Chromosome tracing with OligoFISSEQ Marc A. Marti-Renom CNAG-CRG - PowerPoint PPT Presentation

Chromosome tracing with OligoFISSEQ Marc A. Marti-Renom CNAG-CRG · ICREA Huy Nguyen Shyamtanu Chattoraj David Castillo in collaboration with the Wu Lab (HMS) Nature Methods (2020) 17 p822

OligoFISSEQ Number of cells OligoSTORM Targets per cell

OligoFISSEQ HIT bridge 5’ 3’ sites LIT primer site SIT primer site LIT barcode SIT barcode Genome homology

From tens of kb to Mb OligoFISSEQ Min. of few 100s oligos/target At least a Mb between targets

OligoFISSEQ HIT bridge 5’ 3’ sites LIT primer site SIT primer site LIT barcode SIT barcode Genome homology L igation based I dentification of T argets S ynthesis based I dentification of T argets H ybridization based I dentification of T argets T G 1st Base A C T G A 2nd Base T G N N N Z Z Z C A C T G Pol T 3’ OH 3’ PO4 barcode A barcode B 5’ 5’ ACTGTGAATCGC ATCA Ligase T 3’ TGNNNZZZ barcode A barcode B 3’ 5’ ATCA 5’ ACTGTGAATCGC Z Z Z TGNNN 3’ OH 3’ barcode A barcode B 5’ 5’ ACTGTGAATCGC ATCA 90.8 ± 5.6% 92.1± 5.7% 91.6 ± 3.8% 10 µm 10 µm

OligoFISSEQ scales exponentially! Sequential hybridization Barcode sequencing # of targets = F*N # of targets = F N F = # of fluorophores F = # of fluorophores N = # of seq. rounds N = # of seq. rounds

OligoFISSEQ scales exponentially! Sequential hybridization vs. Barcode sequencing 18000 16,384 Number of targets 13500 6,000 9000 Genome coverage @500kb 4500 4,096 360 1,024 256 4 16 64 24 0 1 2 3 4 5 6 7 Number of rounds

Proof-of-principle chr2 chr3 chr5 chr16 chr19 chrX 600kb-1Mb/target (876 kb average) 5,000 oligos/target 7-70Mb between targets

Detecting a given target Round 1 Round 2 Round 3 Round 4 2 3 5 16 19 X

OligoFISSEQ "Manhattan plot”

In OligoFISSEQ every pixel matters & make “patches” 4 rounds / 4 channels 75 pixels ~20 𝞶 m 0.265 𝞶 m/pixel 2qR2 chr2

In OligoFISSEQ every pixel matters & make “patches” 2pR1 2pR2 2pR3 2qR1 2qR2 2qR3 chr2 2pR1 2pR2 2qR2 2qR3 2qR1 2pR3

OligoFISSEQ barcode efficiency 5 𝞶 m 5 𝞶 m

OligoFISSEQ is high throughput! ~2 days of image acquisition ~1,000 cells ~5,000 complete chromosomes ~150 cells with complete chromosomes 10 𝞶 m

Single cell homolog resolved tracing of chromosomes Cell #1. 97% detection - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 1 1 1 1 1 2 2 2 2 2 2 1 1 1 1 1 1 2 2 2 2 2 2 1 1 1 1 1 1 2 2 2 2 2 2 1 1 1 1 1 1 2 2 2 2 2 2 1 1 1 1 1 1 2 2 2 2 2 2 1 1 1 1 1 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R p p p q q q p p p q q q p p p q q q p p p q q q p p p q q q p p p q q q p p p q q q p p p q q q p p p q q q p p p q q q p p p q q q 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 9 9 9 9 9 9 9 9 9 9 9 9 X X X X X X 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Do OligoFISSEQ tracing maps show known features? Hi-C contact maps & Radial position of chromosomes OligoFISSEQ n=1,108 n=691 r 2 = 0.71 2pR1 2pR2 2pR3 2qR1 2qR2 2qR3 3pR1 3pR2 3pR3 3qR1 3qR2 3qR3 5pR1 5pR2 5pR3 5qR1 5qR2 5qR3 16pR1 16pR2 16pR3 16qR1 16qR2 16qR3 19pR1 19pR2 19pR3 19qR1 19qR2 19qR3 XpR1 XpR2 XpR3 XqR1 XqR2 XqR3 Hi-C 2 3 5 16 19 X n = 1x10 6 + cells Are the chromosomes randomly located inside the nucleus? Are there preferred configurations in the cell population?

OligoFISSEQ tracing of (almost) entire chromosomes 46 Plex in chromosome X chrX 5 rounds 445 kb/probe 2,000 Oligopaints/probe 2 Mb between loci 1 𝞶 m 5 𝞶 m

OligoFISSEQ tracing of (almost) entire chromosomes 46 Plex in chromosome X n=146

OligoFISSEQ tracing of (almost) entire chromosomes 46 Plex in chromosome X Cluster 1 (n=156) Cluster 2 (n=20)

OligoFISSEQ beyond chromosome tracing

OligoFISSEQ pipelined with OligoSTORM chr2 1 2 3 4 OligoSTROM OligoFISSEQ Decoding Mapping 1 round 2 round OligoFISSEQ OligoSTROM (2h/round) (3h/round)

Efficiency 0.0 1.0 HESS: 97 oligos, 11kb 1 ABL: 454 oligos, 45kb 9 FLI1: 499 oligos, 39kb 11 MMP2: 297 oligos, 27kb 16 BCR: 1,015 oligos, 100kb 22 DXZ4: 617 oligos, 136kb X OligoFISSEQ for multiple loci detection 72% 100%

OligoFISSEQ + protein immunofluorescence � -tubulin GAPDH Tomm20 WGA 2 3 5 16 19 X

OligoFISSEQ HIT bridge 5’ 3’ sites • Is a set of technologies for in-situ genome mapping LIT primer site SIT primer site LIT barcode SIT barcode • Is highly versatile: mainstreet and backstreet Genome homology • Used with wide-field microscopy permits the analysis of thousands of cells. • Identifies sub-clusters with specific conformational characteristics • Can be pipelined with other approaches OligoSTORM • Protein immunofluorescence • RNA… •

http://marciuslab.org @marciuslab http://3DGenomes.org @mamartirenom Marco Di Stefano Irene Farabella David Castillo Mike Goodstadt Yasmina Cuartero Juan A. Rodriguez Silvia Galan Rodrigo Jara Iana Kim Maria Marti-Marimon Francesca Mugianesi Julen Mendieta Aleksandra Sparavier In collaboration with the Wu Lab – Ting Wu, Huy Nguyen & Shyamtanu Chattoraj .: Our current sponsors :.