Benchmarked sequencing methods reveal hidden mosaic variation in the human brain

Abstract

Mosaic variants (MVs) record cellular lineages, but their recovery is strongly method-dependent. Here, we benchmarked four duplex sequencing methods alongside bulk and single-neuron genome sequencing in matched human brain tissue. The methods differed in genome representation, MV yield and mutational profiles, with limited caller-level concordance. However, read-level interrogation revealed substantially greater cross-method molecular support. To systematically recover this evidence, we developed Read-level Integration for Variant Evidence Recovery (RIVER), expanding the pool of cross-method-supported candidates by approximately two orders of magnitude relative to caller-level overlap. Orthogonal sequencing at ~10,000x across brain and peripheral tissues validated approximately half of the tested RIVER-rescued candidates. MVs recovered from a single brain region resolved broadly distributed and regionally restricted clones and provided lineage information among individual neurons. These findings show that RIVER substantially expands the pool of endogenous lineage markers available to investigate human development, aging, disease-associated mosaicism and clonal evolution.

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BibTeX RIS

BibTeX

@misc{weng2026benchmarked,
  title = {{Benchmarked sequencing methods reveal hidden mosaic variation in the human brain}},
  author = {J. Weng and D. H. Cha and J. Chun and P. Santamarina-Ojeda and Y. Cheng and R. Howarth and Y. Xu and X. Huangfu and P.-J. Wang and S. Jung and J. Ryu and Y. Noh and S. Lee and X. Yang and Y. S. Ju and L. B. Alexandrov and C. Chung and J. G. Gleeson},
  howpublished = {bioRxiv},
  year = {2026},
  month = oct,
  doi = {10.64898/2026.10.11.758380},
  url = {https://www.biorxiv.org/content/10.64898/2026.10.11.758380v1},
  note = {CC BY},
}

RIS

TY  - UNPB
TI  - Benchmarked sequencing methods reveal hidden mosaic variation in the human brain
AU  - J. Weng
AU  - D. H. Cha
AU  - J. Chun
AU  - P. Santamarina-Ojeda
AU  - Y. Cheng
AU  - R. Howarth
AU  - Y. Xu
AU  - X. Huangfu
AU  - P.-J. Wang
AU  - S. Jung
AU  - J. Ryu
AU  - Y. Noh
AU  - S. Lee
AU  - X. Yang
AU  - Y. S. Ju
AU  - L. B. Alexandrov
AU  - C. Chung
AU  - J. G. Gleeson
PY  - 2026
DA  - 2026/10/11
PB  - bioRxiv
DO  - 10.64898/2026.10.11.758380
UR  - https://www.biorxiv.org/content/10.64898/2026.10.11.758380v1
AB  - Mosaic variants (MVs) record cellular lineages, but their recovery is strongly method-dependent. Here, we benchmarked four duplex sequencing methods alongside bulk and single-neuron genome sequencing in matched human brain tissue. The methods differed in genome representation, MV yield and mutational profiles, with limited caller-level concordance. However, read-level interrogation revealed substantially greater cross-method molecular support. To systematically recover this evidence, we developed Read-level Integration for Variant Evidence Recovery (RIVER), expanding the pool of cross-method-supported candidates by approximately two orders of magnitude relative to caller-level overlap. Orthogonal sequencing at ~10,000x across brain and peripheral tissues validated approximately half of the tested RIVER-rescued candidates. MVs recovered from a single brain region resolved broadly distributed and regionally restricted clones and provided lineage information among individual neurons. These findings show that RIVER substantially expands the pool of endogenous lineage markers available to investigate human development, aging, disease-associated mosaicism and clonal evolution.
ER  -

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