Orthocoding for Spatial Sequencing

空间排序的正交编码

基本信息

批准号：
10191664
负责人：
Polly Morrell Fordyce
金额：
$ 39.43万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-10 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10191664
关键词：
3-Dimensional Address Bar Codes Cell Nucleus Cell Size Cell physiology Cells Complementary DNA Coupled DNA Detection Development Development Plans Diffuse Diffusion Dissociation Engineering Epigenetic Process Equipment Experimental Designs Failure Foundations Gene Expression Genes Genome Homeostasis Image In Situ Laboratories Life Location Measurement Measures Messenger RNA Methods Modification Molecular Molecular Biology Oligonucleotides Organism Positioning Attribute Property Protein Isoforms Proteins Protocols documentation RNA Research Personnel Resolution Resources Sampling Solid Statistical Algorithm Statistical Data Interpretation System Techniques Technology Testing Time Tissue Sample Tissues Transcript Variant base cost data standards deep sequencing design droplet sequencing experimental study high dimensionality human disease innovation novel prototype receptor single-cell RNA sequencing spatial relationship theories transcriptomics

项目摘要

Project Summary The 3D spatial context of a cell determines which genes and RNA isoforms it expresses, enabling specialized cell functions fundamental to multicellular life. In typical single-cell RNA-seq (scRNA-seq), the first step of cell dissociation erases the spatial context of the cell. This flaw creates an urgent need for a technology that has the same throughput of scRNA-seq but also encodes the cells’ spatial context. Although a new wave of spatial transcriptomic technologies based on sequencing has emerged recently, all suffer from severe limitations: low efficiency (~1-2% of the Drop-Seq efficiency), providing 2D resolution only, failure to discriminate cell boundaries and requiring specialized or expensive equipment. These limitations are intrinsic and result from their shared reliance on cDNA synthesis in situ by from a solid support. Imaging-based technologies have higher spatial resolution but require more equipment, time for protocol execution, have limited gene measurement throughput, and cannot profile RNA isoforms or other sequence variants. To overcome these limitations in state-of-the-art spatial transcriptomic methods, we propose to develop Orthocode, an innovative paradigm for statistically-driven spatial transcriptomics, grounded in proof-of-principle molecular experiments, and cutting-edge statistical theory. Orthocode achieves > 50x or higher sensitivity compared to current approaches by encoding and recovering spatial information from simple, inexpensive and efficient molecular biology protocols. The experimental Orthocode protocol has two steps: 1) a pool of two types of “location-encoding oligos” (a) barcoded emitter oligos produce copies of themselves that diffuse locally and (b) “receptors” record the barcodes of nearby emitters are coupled to cells; 2) cells coupled to location- encoding oligos that have together record the spatial position of the cell, are isolated and input into scRNA-seq workflows, eg. Drop-seq and sequenced. Orthocode then employs a rigorous statistical analysis of the barcode profiles of location encoding oligos to triangulate the location of each sequenced cell. This rigorously reasoned experimental design and prototype development builds Orthocode from the simplest test systems to prototypes that will allow unprecedented spatial transcriptomic resolution in tissues to address a critical unmet need in biomedicine. The Orthocode paradigm can be generalized beyond RNA profiling to spatial measurements of proteins, DNA and epigenetic modifications and is a potential breakthrough innovation in deep-sequencing based spatial ‘omics.

