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In situ Sequencing : Cell Atlas, BRAIN Initiative�13-Feb-2018 10-11:30 AM Biophysics 242 George Church

LSRF

NHGRINIGMS

Azco

OppenheimerFoundation

PersonalGenomes.org

Lipper Foundation

v.ht/PHNc

Biophysics 242: Transformative Biotechnologies (R-W-A) Refactored

Jan 23 (1) Exponential, Logistic, Prioritizing global challenges Jan 30 (2) Sequencing & understanding biosphere omes, 3D molecular design Feb 6 (3) Genome Edit/Write (beyond CRISPR), Codon Recoding Feb 13 (4) In situ Sequencing : Mammalian Cell Atlas & BRAIN Initiative Feb 20 (5) Epigenetic programming, signaling pathways, SynEvoDevo Feb 27 (6) Microbiomes: therapeutics, diagnostics, nanopores Mar 6 (7) Synthetic Organs for VUS & Transplantation Mar 20 (8) Aging Reversal Mar 27 (9) Global Warming Apr 3 (10) Germline editing & H. sapiens 2.0

3

In situ-specific issues

Signal amplification: multi-probes, HCR, RCA, PCR Barcode sequence: SbH, SbL, SbS Resolution (nm): Storm, DNA Paint, Expansion Resolution (bp): SbH:HOPS or SbL Occupancy 3D coordinates

Omni Omics in situ�

Genomes (4D nucleome, ATAC-Seq)�Transcriptome (mods & splicing)Proteome MetabolomeMicrobiome/viromeImmunome

Inference of subcellular & inter-cellular interactions -vs- direct observation (& manipulation)

5

SbH & SbS Linear vs

Exponential (combinatorial)

20*N bpN rounds 4N spots

N bp

6

.

7

Sequencing by Hybridization/Ligation SbH/SbL

8

High-Throughput Sequencing Technologies Reuter, Spacek, Snyder (2015) Molecular Cell

FISSEQ: 3D RNA

Fluorescent in situ sequencing

Lee, Daugharthy, et al. Science 2014

Mitra&ChurchNAR1999;MitraetalAn.Bioch2003

Automated High-Throughput Imaging &

Enzymatics

Simultaneously process sixteen 10x10 mm x 10mm tissue samples.

5 Gpixels/sec

Multi-axisstages&thermocyclers

FluidicsFluidics

Objective

SpinningDiskConfocal

Base call quality

Cell atlas�Subcellular in situ sequencing

112

Lee, Daugharthy, .. Church Science 2014, Nature Protocols. 2015

Fibronectin mRNA FISSEQ alternative splicing

Lee,Daugharthy,etal.Science2014

Targeted in-situ�human

cells�5x105 cells/

sample

A C

50 μm

50 μm

50 μm

C

mRNA (3D Z-stack)

22-nt miRNA

Micro-exon 9 nt Splice JunctionsIyer, Punthambaker,

Liu, Jindal, Church(unpublished)

STORM images of the ~300 kb BX-C of Drosophila. Top right: STORM image corresponding to the conventional FISH image to the left. 1 & 2, enlarged insets.

Super-resolution DNA: Wu, Yin, Zhuang Labs

Oligopaints Beliveau,Wu, et al. PNAS 2012

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Homologue-specific OligoPaints (HOPs)

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Chemical ligation X= haloacetyl, tosyl, iodo, dabsyl electrophiles

NH2 Nucleophile

Rouhanifard..Raj2017clampFISH

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Hybridization chain reaction HCR

HCR-ExFISH �

Thy1-YFP �Gad1 mRNA

Scale bar: 500 μm 2.9x expan

Chen, Wassie, Cote, Sinha, Alon, Asano, Daugharthy, Chang, Marblestone, Church, Raj, Boyden. Nanoscale imaging of RNA with expansion microscopy. Nat Methods. 2016

Expansion microscopy of proteins�(also ExSeq, ExFISH)

Chen, et al, Science Jan-2015

Bassoon&Homer1

Ab

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Protein in situ multiplexing with DNA-Exchange

Wang, Woehrstein, Donoghue, Dai, Avendaño, Schackmann, Zoeller, Wang, Tillberg, Park, Lapan, Boyden, Brugge, Kaeser, Church, Agasti, Jungmann, Yin (2017)

9 Metabolite Sensor-interface strategies

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(I) mRNA binding(H) tRNA-ribosome(G) riboswitches

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(F) protein stability

Sensor-interface (F)

Justin Feng, Jester, Tinberg, Mandell, Antunes, Chari, Morey, Rios, Medford, Church, Fields, Baker. Submitted

Sensor-interface Taylor ND, Garruss AS, Moretti R, Chan S, Arbing M, Cascio D, Rogers RK, Isaacs FJ, Kosuri S, Baker D, Fields S, Church GM, Raman S (2015)

Sucralose

Sucralose �Sensor�

(from LacI)

1 color. 10K RNAs = 1250 brains 8probesperbrain25µmsections.

