Robust and accessible in situ sequencing with an open-source automated sequencer
- 1Allen Institute, Brain Science, Seattle, WA, USA
Abstract
Targeted in situ sequencing underlies many spatial transcriptomic, barcoded connectomic, and multiplexed perturbation techniques. Each experiment requires many cycles of chemical reactions and imaging that are time-consuming and error-prone to perform manually, and few commercial platforms can sequence highly diverse RNA barcodes in situ at cellular resolution across large tissue sections. Here we present Seq-o-Matic, an open-source automated in situ sequencer built entirely from commercially available components, which integrates fluidics, heated incubation, and imaging of up to three continuous 18 mm x 50 mm areas on standard microscope slides. Relative to manual sequencing, Seq-o-Matic reduced hands-on time 6- to 17.5-fold and total run time about 2.6-fold. Across three runs spanning two mouse strains and a neonatal marmoset, sequencing quality, measured by signal retention, channel specificity, and phasing, was similar to that of four manually sequenced mouse brains. In four mouse hemispheres sequenced in separate runs (two C57BL/6J, one NOD, and one WSB), consensus cell types were present in every brain and corresponded to those of a manually sequenced C57BL/6J brain. Within cortical excitatory neurons, the two C57BL/6J brains were more similar to each other than were brains of different strains, suggesting that technical variation between runs was small relative to strain differences. Seq-o-Matic thus makes consistent, high-throughput in situ sequencing accessible to laboratories without specialized in situ sequencing expertise.
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BibTeX
@misc{zhang2026robust,
title = {{Robust and accessible in situ sequencing with an open-source automated sequencer}},
author = {A. Zhang and Z. Maltzer and J. Lapka and S. Cheng and A. Guthrie and C. Slaughterbeck and A. Y. Fan and N. Goswami and M. C. P. Rue and M. Majeed and D. M. Maharjan and J. Ariza and A. P. Ayala and M. Bixby and C. T. Dickson and S. D. Hastings and A. Huang and T. E. O'Neill and A. A. Oyama and C. Shi and J. Waters and Y.-C. Wu and R. Galluccio and H. Zhan and J. A. Bourne and G. Horwitz and J. M. Kebschull and I. R. Wickersham and P. Rudebeck and A. Cahoon and X. Chen},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.18.759961},
url = {https://www.biorxiv.org/content/10.64898/2026.10.18.759961v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Robust and accessible in situ sequencing with an open-source automated sequencer
AU - A. Zhang
AU - Z. Maltzer
AU - J. Lapka
AU - S. Cheng
AU - A. Guthrie
AU - C. Slaughterbeck
AU - A. Y. Fan
AU - N. Goswami
AU - M. C. P. Rue
AU - M. Majeed
AU - D. M. Maharjan
AU - J. Ariza
AU - A. P. Ayala
AU - M. Bixby
AU - C. T. Dickson
AU - S. D. Hastings
AU - A. Huang
AU - T. E. O'Neill
AU - A. A. Oyama
AU - C. Shi
AU - J. Waters
AU - Y.-C. Wu
AU - R. Galluccio
AU - H. Zhan
AU - J. A. Bourne
AU - G. Horwitz
AU - J. M. Kebschull
AU - I. R. Wickersham
AU - P. Rudebeck
AU - A. Cahoon
AU - X. Chen
PY - 2026
DA - 2026/10/18
PB - bioRxiv
DO - 10.64898/2026.10.18.759961
UR - https://www.biorxiv.org/content/10.64898/2026.10.18.759961v1
AB - Targeted in situ sequencing underlies many spatial transcriptomic, barcoded connectomic, and multiplexed perturbation techniques. Each experiment requires many cycles of chemical reactions and imaging that are time-consuming and error-prone to perform manually, and few commercial platforms can sequence highly diverse RNA barcodes in situ at cellular resolution across large tissue sections. Here we present Seq-o-Matic, an open-source automated in situ sequencer built entirely from commercially available components, which integrates fluidics, heated incubation, and imaging of up to three continuous 18 mm x 50 mm areas on standard microscope slides. Relative to manual sequencing, Seq-o-Matic reduced hands-on time 6- to 17.5-fold and total run time about 2.6-fold. Across three runs spanning two mouse strains and a neonatal marmoset, sequencing quality, measured by signal retention, channel specificity, and phasing, was similar to that of four manually sequenced mouse brains. In four mouse hemispheres sequenced in separate runs (two C57BL/6J, one NOD, and one WSB), consensus cell types were present in every brain and corresponded to those of a manually sequenced C57BL/6J brain. Within cortical excitatory neurons, the two C57BL/6J brains were more similar to each other than were brains of different strains, suggesting that technical variation between runs was small relative to strain differences. Seq-o-Matic thus makes consistent, high-throughput in situ sequencing accessible to laboratories without specialized in situ sequencing expertise.
ER -