Variants in cis-regulatory elements link the noncoding genome to human brain pathology; however, detailed analytic tools for understanding the association between cell-level brain pathology and noncoding variants are lacking. CWAS-Plus, adapted from a Python package for category-wide association testing (CWAS) employs both whole-genome sequencing and user-provided functional data to enhance noncoding variant analysis, with a faster and more efficient execution of the CWAS workflow. Here, we used single-nuclei assay for transposase-accessible chromatin with sequencing to facilitate CWAS-guided noncoding variant analysis at cell-type specific enhancers and promoters. Examining autism spectrum disorder whole-genome sequencing data (n = 7,280), CWAS-Plus identified noncoding de novo variant associations in transcription factor binding sites within conserved loci. Independently, in Alzheimer's disease whole-genome sequencing data (n = 1,087), CWAS-Plus detected rare noncoding variant associations in microglia-specific regulatory elements. These findings highlight CWAS-Plus's utility in genomic disorders and scalability for processing large-scale whole-genome sequencing data and in multiple-testing corrections. CWAS-Plus and its user manual are available at https://github.com/joonan-lab/cwas/ and https://cwas-plus.readthedocs.io/en/latest/, respectively.

The authors have declared no competing interest.

This study was supported by grants from the National Research Foundation (NRF) of Korea (NRF-2020R1C1C1003426 and NRF-2021M3E5D9021878 to J.-Y.A; NRF-2021M3A9E4080784 to I.B.K.), the Korea Health Technology R&D Project through the Korea Health Industry Development Institute and Korea Dementia Research Center, funded by the Ministry of Health & Welfare and Ministry of Science and ICT, Republic of Korea (grant number : HU22C0042), and Korea University (to J.-Y.A.), SFARI (#606289 to D.M.W.), and the Brain and Behavior Research Foundation (BBRF, #29815 to D.M.W.). This work was supported by the Korea Bio Data Station with computing resources including technical support. Y.K., I.G.K., H.L. and J.H.K. received a scholarship from the Brain Korea FOUR education program. Y.K. received a scholarship from Seoul Broadcasting System foundation scholarship program.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Institutional Review Board of Korea University gave ethical approval for this work (approval number: KUIRB-2022-0409-03). The Institutional Review Board of Rush University Medical Center gave ethical approval for this work.

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

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The source code of CWAS-Plus is available via a GitHub repository (https://github.com/joonan-lab/cwas/) and Zenodo (https://doi.org/10.5281/zenodo.10795678), with a user manual (https://cwas-plus.readthedocs.io/en/latest/). Both repositories are released under the MIT license. The scripts to produce the annotation dataset, along with the burden test results from the CWAS analyses conducted in this study, are available via Zenodo (https://doi.org/10.5281/zenodo.10814080). The Zenodo repository is released under the Creative Commons Attribution 4.0 International license. CWAS-Plus is written in python (version≥3.9), and easy to install and use in Linux or Mac OS. De novo variants were obtained from the pVCF file, accessible with approval from the Simons Foundation Autism Research Initiative (SFARI Base; https://sfari.org/resources/sfari-base). ROSMAP WGS data can be requested at the AD Knowledge Portal under accession code syn10901595 (https://www.synapse.org/#!Synapse:syn10901595; see https://adknowledgeportal.synapse.org/Data%20Access for data access instructions).