TY - JOUR
T1 - High-Throughput Single-Cell Sequencing with Linear Amplification
AU - Yin, Yi
AU - Jiang, Yue
AU - Lam, Kwan Wood Gabriel
AU - Berletch, Joel B.
AU - Disteche, Christine M.
AU - Noble, William S.
AU - Steemers, Frank J.
AU - Camerini-Otero, R. Daniel
AU - Adey, Andrew C.
AU - Shendure, Jay
N1 - Funding Information:
The raw data are deposited with the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/PRJNA511715). We thank G. Bonora, N. Kleckner, and members of the Shendure lab for helpful discussions. We thank C. Chen, D. Xing, J. Cao, and M. Spielmann for helpful technical suggestions; A. Leith for exceptional assistance in flow sorting; and T. Reh's lab for sharing the NIH/3T3 cell line. This work was funded by grants from the NIH (DP1HG007811 and R01HG006283 to J.S.; R35GM124704 to A.C.A.; DK107979 to J.S. W.S.N. and C.M.D.; and GM046883 to C.M.D.), NIDDK Intramural Research Program to R.D.C.-O. and the Paul G. Allen Frontiers Group (Allen Discovery Center grant to J.S.). Y.Y. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2248-16). J.S. is an investigator of the Howard Hughes Medical Institute. Y.Y. developed techniques and performed the experiments; Y.Y. and Y.J. performed computational analyses; K.-W.G.L. and R.D.C.-O. provided (B6 ? Cast) F1 mouse nuclei and helpful discussions; J.B.B. and C.M.D. provided (B6 ? Spret) F1 mice; W.S.N. provided advice on the analyses; Y.Y. and J.S. wrote the paper with input from A.C.A. and all of the authors; and J.S. supervised the work. F.J.S. declares competing financial interests in the form of stock ownership and paid employment by Illumina. One or more embodiments of one or more patents and patent applications by the University of Washington and Illumina may encompass the methods, reagents, and data disclosed in this manuscript.
Funding Information:
The raw data are deposited with the Sequence Read Archive ( https://www.ncbi.nlm.nih.gov/sra/PRJNA511715 ). We thank G. Bonora, N. Kleckner, and members of the Shendure lab for helpful discussions. We thank C. Chen, D. Xing, J. Cao, and M. Spielmann for helpful technical suggestions; A. Leith for exceptional assistance in flow sorting; and T. Reh’s lab for sharing the NIH/3T3 cell line. This work was funded by grants from the NIH ( DP1HG007811 and R01HG006283 to J.S.; R35GM124704 to A.C.A.; DK107979 to J.S., W.S.N., and C.M.D.; and GM046883 to C.M.D.), NIDDK Intramural Research Program to R.D.C.-O., and the Paul G. Allen Frontiers Group (Allen Discovery Center grant to J.S.). Y.Y. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation ( DRG-2248-16 ). J.S. is an investigator of the Howard Hughes Medical Institute.
Publisher Copyright:
© 2019 Elsevier Inc.
Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.
PY - 2019/11/21
Y1 - 2019/11/21
N2 - Conventional methods for single-cell genome sequencing are limited with respect to uniformity and throughput. Here, we describe sci-L3, a single-cell sequencing method that combines combinatorial indexing (sci-) and linear (L) amplification. The sci-L3 method adopts a 3-level (3) indexing scheme that minimizes amplification biases while enabling exponential gains in throughput. We demonstrate the generalizability of sci-L3 with proof-of-concept demonstrations of single-cell whole-genome sequencing (sci-L3-WGS), targeted sequencing (sci-L3-target-seq), and a co-assay of the genome and transcriptome (sci-L3-RNA/DNA). We apply sci-L3-WGS to profile the genomes of >10,000 sperm and sperm precursors from F1 hybrid mice, mapping 86,786 crossovers and characterizing rare chromosome mis-segregation events in meiosis, including instances of whole-genome equational chromosome segregation. We anticipate that sci-L3 assays can be applied to fully characterize recombination landscapes, to couple CRISPR perturbations and measurements of genome stability, and to other goals requiring high-throughput, high-coverage single-cell sequencing.
AB - Conventional methods for single-cell genome sequencing are limited with respect to uniformity and throughput. Here, we describe sci-L3, a single-cell sequencing method that combines combinatorial indexing (sci-) and linear (L) amplification. The sci-L3 method adopts a 3-level (3) indexing scheme that minimizes amplification biases while enabling exponential gains in throughput. We demonstrate the generalizability of sci-L3 with proof-of-concept demonstrations of single-cell whole-genome sequencing (sci-L3-WGS), targeted sequencing (sci-L3-target-seq), and a co-assay of the genome and transcriptome (sci-L3-RNA/DNA). We apply sci-L3-WGS to profile the genomes of >10,000 sperm and sperm precursors from F1 hybrid mice, mapping 86,786 crossovers and characterizing rare chromosome mis-segregation events in meiosis, including instances of whole-genome equational chromosome segregation. We anticipate that sci-L3 assays can be applied to fully characterize recombination landscapes, to couple CRISPR perturbations and measurements of genome stability, and to other goals requiring high-throughput, high-coverage single-cell sequencing.
KW - DNA repair
KW - chromosome segregation
KW - double-strand break
KW - homologous recombination
KW - infertility
KW - linear amplification
KW - meiotic crossover
KW - mouse
KW - single-cell combinatorial indexing
KW - single-cell sequencing
UR - http://www.scopus.com/inward/record.url?scp=85074992710&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85074992710&partnerID=8YFLogxK
U2 - 10.1016/j.molcel.2019.08.002
DO - 10.1016/j.molcel.2019.08.002
M3 - Article
C2 - 31495564
AN - SCOPUS:85074992710
VL - 76
SP - 676-690.e10
JO - Molecular Cell
JF - Molecular Cell
SN - 1097-2765
IS - 4
ER -