---
title: "Calibration of AVI and SpliceAI2 as PP3/BP4 Evidence for Missense, Noncoding, and Synonymous Varia"
id: "biorxiv-3-calibration-of-alphagenome-variant-impact-and-spliceai2-scores-as-pp3-bp4"
canonical_url: "https://medichelpline.com/clinical-feed/biorxiv-3-calibration-of-alphagenome-variant-impact-and-spliceai2-scores-as-pp3-bp4"
content_type: "clinical_feed_article"
specialty: "General"
source_name: "bioRxiv (Biomedical Preprints)"
source_url: "https://www.biorxiv.org/content/10.64898/2026.10.21.760715v1?rss=1"
published_at: "2026-10-24T12:00:00.000Z"
evidence_level: "Verified Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Calibration of AVI and SpliceAI2 as PP3/BP4 Evidence for Missense, Noncoding, and Synonymous Varia
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/biorxiv-3-calibration-of-alphagenome-variant-impact-and-spliceai2-scores-as-pp3-bp4
- **Specialty:** [General](https://medichelpline.com/clinical-feed/general.md)
- **Primary Source:** bioRxiv (Biomedical Preprints)
- **Source URL:** [Original Journal Publication](https://www.biorxiv.org/content/10.64898/2026.10.21.760715v1?rss=1)
- **Published At:** 2026-10-24T12:00:00.000Z
- **Evidence Rating:** Verified Feed
## Executive GIST (TL;DR)
- The authors calibrated the **AlphaGenome Variant Impact (AVI)** score for missense variants and for six non-coding and synonymous variant classes, and calibrated **SpliceAI2** for the same six classes. - For missense variants, AVI produced evidence strength between moderate and strong in both pathogenic and benign directions; it did not exceed the performance of its component **AlphaMissense**, investigators reported. - AVI reached strong pathogenic-level evidence in the three classes immediately adjacent to exons, moderate evidence in proximal introns and other synonymous sites, and supporting evidence (but at an unstable threshold) in deep introns. - SpliceAI2 achieved similar calibrated levels overall but performed one evidence level lower at near-canonical positions and produced no calibrated level in deep introns, the cohort showed. - Validation against later **ClinVar** classifications found no reached level that later failed validation, although 14 of 30 benign evidence levels were not tested because of insufficient data. - Using population variants as controls changed thresholds: with population controls, both AVI and SpliceAI2 attained supporting and moderate pathogenic evidence across all six classes, with stricter thresholds near exons and at other synonymous sites, and lower thresholds in proximal and deep introns. - The authors observed a roughly 60-fold change in ClinVar likely benign classifications at 20 nucleotides from the exon without an accompanying change in measured splicing. - AVI, a genome-wide score, had similar discrimination to the splicing-specific **SpliceAI2** in four classes; SpliceAI2 discriminated better in two classes and in a splicing assay, and showed greater sensitivity at 99% specificity in four classes. - The study team noted that both scores can be used as calibrated evidence for non-coding and synonymous variants, but recommended that thresholds depend on whether one uses ClinVar likely benign calls or population variants as the benign comparator. - The source did not report detailed numeric thresholds, specific dataset sizes, or full methodological parameters in the abstract; those details are in the full preprint and supplementary material.
