## 4 Discussion
Our data show that after the final round of fFcRn FACS sorting, the frequency of GFP\({}^{+}\)APC\({}^{+}\) cells in the control group increased. A similar result was found in the isolation of FIPV nucleocapsid-specific VNARs, showing that the enrichment of off-target clones is designed to happen in the end. To avoid this phenomenon, a more rigid negative selection during MACS or changing fluorescent dye between rounds of panning could be the solution. However, it means more delays and costs in the development process. We thought to speculate that if the NGS assay can be applied to compare the sorted sequences in every round of the control group, the unwanted sequences can be excluded from sorted candidates easily without needing more work and changing reagents. Biopanning is a standard process for screening affinity clones from the phage antibody library. In each round of screening, the bound phage must be stripped from the antigen-coated ELISA plate. Many strongly bound phages cannot be detached from the antigen and disappear in the next round of screening. In addition, after multiple rounds of biopanning, the screened clones can be dominated by phages with moderate affinity or rapid replication. Usually, \(10^{4-6}\) phages were panned out, but only 100-1000 phages will be hand-selected for sequencing, and the opportunity to find high-affinity clones is diminished [41]. Although biopanning by FACS does not lose the strong bound clones during sorting, the sorted populations may still be occupied by the fast-replication clones. In this study, all sorted yeasts were sequenced by NGS, so the influence of fast-replication clones could be reduced. The study of [PERSON] et al. combined two analysis methods (sequence frequency > 10 and amplification factor > 10) to narrow the 2.7 \(\times\)\(10^{5}\) VHH phage clones down to 5173 clones. In total, 28 clones were selected for expression and validation for antigen binding, and 25 of 28 clones were high-affinity binders. However, the affinity of VHH phage clones is not highly correlated with the sequence frequency and amplification factor [57]. Another method to remove fast-propagating sequences or biased sequences is the bioinformatics processing tools such as the pLogo analysis to extract specific-binding sequences. In our platform, two antigens were screened for high-affinity binders, and the results of the NGS sequencing were grouped according to the sequence frequency and amplification factor analysis. Two of each most abundant and enriched antigen-specific CDR3 sequences are selected for further validation. Interestingly, these sequences are identical or highly consistent to the pLogo extracted motif (V6 sequence KSRRKLERNRC-RTRRPCPIRSSR versus 23-mer motif KSRRKLERNRCRTRRPCIRSSR; V5 sequence RQRESTSCKRKRCRVRST versus 20-mer motif RQRESTSCKRKRCRVIST; V3 sequence KRYGKCRCRKTCAPSVVL versus 18-mer motif KRYGKCRRRTCASPSVL; and V2 sequence RRRGRCPRRCATVGRP versus 16-mer motif RRRGRCRPCRATVGWP). The frequent occurrence of arginine and proline in the CDR3 motif may be due to the influence of frequency bias. But arginine and proline can be served as protecting osmolytes to stabilize protein folding and increase protein solubility, inhibiting protein aggregation [58]. Thus, the retained arginine and proline in the consensus CDR3 motif might be the result of a secondary selection pressure other than the target-binding. The VNAR protein structure with medium affinity may be robust compared to the high-affinity one and then stably presented on the yeast surface during biopanning. Although it seems that the result of biopanning is affected by the translation-based bias, the cell-based fluorescent assay results verified that the selected VNAR-displaying clones are able to bind to their ligand. In the end, their affinity needs to be determined after being solely expressed to verify the readiness of the grouping strategy.