snPATHO-seq, a versatile FFPE single-nucleus RNA sequencing method to unlock pathology archives

Taopeng Wang; Michael J Roach; Kate Harvey; Javier Escudero Morlanes; Beata Kiedik; Ghamdan Al-Eryani; Alissa Greenwald; Nikolaos Kalavros; Felipe Segato Dezem; Yuling Ma; Yered H Pita-Juarez; Kellie Wise; Cyril Degletagne; Anna Elz; Azi Hadadianpour; Jack Johanneson; Fiona Pakiam; Heeju Ryu; Evan W Newell; Laurie Tonon; Andrew Kohlway; Tingsheng Drennon; Jawad Abousoud; Ryan Stott; Paul Lund; Jens Durruthy; Andres F Vallejo; Wenyan Li; Robert Salomon; Dominik Kaczorowski; Joanna Warren; Lisa M Butler; Sandra O'Toole; Jasmine Plummer; Ioannis S Vlachos; Joakim Lundeberg; Alexander Swarbrick; Luciano G Martelotto

doi:10.1038/s42003-024-07043-2

snPATHO-seq, a versatile FFPE single-nucleus RNA sequencing method to unlock pathology archives

Commun Biol. 2024 Oct 16;7(1):1340. doi: 10.1038/s42003-024-07043-2.

Authors

Taopeng Wang^#^{1

2}, Michael J Roach^#^{3

4

5}, Kate Harvey¹, Javier Escudero Morlanes⁶, Beata Kiedik^{1

2}, Ghamdan Al-Eryani^{1

2}, Alissa Greenwald⁷, Nikolaos Kalavros^{8

9

10

11}, Felipe Segato Dezem¹², Yuling Ma^{8

9

10

11}, Yered H Pita-Juarez^{8

9

10}, Kellie Wise^{3

4}, Cyril Degletagne¹³, Anna Elz¹⁴, Azi Hadadianpour¹⁴, Jack Johanneson¹⁴, Fiona Pakiam¹⁴, Heeju Ryu¹⁵, Evan W Newell^{14

15}, Laurie Tonon^{13

16}, Andrew Kohlway¹⁷, Tingsheng Drennon¹⁷, Jawad Abousoud¹⁷, Ryan Stott¹⁷, Paul Lund¹⁷, Jens Durruthy¹⁷, Andres F Vallejo¹⁸, Wenyan Li^{19

20}, Robert Salomon^{19

20}, Dominik Kaczorowski²¹, Joanna Warren²¹, Lisa M Butler^{4

22}, Sandra O'Toole^{1

2

23

24

25}, Jasmine Plummer¹², Ioannis S Vlachos^{8

9

10

11}, Joakim Lundeberg⁶, Alexander Swarbrick^{26

27}, Luciano G Martelotto^{28

29}

Affiliations

¹ Cancer Ecosystems Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.
² School of Clinical Medicine, Faculty of Medicine and Health, UNSW Sydney, Sydney, NSW, Australia.
³ Adelaide Centre for Epigenetics, University of Adelaide, Adelaide, SA, Australia.
⁴ South Australian Immunogenomics Cancer Institute, University of Adelaide, Adelaide, SA, Australia.
⁵ College of Science and Engineering, Flinders University, Adelaide, SA, Australia.
⁶ KTH Royal Institute of Technology, Science for Life Laboratory, Solna, Sweden.
⁷ Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
⁸ Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA.
⁹ Harvard Medical School, Boston, MA, USA.
¹⁰ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
¹¹ Spatial Technologies Unit, Harvard Medical School Initiative for RNA Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA.
¹² Center for Spatial Omics, St. Jude Children's Research Hospital, Memphis, TN, USA.
¹³ CRCL Core facilities, Centre de Recherche en Cancérologie de Lyon (CRCL) INSERM U1052-CNRS UMR5286, Université de Lyon, Université Claude Bernard Lyon, Centre Léon Bérard, Lyon, France.
¹⁴ Fred Hutch Innovation Lab, Fred Hutchinson Cancer Center, Seattle, WA, USA.
¹⁵ Vaccine and Infectious Disease Division, Immunotherapy Integrated Research Center, Fred Hutchinson Cancer Center, Seattle, WA, USA.
¹⁶ Fondation Synergie Lyon Cancer, Plateforme de Bioinformatique Gilles Thomas, Centre Léon Bérard, Lyon, France.
¹⁷ 10X Genomics, Pleasanton, CA, USA.
¹⁸ Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, Faculty of Medicine, University of Southampton, Southampton, UK.
¹⁹ Children's Cancer Institute, UNSW Lowy Cancer Research Centre, Kensington, NSW, Australia.
²⁰ School of Clinical Medicine, UNSW Medicine & Health, UNSW Sydney, Kensington, NSW, Australia.
²¹ Cellular Genomics Platform, Garvan Institute of Medical Research, Sydney, NSW, Australia.
²² Solid Tumour Program, Precision Cancer Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, SA, Australia.
²³ Department of Tissue Pathology and Diagnostic Oncology, NSW Health Pathology, Royal Prince Alfred Hospital, Sydney, NSW, Australia.
²⁴ Sydney Medical School, University of Sydney, Sydney, NSW, Australia.
²⁵ School of Medicine, University of Western Sydney, Sydney, NSW, Australia.
²⁶ Cancer Ecosystems Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia. a.swarbrick@garvan.org.au.
²⁷ School of Clinical Medicine, Faculty of Medicine and Health, UNSW Sydney, Sydney, NSW, Australia. a.swarbrick@garvan.org.au.
²⁸ Adelaide Centre for Epigenetics, University of Adelaide, Adelaide, SA, Australia. luciano.martelotto@adelaide.edu.au.
²⁹ South Australian Immunogenomics Cancer Institute, University of Adelaide, Adelaide, SA, Australia. luciano.martelotto@adelaide.edu.au.

^# Contributed equally.

Abstract

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3' and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization.

MeSH terms

Formaldehyde / chemistry
Gene Expression Profiling / methods
Humans
Paraffin Embedding* / methods
Sequence Analysis, RNA* / methods
Single-Cell Analysis* / methods
Tissue Fixation* / methods
Transcriptome
Workflow

Substances

Formaldehyde

Grants and funding

APP2004774/Department of Health | National Health and Medical Research Council (NHMRC)