High-Throughput Single-Cell Sequencing with Linear Amplification

Yi Yin; Yue Jiang; Kwan-Wood Gabriel Lam; Joel B Berletch; Christine M Disteche; William S Noble; Frank J Steemers; R Daniel Camerini-Otero; Andrew C Adey; Jay Shendure

doi:10.1016/j.molcel.2019.08.002

High-Throughput Single-Cell Sequencing with Linear Amplification

Mol Cell. 2019 Nov 21;76(4):676-690.e10. doi: 10.1016/j.molcel.2019.08.002. Epub 2019 Sep 5.

Authors

Affiliations

¹ Department of Genome Sciences, University of Washington, Seattle, WA, USA. Electronic address: [email protected].
² Seattle, WA, USA.
³ Genetics and Biochemistry Branch, NIDDK, NIH, Bethesda, MD, USA.
⁴ Department of Pathology, University of Washington, Seattle, WA, USA.
⁵ Department of Genome Sciences, University of Washington, Seattle, WA, USA.
⁶ Illumina, San Diego, CA, USA.
⁷ Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA; Knight Cardiovascular Institute, Portland, OR, USA.
⁸ Department of Genome Sciences, University of Washington, Seattle, WA, USA; Allen Discovery Center for Cell Lineage Tracing, Seattle, WA, USA; Brotman Baty Institute for Precision Medicine, Seattle, WA, USA; Howard Hughes Medical Institute, Seattle, WA, USA. Electronic address: [email protected].

Abstract

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.

Keywords: DNA repair; chromosome segregation; double-strand break; homologous recombination; infertility; linear amplification; meiotic crossover; mouse; single-cell combinatorial indexing; single-cell sequencing.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Chromosome Segregation
Gene Expression Profiling*
High-Throughput Nucleotide Sequencing*
Male
Meiosis / genetics
Mice
Nucleic Acid Amplification Techniques*
Proof of Concept Study
Sequence Analysis, DNA*
Sequence Analysis, RNA*
Single-Cell Analysis / methods*
Spermatozoa / physiology
Transcriptome
Whole Genome Sequencing*
Workflow

Abstract

Publication types

MeSH terms

Grants and funding