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Ali Babazade Gazi University, Faculty of Medicine, Department of Medical Genetics, Ankara, Turkey * [Find this author on Google Scholar](https://www.biorxiv.org/lookup/google-scholar?link_type=googlescholar&gs_type=author&author%5B0%5D=Ali%2BBabazade%2B "Open in new tab") * [Find this author on PubMed](https://www.biorxiv.org/lookup/external-ref?access_num=Babazade%20A&link_type=AUTHORSEARCH "Open in new tab") * [Search for this author on this site](https://www.biorxiv.org/search/author1%3AAli%2BBabazade%2B) * [ORCID record for Ali Babazade](http://orcid.org/0009-0004-4473-3932 "Open in new tab") Yarkin Dolas Gazi University, Faculty of Medicine, Department of Medical Genetics, Ankara, Turkey * [Find this author on Google Scholar](https://www.biorxiv.org/lookup/google-scholar?link_type=googlescholar&gs_type=author&author%5B0%5D=Yarkin%2BDolas%2B "Open in new tab") * [Find this author on PubMed](https://www.biorxiv.org/lookup/external-ref?access_num=Dolas%20Y&link_type=AUTHORSEARCH "Open in new tab") * [Search for this author on this site](https://www.biorxiv.org/search/author1%3AYarkin%2BDolas%2B) * [ORCID record for Yarkin Dolas](http://orcid.org/0000-0002-2467-5569 "Open in new tab") Tarik Duzenli Gazi University, Faculty of Medicine, Department of Medical Genetics, Ankara, Turkey * [Find this author on Google Scholar](https://www.biorxiv.org/lookup/google-scholar?link_type=googlescholar&gs_type=author&author%5B0%5D=Tarik%2BDuzenli%2B "Open in new tab") * [Find this author on PubMed](https://www.biorxiv.org/lookup/external-ref?access_num=Duzenli%20T&link_type=AUTHORSEARCH "Open in new tab") * [Search for this author on this site](https://www.biorxiv.org/search/author1%3ATarik%2BDuzenli%2B) * [ORCID record for Tarik Duzenli](http://orcid.org/0000-0002-3651-9467 "Open in new tab") Ozan Vural Gazi University, Faculty of Medicine, Department of Medical Genetics, Ankara, Turkey * [Find this author on Google Scholar](https://www.biorxiv.org/lookup/google-scholar?link_type=googlescholar&gs_type=author&author%5B0%5D=Ozan%2BVural%2B "Open in new tab") * [Find this author on PubMed](https://www.biorxiv.org/lookup/external-ref?access_num=Vural%20O&link_type=AUTHORSEARCH "Open in new tab") * [Search for this author on this site](https://www.biorxiv.org/search/author1%3AOzan%2BVural%2B) * [ORCID record for Ozan Vural](http://orcid.org/0009-0009-1420-9263 "Open in new tab") Ceren Furtana Gazi University, Faculty of Medicine, Department of Medical Genetics, Ankara, Turkey * [Find this author on Google Scholar](https://www.biorxiv.org/lookup/google-scholar?link_type=googlescholar&gs_type=author&author%5B0%5D=Ceren%2BFurtana%2B "Open in new tab") * [Find this author on PubMed](https://www.biorxiv.org/lookup/external-ref?access_num=Furtana%20C&link_type=AUTHORSEARCH "Open in new tab") * [Search for this author on this site](https://www.biorxiv.org/search/author1%3ACeren%2BFurtana%2B) Mehmet Ali Ergun Gazi University, Faculty of Medicine, Department of Medical Genetics, Ankara, Turkey * [Find this author on Google Scholar](https://www.biorxiv.org/lookup/google-scholar?link_type=googlescholar&gs_type=author&author%5B0%5D=Mehmet%2BAli%2BErgun%2B "Open in new tab") * [Find this author on PubMed](https://www.biorxiv.org/lookup/external-ref?access_num=Ergun%20MA&link_type=AUTHORSEARCH "Open in new tab") * [Search for this author on this site](https://www.biorxiv.org/search/author1%3AMehmet%2BAli%2BErgun%2B) * [ORCID record for Mehmet Ali Ergun](http://orcid.org/0000-0001-9696-0433 "Open in new tab") * For correspondence: aliergun@gazi.edu.tr * [Abstract](https://www.biorxiv.org/content/10.64898/2026.10.21.760715v1)[](https://www.biorxiv.org/panels_ajax_tab/biorxiv_tab_art/node:5899771/1) * [Info/History](https://www.biorxiv.org/content/10.64898/2026.10.21.760715v1.article-info)[](https://www.biorxiv.org/panels_ajax_tab/biorxiv_tab_info/node:5899771/1) * [Metrics](https://www.biorxiv.org/content/10.64898/2026.10.21.760715v1.article-metrics)[](https://www.biorxiv.org/panels_ajax_tab/article_tab_metrics/node:5899771/1) * [Supplementary