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.


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