AllenInst.goo.gl/CF5x4160,448

Rosetta BRAIN: Integration of data types

Activity (tickertape/Ca++ imaging) Behavior, perception, memory (video) Connectome (synapse Ab + ID)Developmental lineage (ID)Expression (RNA, protein: ID)FISSEQ: Serial section & Organoids

Marblestone et al. Physical Principles for Scalable Neural Recording. ArXiv. 2013 Conneconomics bioRxiv 2013. Rosetta Brains ArXiv. 2014

Connectome: Accuracy of assigning neurite to cell body drops exponentially with distance in serial reconstruction.

Barcode-based reconstruction is distance independent.

Theoretical Diversity ~ 200 colors

Multiplexing neuron tracing with brainbow

Livet, Lichtman et al., 2007

Resolvable colors <10

Cepko et al. Science 1992, Methods Enzym 2000. 4^N Zador et al. PLoS Bio 2012 2NN!

MAPseq: Multiplexed Analysis of Projections by sequencing BOINC: Barcoding Of Individual Neuronal Connectivity

Previous Barcoding methods

Random sequences can uniquely label neurons

RandomSequence

A T G C G C T A T …

Length Diversity

1 4

2 16

10 106

30 1018

N 4N

109 barcodes are sufficient to label all 107 neurons in mouse cortex

Reza Kalhor � Prashant Mali, Nature Methods 2016

Rapidly evolving homing CRISPR barcodes.

Connectome & Development: Evolving Barcode IDs

Kalhor, Mali, Church Nature Methods 2016

Observed bit density

Next-Gen Sequencing “images” DNA sequences

Cluster 1: Cluster 2: Cluster 3: Cluster 4:

Conventional Sequencing

In situ Sequencing

Cluster 1: Cluster 2: Cluster 3: Cluster 4:

Conventional Sequencing

Lee, Church et al. 2015

Next-Gen Sequencing “images” DNA sequences

Chen, Zador et al

In situ sequencing

In situ sequencing

Neuron1

Neuron2

A

G

Neuron1

Neuron2

ATCG

XiaoyinChen

Neuron1

Neuron2

AG

GT

Neuron1

Neuron2

ATCGIn situ sequencing

Chen, Zador et al

Neuron1

Neuron2

AGC

GTG

Neuron1

Neuron2

ATCGIn situ sequencing

Chen, Zador et al

Neuron1

Neuron2

AGCG

GTGC

Neuron1

Neuron2

ATCG

Neuron1

Neuron2

ATCGIn situ sequencing

Chen, Zador et al

Neuron1

Neuron2

AGCGT

GTGCA

Neuron1

Neuron2

ATCG

Neuron1

Neuron2

ATCGIn situ sequencing

Chen, Zador et al

Neuron1

Neuron2

AGCGTG

GTGCAT

Neuron1

Neuron2

ATCG

Neuron1

Neuron2

ATCGIn situ sequencing

Chen, Zador et al

Neuron1

Neuron2

AGCGTGT

GTGCATA

Neuron1

Neuron2

ATCG

Neuron1

Neuron2

ATCGIn situ sequencing

Chen, Zador et al

Neuron1

Neuron2

AGCGTGTT

GTGCATAA

Neuron1

Neuron2

ATCG

Neuron1

Neuron2

ATCGIn situ sequencing

Chen, Zador et al

Kebschull, … Zador (2016) Neuron 91:975. High-Throughput Mapping of Single-Neuron Projections by Sequencing of Barcoded RNA.

Major projections of 2 (randomly selected) ... Up to 500,000 neurons

Long range Connectome & Developmental Lineage via barcode sequencing

Omni Omics in situ�

Genomes (4D nucleome, ATAC-Seq)�Transcriptome (mods & splicing)Proteome MetabolomeMicrobiome/viromeImmunome

Inference of subcellular & inter-cellular interactions -vs- direct observation (& manipulation)

48

Problems #4 Paul Reginato & Yu Wang 4A. What can the human cell atlas be used for? 4B. What methods can be used to obtain the data necessary for the atlas? 4C. What do you think about the idea of 'cell types'? 4D. Next gen sequencing is currently used to query biological specimens in many different ways, using many different tricks. Think of at least two applications of in situ sequencing other than basic transcriptome, genome, or proteome sequencing. Some existing ideas that serve as examples: •  DARPA MICrONs v.ht/P0rU •  Molecular tickertape to record neural activity

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