title: DNA sequencing using polymerase substrate-binding kinetics.
abstract: Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications.
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Michael John Robert
authors.initials: MJ
authors.lastname: Previte
authors.affiliation: Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Chunhong
authors.initials: C
authors.lastname: Zhou
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Kellinger
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Rigo
authors.initials: R
authors.lastname: Pantoja
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Cheng-Yao
authors.initials: CY
authors.lastname: Chen
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Shi
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: BeiBei
authors.initials: B
authors.lastname: Wang
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Amirali
authors.initials: A
authors.lastname: Kia
authors.affiliation: Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Sergey
authors.initials: S
authors.lastname: Etchin
authors.affiliation: Bioinformatics, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: John
authors.initials: J
authors.lastname: Vieceli
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Ali
authors.initials: A
authors.lastname: Nikoomanzar
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Bomati
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Gloeckner
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Ronaghi
authors.affiliation: Protein Engineering, Illumina Inc., 5200 Research Place, San Diego, California 92122, USA.
authors.forename: Molly Min
authors.initials: MM
authors.lastname: He
mesh_terms: D010584:Bacteriophage phi X 174 / Q000235:genetics; D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004867:Equipment Design; D016679:Genome, Viral; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation* / Q000379:methods*; D007700:Kinetics; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000235:genetics*; D011108:Polymers; D017422:Sequence Analysis, DNA
chemical_list: D009711:Nucleotides; D011108:Polymers; D004247:DNA; D004259:DNA-Directed DNA Polymerase