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

Published: 07-12-2020| Version 3 | DOI: 10.17632/4rsz3snvk5.3
Contributors:
Ajit Singh,
Somesh Banerjee,
Jagveer Rawat,
Sushila Maan,
Nitish Bansal,
Savita Budania

Description

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.

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