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Developing a one-tube circularized ligation product sequencing (CLP-seq) method for the mapping of 3D genome architecture in small cell populations or single cells.

Award Number: R01HG009658
ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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The 3D architecture of mammalian genome plays a key role in transcription regulation. Through DNA looping, non-coding cis-regulatory elements may regulate target genes from hundreds of kilobases away. Because of this complexity, generating a comprehensive map of long-range DNA looping interactions will greatly facilitate our understanding of genome functions. Our previous work for the first time demonstrated that it is feasible to map the 3D genome in mammalian cells with 3~5 billion Hi-C reads, at a resolution of 5-10kb. At this resolution, interactions between individual cis-regulatory elements can be revealed. Recently, single cell Hi-C approach has also been tested to reveal cell-to-cell variability of chromosome structures. The fast growing field of 3D genome research calls for 3D genome maps in a variety of cell or tissue types under different physiological or pathogenic perturbations. In order to achieve broad applicability, 3D genome mapping technology must address the following challenges: (i) Ability to assay rare bio-samples; (ii) Generating high-quality library for deep sequencing at a level of several billion reads; (iii) The ability to analyze a large number of single cells for the analyses of complex tissue or cellular heterogeneity. However, the library quality from Hi-C and its derivatives is usually poor when the amount of starting material is small. The overall goal of this proposal is to develop a simple and efficient 3C-seq method (Circularized Ligation Products sequencing, or CLP-seq) to generate high-quality libraries suitable for ultra-deep sequencing from a small number of cells. In contrast to Hi-C and its derivatives, CLP-seq is unique because it enriches ligation junction products through a series of enzymatic reactions without the need for biotin labeling and pull-down. From a pilot experiment, we estimate that this new method requires less than 1% of cells as starting material to reach sequencing depth at that level of a billion reads (over 100-fold improvement over Hi-C). Furthermore, because CLP- seq avoids biotin labeling and pull-down, it is amenable to the development of a one-tube single cell CLP- seq protocol (scCLP-seq) for massive scalable single cell analysis. In this project, we will establish and optimize these new technologies, and as proof-of-principle, also produce a significant amount of valuable data resources with these methods in the following three aims. In aim 1, we will optimize CLP-seq protocol to generate high-complexity libraries for ultra-deep sequencing from small cell populations or rare human tissues. In aim 2, we will develop a full-package CLP-seq data analysis pipeline to detect and visualize DNA looping interactions at kilobase resolution. We will generate kilobase-resolution 3D genome maps in a few difficult human tissues and perform preliminary functional annotation of non- coding GWAS SNPs in relevant human diseases. In aim 3, we will further develop a one-tube scCLP- seq protocol for simultaneous analysis of hundreds of single cells. As test cases, we will generate 3D genome data in hundreds of single cells from human islet tissues, and explore strategies to perform subpopulation analysis using clustering methods. We believe this technology advance will expand the field of 3D genome study and eventually benefit our understanding of genome functions and human diseases.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2023 ( Subtotal = $559,622 )
2023	2023	CASE WESTERN RESERVE UNIVERSITY	10900 EUCLID AVE	CLEVELAND	OH	44106	CUYAHOGA	USA	Human Genome Research	000	7	5/15/2023	NON-COMPETING CONTINUATION	$559,622
2023	2022	CASE WESTERN RESERVE UNIVERSITY	10900 EUCLID AVE	CLEVELAND	OH	44106	CUYAHOGA	USA	Human Genome Research	002	6	6/21/2023	COMPETING CONTINUATION	$89,194
2023	2021	CASE WESTERN RESERVE UNIVERSITY	10900 EUCLID AVE	CLEVELAND	OH	44106	CUYAHOGA	USA	Human Genome Research	001	5	6/21/2023	NON-COMPETING CONTINUATION	-$89,194
														Subtotal = $559,622

Issue Date FY: 2022 ( Subtotal = $621,802 )
2022	2022	CASE WESTERN RESERVE UNIVERSITY	10900 EUCLID AVE	CLEVELAND	OH	44106	CUYAHOGA	USA	Human Genome Research	001	6	8/3/2022	COMPETING CONTINUATION	$559,622
2022	2022	CASE WESTERN RESERVE UNIVERSITY	10900 EUCLID AVE	CLEVELAND	OH	44106	CUYAHOGA	USA	Human Genome Research	002	6	9/15/2022	COMPETING CONTINUATION	$62,180
2022	2021	CASE WESTERN RESERVE UNIVERSITY	10900 EUCLID AVE	CLEVELAND	OH	44106	CUYAHOGA	USA	Human Genome Research	000	5	7/26/2022	NON-COMPETING CONTINUATION	$0
														Subtotal = $621,802

Issue Date FY: 2021 ( Subtotal = $464,113 )
2021	2021	CASE WESTERN RESERVE UNIVERSITY	10900 EUCLID AVE	CLEVELAND	OH	44106	CUYAHOGA	USA	Human Genome Research	000	5	6/23/2021	NON-COMPETING CONTINUATION	$464,113
														Subtotal = $464,113

Issue Date FY: 2020 ( Subtotal = $464,113 )
2020	2020	CASE WESTERN RESERVE UNIVERSITY	10900 EUCLID AVE	CLEVELAND	OH	44106	CUYAHOGA	USA	Human Genome Research	000	4	5/22/2020	NON-COMPETING CONTINUATION	$464,113
														Subtotal = $464,113

Issue Date FY: 2019 ( Subtotal = $461,230 )
2019	2019	CASE WESTERN RESERVE UNIVERSITY	10900 EUCLID AVE	CLEVELAND	OH	44106	CUYAHOGA	USA	Human Genome Research	000	3	6/28/2019	NON-COMPETING CONTINUATION	$461,230
														Subtotal = $461,230

Issue Date FY: 2018 ( Subtotal = $461,230 )
2018	2018	CASE WESTERN RESERVE UNIVERSITY	10900 EUCLID AVE	CLEVELAND	OH	44106	CUYAHOGA	USA	Human Genome Research	000	2	6/29/2018	NON-COMPETING CONTINUATION	$461,230
														Subtotal = $461,230

Issue Date FY: 2017 ( Subtotal = $456,906 )
2017	2017	CASE WESTERN RESERVE UNIVERSITY	10900 EUCLID AVE	CLEVELAND	OH	44106	CUYAHOGA	USA	Human Genome Research	000	1	8/9/2017	NEW	$456,906
														Subtotal = $456,906

Grand Total All Awards = $3,489,016

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Developing a one-tube circularized ligation product sequencing (CLP-seq) method for the mapping of 3D genome architecture in small cell populations or single cells.

Award Number: R01HG009658

ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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