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Proc Natl Acad Sci U S A. 2017 Nov 21;114(47):12512-12517. doi: 10.1073/pnas.1707609114. Epub 2017 Oct 24.

Ultraaccurate genome sequencing and haplotyping of single human cells.

Author information

1
Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093.
2
Department of Computer Science and Engineering, University of California, San Diego, La Jolla, CA 92093.
3
Department of Electrical and Computer Engineering, University of California, San Diego, La Jolla, CA 92093.
4
Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093.
5
Department of Computer Science and Engineering, University of California, San Diego, La Jolla, CA 92093; vbafna@cs.ucsd.edu x2huang@ucsd.edu kzhang@eng.ucsd.edu.
6
Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093; vbafna@cs.ucsd.edu x2huang@ucsd.edu kzhang@eng.ucsd.edu.

Abstract

Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10-8 and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs.

KEYWORDS:

haplotyping; microfluidics; mutation detection; single-cell sequencing

Comment in

PMID:
29078313
PMCID:
PMC5703283
DOI:
10.1073/pnas.1707609114
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

Conflict of interest statement: X.H. and K.Z. are listed as inventors for a patent application related to the method disclosed in this manuscript. K.Z. is a cofounder and equity holder of Singlera Genomics Inc. V. Bafna is a cofounder, has an equity interest, and receives income from Digital Proteomics, LLC. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. Digital Proteomics, LLC was not involved in the research presented here.

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