{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T13:19:56Z","timestamp":1790169596080,"version":"4.1.0"},"posted":{"date-parts":[[2026,2,15]]},"group-title":"Biochemistry","reference-count":70,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,15]],"date-time":"2026-02-15T00:00:00Z","timestamp":1771113600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"Natural Sciences and Engineering Council of Canada","award":["RGPIN-2022-04484 to R.D."],"award-info":[{"award-number":["RGPIN-2022-04484 to R.D."]}]},{"name":"Natural Sciences and Engineering Council of Canada","award":["Accelerator Supporting to R.D."],"award-info":[{"award-number":["Accelerator Supporting to R.D."]}]},{"DOI":"10.13039\/501100000024","name":"Canadian Institutes of Health Research","doi-asserted-by":"crossref","award":["CIHR FRN: 168961 to R.D."],"award-info":[{"award-number":["CIHR FRN: 168961 to R.D."]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100004489","name":"Mitacs","doi-asserted-by":"crossref","award":["Elevate Scholarhip to G.M.L"],"award-info":[{"award-number":["Elevate Scholarhip to G.M.L"]}],"id":[{"id":"10.13039\/501100004489","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000196","name":"Canada Foundation for Innovation","doi-asserted-by":"crossref","award":["New Leader Opportunity to R.D."],"award-info":[{"award-number":["New Leader Opportunity to R.D."]}],"id":[{"id":"10.13039\/501100000196","id-type":"DOI","asserted-by":"crossref"}]},{"name":"National Institute of General Medical Sciences of the National Institutes of Health","award":["R01GM145011 and F31GM155953 to F.B.M"],"award-info":[{"award-number":["R01GM145011 and F31GM155953 to F.B.M"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,2,15]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Genetically encoded (GE) libraries enable identification of high-affinity ligands for diverse molecular targets through iterative\n                  <jats:italic>in vitro<\/jats:italic>\n                  selection and DNA sequencing or next-generation sequencing (NGS). Despite their impact in therapeutic development, a systematic framework for evaluating reproducibility in GE-molecular discoveries remains limited. To aid such analysis, we introduce the concept of baseline response, which reproducibly partitions active and inactive members of\n                  <jats:italic>in vitro<\/jats:italic>\n                  selection. The baseline response is provided by spiking a random DNA-barcoded population. We calibrated the baseline concept using Bioconductor EdgeR differential enrichment (DE) analysis of NGS of phage-displayed selection on oligosaccharide chitin and hepatitis virus NS3a* protease as model targets. We further show that mixing discovery campaigns also offers an effective baseline: chitin-enriched peptides serve as a baseline for DE-analysis of NS3a* selection and NS3a*-enriched peptides serve as a baseline for chitin binders. We applied baseline-stratified DE-analysis to 66 parallel selections performed in 3\u20135 replicates across 22 extracellular targets, including HER1-3, EpCAM, CAIX, PD-L1, and eight integrin receptors. Automated DE-analysis across hundreds of NGS files produced hits validated in a secondary screen and yielded synthetic macrocyclic ligands with mid-nanomolar affinity confirmed in 2\u20133 biophysical assays. For PD-L1, we further demonstrated how baseline-calibrated NGS data provide decision-enabling information for optimization of peptide macrocycles to yield potent single-digit nanomolar ligands for the cell-surface receptor. We anticipate that baseline-based analyses of NGS data from\n                  <jats:italic>in vitro<\/jats:italic>\n                  selection procedures will offer a scalable framework for reproducible hit discovery and standardized analysis across diverse\n                  <jats:italic>in vitro<\/jats:italic>\n                  selection campaigns.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>\n                    Genetically encoded selection technologies such as phage, mRNA and ribosome display, have produced FDA-approved therapeutics and numerous clinical candidates. Yet reproducibility in such\n                    <jats:italic>in vitro<\/jats:italic>\n                    discovery systems is rarely evaluated against a defined experimental baseline. Here, we establish a universal baseline by spiking unrelated, DNA-barcoded peptide sequences into selection libraries and quantifying their binding alongside target-enriched populations. This composition-agnostic strategy enables rigorous normalization, confidence assessment, and cross-target comparison of molecular discovery outcomes. Our framework introduces practical standards for reproducibility and statistical benchmarking across genetically encoded display platforms.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.02.14.705946","type":"posted-content","created":{"date-parts":[[2026,2,16]],"date-time":"2026-02-16T03:35:12Z","timestamp":1771212912000},"source":"Crossref","is-referenced-by-count":1,"title":["Universal Baseline for\n                  <i>in vitro<\/i>\n                  Selection of Genetically Encoded Libraries"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-3181-3563","authenticated-orcid":false,"given":"Kejia","family":"Yan","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3268-6081","authenticated-orcid":false,"given":"Guilherme 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