含VHH的标志性氨基酸序列。对3条一致性重叠群序列进行核苷酸BLAST（Blastn）分析显示，其与靶向大肠杆菌脂多糖、伊氏锥虫可变表面糖蛋白以及其他多种人畜共患病原重要抗原的纳米抗体序列具有同源性。抗脂多糖纳米抗体克隆与NGS序列读段高度相似。本次NGS测序数据辅助成功分离得到抗伊氏锥虫RoTat1.2可变表面糖蛋白纳米抗体克隆。该方法可用于筛选其他纳米抗体，以及为纳米抗体重构及基于纳米抗体的药物设计提供模板。