Dataset Information

High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library.

ABSTRACT: In antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have technical limitations, such as low process throughput from the laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform for the identification and retrieval of clones in the library, TrueRepertoire™. This new platform provides highly accurate sequences of the clones with linkage information between heavy and light chains of the antibody fragment. Additionally, the physical DNA of clones can be retrieved in high throughput based on the sequence information. We validated the high accuracy of the sequences and demonstrated that there is no platform-specific bias. Moreover, the applicability of TrueRepertoire™ was demonstrated by a phage-displayed single-chain variable fragment library targeting human hepatocyte growth factor protein.

SUBMITTER: Noh J

PROVIDER: S-EPMC6512904 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library.

Noh Jinsung J Kim Okju O Jung Yushin Y Han Haejun H Kim Jung-Eun JE Kim Soohyun S Lee Sanghyub S Park Jaeseong J Jung Rae Hyuck RH Kim Sang Il SI Park Jaejun J Han Jerome J Lee Hyunho H Yoo Duck Kyun DK Lee Amos C AC Kwon Euijin E Ryu Taehoon T Chung Junho J Kwon Sunghoon S

mAbs 20190212 3

In antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have technical limitations, such as low process throughput from the laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform fo ...[more]

PMID: 30735467

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Project description:Next-generation sequences (NGS) dataset of nanobody (Nb) clones in a phage display library (PDL) is of immense value as it serves in many different ways, such as: i). estimating the library size, ii). improving selection and identification of Nbs, iii). informing about frequency of V gene families, diversity and length of CDRs, iv). high resolution analysis of natural and synthetic libraries, etc. [1], [2], [3]. We used a fraction of our previously constructed PDL of Nbs derived from an E. coli lipopolysaccharide-immunized Indian desert camel in order to obtain the dataset of NGS reads of Nbs. 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 [6]. 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 comprised of a total read count of 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. The raw data of NGS reads was submitted to NCBI Sequence Reads Archive accessible at URL: https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA516512 (dataset ref. [7]), and after analysis deposited in Mendeley Datasets repository, which is accessible at URL: [https://data.mendeley.com/datasets/4rsz3snvk5/3] (dataset ref. [8]). The sequence reads were analyzed by bioinformatics tools [9], [10], [11], [12]. The assembled consensus contigs revealed Nb orthologs of diverse Ag-specificities, including those isolated by conventional panning and Sanger-sequenced functional Nbs. Contig 1 CDR1-3 matched to those of anti-Trypanosoma evansi RoTat1.2 variant surface glycoprotein (VSG), while Contig 2 CDR1-3 matched to those of anti-LPS Nb clones isolated from the library. Contig 3 was however incomplete and lacked CDR3. Despite lacking the depth, the NGS data is a useful guide for selection of antigen-specific Nbs from the library, as demonstrated by anti-T. evansi VSG Nbs, and provides templates for Nb-based diagnostic reagents and therapeutic agents.

Dataset Information

High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library.

Publications

High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library.

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