Construction and next-generation sequencing analysis of a large phage-displayed V(NAR) single-domain antibody library from six naïve nurse sharks

Feng, Mingqian; Bian, Hejiao; Wu, Xiaolin; Fu, Tianyun; Fu, Ying; Hong, Jessica; Fleming, Bryan D; Flajnik, Martin F; Ho, Mitchell

NGS of the library was generated from shark V NAR insert DNA digested out of plasmid library using XhoI and MscI restriction enzymes (New England Biolabs). The shark V NAR library was excised out from the vector with restriction enzyme (XhoI and MscI) and gel purified. The insert fragments were ligated to Illumina adaptor and sequenced on Illumina MiSeq with 2 × 250 bp paired-end reads using both NEBNext Ultra II DNA library preparation kit and Kappa Hyper Prep library preparation kit. Paired-end reads were merged to cover the full length of the insert using FLASH [42] . Merged sequence reads were then analyzed with custom Perl Scripts. All DNA sequences were oriented and translated to protein sequences. Sequences with stop codons were removed from further analysis. We also required the amino acid sequences to start with "RV" at the N-terminus and end with "XXXGTXXTVNS" at the C-terminus to be considered as full-length V NAR s. After the selection with these criteria, there are almost 2 million V NAR amino acid sequences from this experiment, with more than 1 million unique V NAR sequences. We aligned all V NAR sequences by anchoring the constant regions and allow CDR3 regions to have variable lengths from 0 to 40 amino acids. We then calculated the variability according to methods described in Wu and Kabat [43] . We further classified V NAR sequences into different subtypes based on residue numbers and positions. Type I and Type II/III sequences were also analyzed separately for the CDR3 lengths, total cysteine numbers, and CDR3 cysteine numbers.