项目摘要细胞的3D空间上下文确定其表达的基因和RNA同工型，使得专门的细胞功能是多细胞寿命的基础。在典型的单细胞RNA-seq（SCRNA-Seq）中，第一个细胞解离的步骤消除了细胞的空间环境。这个缺陷迫切需要技术它具有SCRNA-SEQ相同的吞吐量，但也编码了细胞的空间环境。虽然是新浪潮最近出现了基于测序的空间转录组技术，所有这些都遭受了严重的困扰局限性：低效率（约1-2％的滴度序列效率），仅提供2D分辨率，未能区分细胞边界，需要专业或昂贵的设备。这些限制是固有的并由他们的共同继电器在cDNA合成上的共同继电器所产生的稳固支持。基于成像技术具有较高的空间分辨率，但需要更多的设备，协议执行时间，具有有限的基因测量吞吐量，无法谱图RNA同工型或其他序列变体。为了克服最先进的空间转录方法中的这些限制，我们建议开发 Orthocode，一种用于统计驱动的空间转录组学的创新范式，基于原理证明分子实验和尖端的统计理论。矫置的灵敏度> 50倍或更高的灵敏度与当前方法相比，通过从简单，廉价和有效的分子生物学方案。实验矫置协议有两个步骤：1）两个池 “位置编码的寡聚物”（a）条形码发射极寡核酸的副本，这些副本本地分散了（b）“受体”记录附近发射器的条形码与细胞耦合； 2）耦合到位置的细胞 - 编码与电池的空间位置共同记录的寡聚物是分离的，并输入到scrna-seq中工作流程，例如。 drop-seq并测序。然后，Orthocode对条形码进行了严格的统计分析编码寡聚的位置轮廓，以三角测量每个测序单元的位置。这种严格的理由实验设计和原型开发建立了从最简单的测试系统到原型的矫形器这将允许组织中前所未有的空间转录分辨率，以满足关键的未满足需求生物医学。可以将矫形码范式推广到RNA分析以外的空间测量蛋白质，DNA和表观遗传学修饰，是深层序列的潜在突破性创新基于空间的“ Omics”。

项目成果

期刊论文数量（18）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Pre-Cambrian roots of novel Antarctic cryptoendolithic bacterial lineages.

DOI：
10.1186/s40168-021-01021-0
发表时间：
2021-03-19
期刊：
Microbiome
影响因子：
15.5
作者：
Albanese D;Coleine C;Rota-Stabelli O;Onofri S;Tringe SG;Stajich JE;Selbmann L;Donati C
通讯作者：
Donati C

Functional Insights of Salinity Stress-Related Pathways in Metagenome-Resolved Methanothrix Genomes.

DOI：
10.1128/aem.02449-21
发表时间：
2022-05-24
期刊：
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
影响因子：
4.4
作者：
Gagliano, Maria Cristina;Sampara, Pranav;Plugge, Caroline M.;Temmink, Hardy;Sudmalis, Dainis;Ziels, Ryan M.
通讯作者：
Ziels, Ryan M.

Root-associated bacterial communities and root metabolite composition are linked to nitrogen use efficiency in sorghum.

DOI：
10.1128/msystems.01190-23
发表时间：
2024-01-23
期刊：
mSystems
影响因子：
6.4
作者：
通讯作者：

Desert truffle genomes reveal their reproductive modes and new insights into plant-fungal interaction and ectendomycorrhizal lifestyle.

DOI：
10.1111/nph.17044
发表时间：
2021-03
期刊：
The New phytologist
影响因子：
0
作者：
Marqués-Gálvez JE;Miyauchi S;Paolocci F;Navarro-Ródenas A;Arenas F;Pérez-Gilabert M;Morin E;Auer L;Barry KW;Kuo A;Grigoriev IV;Martin FM;Kohler A;Morte A
通讯作者：
Morte A

Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA.

DOI：
10.1093/plphys/kiac128
发表时间：
2022-06-01
期刊：
PLANT PHYSIOLOGY
影响因子：
7.4
作者：
Bewg, W. Patrick;Harding, Scott A.;Engle, Nancy L.;Vaidya, Brajesh N.;Zhou, Ran;Reeves, Jacob;Horn, Thomas W.;Joshee, Nirmal;Jenkins, Jerry W.;Shu, Shengqiang;Barry, Kerrie W.;Yoshinaga, Yuko;Grimwood, Jane;Schmitz, Robert J.;Schmutz, Jeremy;Tschaplinski, Timothy J.;Tsai, Chung-Jui
通讯作者：
Tsai, Chung-Jui