Abstract
Sequencing of a single-cell genome requires DNA amplification, a process prone to introducing bias and errors into the amplified genome. Here we introduce a novel multiple displacement amplification (MDA) method based on the unique DNA primase features of Thermus thermophilus (Tth) PrimPol. TthPrimPol displays a potent primase activity preferring dNTPs as substrates unlike conventional primases. A combination of TthPrimPol's unique ability to synthesize DNA primers with the highly processive Phi29 DNA polymerase (Φ29DNApol) enables near-complete whole genome amplification from single cells. This novel method demonstrates superior breadth and evenness of genome coverage, high reproducibility, excellent single-nucleotide variant (SNV) detection rates with low allelic dropout (ADO) and low chimera formation as exemplified by sequencing HEK293 cells. Moreover, copy number variant (CNV) calling yields superior results compared with random primer-based MDA methods. The advantages of this method, which we named TruePrime, promise to facilitate and improve single-cell genomic analysis.
Highlights
Sequencing of a single-cell genome requires DNA amplification, a process prone to introducing bias and errors into the amplified genome
Conventional archaeal-eukaryotic primase (AEP)-like primases, as human Prim[1], have the three conserved motifs (A, B and C) that form the primase active site, the PrimPols already characterized have the same three conserved motifs and a Zn-finger domain required for the DNA primase activity[34,35,36]
The significant amino acid sequence similarity with pRN1 PrimPol and with the polymerization domain (PolDom) of Mycobacterium tuberculosis LigD, whose threedimensional (3D) structures have been solved[26,43,44], was sufficient to generate a 3D model for TthPrimPol in complex with DNA and nucleotide substrates (Fig. 1b; see Methods for details)
Summary
Sequencing of a single-cell genome requires DNA amplification, a process prone to introducing bias and errors into the amplified genome. A combination of TthPrimPol’s unique ability to synthesize DNA primers with the highly processive Phi[29] DNA polymerase (F29DNApol) enables near-complete whole genome amplification from single cells. This novel method demonstrates superior breadth and evenness of genome coverage, high reproducibility, excellent single-nucleotide variant (SNV) detection rates with low allelic dropout (ADO) and low chimera formation as exemplified by sequencing HEK293 cells. Copy number variant (CNV) calling yields superior results compared with random primer-based MDA methods The advantages of this method, which we named TruePrime, promise to facilitate and improve single-cell genomic analysis. Perhaps the most significant feature of PrimPols, unlike conventional primases, is their ability to carry out the initiation and extension of DNA chains[27,30,31,32]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
