Image Based Transcriptomics: An Overview
- 1. Image based transcriptomics Trace Henry HHMI Janelia Research Campus
- 2. Aproaches for spatial genomics https://www.youtube.com/watch?v=myx8N0zjx48&t=234s
- 3. Direct Measurement multiplex RNA fISH in situ multi- plex protein localization In situ sequencing Aproaches for spatial genomics B-actin MAP2 reported genes reportedcells https://www.youtube.com/watch?v=myx8N0zjx48&t=234s
- 4. Direct Measurement multiplex RNA fISH in situ multi- plex protein localization In situ sequencing Aproaches for spatial genomics Moor and Itzkovitz, Current Opinion Biotech, 2017
- 5. Direct Measurement multiplex RNA fISH in situ multi- plex protein localization In situ sequencing Aproaches for spatial genomics B-actin MAP2 Batish et al., Methods in Mol Bio, 2011
- 6. Direct Measurement multiplex RNA fISH in situ multi- plex protein localization In situ sequencing Aproaches for spatial genomics B-actin MAP2 Spatial Barcoding w/ Superesolution imaging Lubeck and Cai, Nature Methods, 2012
- 7. Direct Measurement multiplex RNA fISH in situ multi- plex protein localization In situ sequencing Aproaches for spatial genomics B-actin MAP2 Battich et al., Nature Methods, 2013 Sequential Methods A: 1 gene = 1 color, many rounds + stripping # genes = F * N
- 8. Direct Measurement multiplex RNA fISH in situ multi- plex protein localization In situ sequencing Aproaches for spatial genomics B-actin MAP2 Sequential Methods B: Barcoding strategy, many rounds + stripping Lubeck et al., Nature Methods, 2014 # genes = FN
- 9. SeqFISH (2016) Shah, Lubeck, et al., Neuron, 2016
- 10. SeqFISH Barcoding strategy 125 genes profiled, 100 of which are barcoded and 25 are identified by serial smHCR hybridizations. Five control genes (Hdx, Vps13c, Zfp715, Fbll1, Slc4a8) were quantified by both techniques. The smHCR round of hybridization of control genes were performed twice to colocalize signal to obtain an absolute count.
- 11. Direct Measurement multiplex RNA fISH in situ multi- plex protein localization In situ sequencing Aproaches for spatial genomics B-actin MAP2 Merfish
- 12. MERFISH (2015) “multiplexed error-robust fluorescence in situ hybridization” Encoding Scheme 1 • 16 bit barcodes • ID 140 unique mRNA species • 80% detection efficiency • ~100 human fibroblast cells in single 18Hr period Chen, Boettiger, et al., Science, 2015
- 13. MERFISH v2 (2016) ~15,000 cells • Much faster than previous version • 40K cells in 18Hr period • 3 major changes: 1. No photobleaching 2. Larger FOV 3. Two color imaging Moffitt, Hao, Wang, et al., PNAS, 2016
- 14. Direct Measurement multiplex RNA fISH in situ multi- plex protein localization In situ sequencing Aproaches for spatial genomics Multiplexed ion beam imaging Angelo et al., Nature Medicine, 2014
- 15. Direct Measurement multiplex RNA fISH in situ multi- plex protein localization In situ sequencing Aproaches for spatial genomics Moor and Itzkovitz, Current Opinion Biotech, 2017 mRNA —> cDNA —> Nanoballs (via RCA) Targeted via Padlock probes (ISS) Unbiased via random hexamers (FISSEQ)
- 16. In situ sequencing (Nillsson) Uses padlock probes to target specific mRNAs, with cDNA synthesis and rolling-circle amplification in situ, followed by sequencing by ligation Ke, et al., Nature Methods, 2013
- 17. Fisseq (2014) “Fluorescent in situ sequencing” (6) 3D in situ RNA sequencing library within the cell (1) A tagged random hexamer primer is used to prime reverse transcriptase to generate modified cDNA fragments in fixed cells or tissues. (2) BS(PEG)9 permanently cross-links the modified cDNA and the cellular protein matrix. (3) cDNA circularization (4) DNA polymerase generates cDNA amplicons (5) amplicons are cosslinked Lee, et al., Science, 2014
- 18. Fisseq (2014): SOLID based sequencing - Each amplicon contains numerous tandem copies of the cDNA template and adapter sequence. - A sequencing primer hybridizes to the adapter sequences in individual amplicons, and fluorescent eight-base probes interrogate the adjacent dinucleotide pair. - After imaging, the three bases attached to a fluorophore are cleaved, generating a phosphorylated 5′ end at the ligation complex suitable for additional ligation cycles interrogating every fifth dinucleotide pairs. - The whole process is repeated using four other sequencing primers with an offset to interrogate intervening base positions Lee, et al., Nature Protocols, 2015
- 19. Fisseq (2014): Technical considerations Extending sequencing primers by one or more bases, one can randomly sample amplicons at 1/4th, 1/16th, and 1/256th of the original density in fibroblasts Due to the size of the generated Nano balls (200-400nm) and technical limitations of optics, only a few hundred molecules can be detected in each cell Also, efficiency of RCA is not uniform across subcellular compartments, especially across different cell types Sensitivity of padlock probes is two orders of magnitude higher than FISSEQ for a given gene Lee, et al., Science, 2014
- 20. Fisseq (2014) Lee, et al., Science, 2014
- 21. Inference Computational Imputation Aproaches for spatial genomics Habib, Li, et al., Science, 2016
- 22. Inference Computational Imputation Aproaches for spatial genomics Satija et al., Nature Biotechnology, 2015
- 23. Gridding/Barcoding Inference Direct Measurement “Spatial Transcriptomics” multiplex RNA fISH in situ multi- plex protein localization Computational Imputation In situ sequencing Aproaches for spatial genomics 1) Tissue Fixation on slide 2) Permeabilization 3) cDNA Synthesis 4) Library Prep and Sequencing Stahl, Salmen, et al., Science 2016
- 24. Stahl, Salmen, et al., Science 2016 “Spatial Transcriptomics”
- 26. Croesetto, et al., Nat Rev Genetics ,2015 Grand Overview
- 27. Things to consider: What genes to pick? Tasic, Menon, et al., Nature Neuroscience, 2016
- 28. How to overcoming crowding issue: Expansion Microscopy Chen, Wassie, et al., Nature Methods, 2016
- 29. How to overcoming crowding issue: Correlation decoding Coskun and Cai, Nature Methods, 2016
- 31. Spatial DE: Identification of spatially variable genes https://www.biorxiv.org/content/early/2017/05/28/143321
- 32. Applications to neuroscience Lein, et al., Science, 2017
- 33. END