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In vivo  phage display is widely used for identification of organ- or disease-specific homing peptides. However, the current  in vivo  phage biopanning approaches fail to assess biodistribution of specific peptide phages across tissues during the screen, thus necessitating laborious and time-consuming post-screening validation studies on individual peptide phages. Here, we adopted bioinformatics tools used for RNA sequencing for analysis of high-throughput sequencing (HTS) data to estimate the representation of individual peptides during biopanning  in vivo . The data from  in vivo  phage screen were analyzed using differential binding—relative representation of each peptide in the target organ versus in a panel of control organs. Application of this approach in a model study using low-diversity peptide T7 phage library with spiked-in brain homing phage demonstrated brain-specific differential binding of brain homing phage and resulted in identification of novel lung- and brain-specific homing peptides. Our study provides a broadly applicable approach to streamline  in vivo  peptide phage biopanning and to increase its reproducibility and success rate.

In vivo phage display: identification of organ-specific peptides using deep sequencing and differential profiling across tissues