material](https://www.biorxiv.org/content/10.64898/2026.10.21.760715v1.supplementary-material)[](https://www.biorxiv.org/panels_ajax_tab/biorxiv_tab_data/node:5899771/1) * [Data/Code](https://www.biorxiv.org/content/10.64898/2026.10.21.760715v1.external-links)[](https://www.biorxiv.org/panels_ajax_tab/article_tab_data_code/node:5899771/1) * [ Preview PDF](https://www.biorxiv.org/content/10.64898/2026.10.21.760715v1.full.pdf+html)[](https://www.biorxiv.org/panels_ajax_tab/biorxiv_tab_pdf/node:5899771/1) ![Loading](https://www.biorxiv.org/sites/all/modules/contrib/panels_ajax_tab/images/loading.gif) ## Abstract Computational predictions can serve as graded evidence in variant classification after calibration. We calibrated the AlphaGenome Variant Impact score (AVI) for missense variants and for six non-coding and synonymous classes, and SpliceAI2 for the six classes. For missense variants, AVI reached evidence between moderate and strong in both directions but did not outperform its component AlphaMissense. AVI reached strong pathogenic evidence in the three classes adjacent to exons. It reached moderate evidence, the highest estimable level, in proximal introns and at other synonymous sites, and supporting evidence, at an unstable threshold, in deep introns. SpliceAI2 reached the same levels, except one level lower at near-canonical positions and none in deep introns. No reached level failed validation on later ClinVar classifications, although 14 of 30 benign levels were not tested. With population variants as controls, both tools reached supporting and moderate pathogenic evidence in all six classes, at stricter thresholds near exons and at other synonymous sites, and at lower thresholds in proximal and deep introns. ClinVar likely benign classifications changed about 60-fold in number at 20 nucleotides from the exon without a change in measured splicing. AVI, a genome-wide score, did not differ detectably in discrimination from the splicing-specific SpliceAI2 in four classes; SpliceAI2 discriminated better in two classes and in a splicing assay, and was more sensitive at 99% specificity in four classes. Both scores provide calibrated evidence for non-coding and synonymous variants, but thresholds depend on whether ClinVar benign classifications or population variants are used for comparison. ### Competing Interest Statement The authors have declared no competing interest. ## Footnotes * Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a [CC-BY-NC-ND 4.0 International license](http://creativecommons.org/licenses/by-nc-nd/4.0/). bioRxiv and medRxiv thank the following for their generous financial support: > The Chan Zuckerberg Initiative, Cold Spring Harbor Laboratory, the Sergey Brin Family Foundation, California Institute of Technology, Centre National de la Recherche Scientifique, Fred Hutchinson Cancer Center, Imperial College London, Massachusetts Institute of Technology, Stanford University, The University of Edinburgh, University of Washington, and Vrije Universiteit Amsterdam. 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[ Download PDF](https://www.biorxiv.org/content/10.64898/2026.10.21.760715v1.full.pdf) Print/Save Options [Download PDF](https://www.biorxiv.org/content/biorxiv/early/2026/10/24/2026.10.21.760715.full.pdf)Full Text & In-line FiguresXML [More Info](https://www.biorxiv.org/about/FAQ#PrintOptions "More Information on Print/Save Options") [Supplementary Material ](https://www.biorxiv.org/content/10.64898/2026.10.21.760715v1.supplementary-material) [ Data/Code](https://www.biorxiv.org/content/early/2026/10/24/2026.10.21.760715.external-links) [ Email](https://www.biorxiv.org/ "Email this Article") [ Share](https://www.biorxiv.org/) Calibration of AlphaGenome Variant Impact and SpliceAI2 scores as PP3/BP4 evidence for missense, non-coding, and synonymous variants Ali Babazade, Yarkin Dolas, Tarik Duzenli, Ozan Vural, Ceren Furtana, Mehmet Ali Ergun bioRxiv 2026.10.21.760715; doi: https://doi.org/10.64898/2026.10.21.760715 This article is a preprint and has not been certified by peer review [[what does this mean?](https://www.biorxiv.org/about/FAQ#unrefereed)]. 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