Single molecule targeted sequencing for cancer gene mutation detection

Abstract

With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis.

Single molecule sequencing of M13 virus genome without amplification

“…The single molecule SBS process has been described elsewhere [31] Each cycle includes terminator incorporation, imaging, cleavage of fluorophore, and capping of residual bonds. The GenoCare platform adopts the total internal fluorescence microscopy (TIRF) for the observation of single molecules.…”

“…Our approach is devised to provide simple operation and high-throughput, unbiased data. Recently, we have demonstrated a direct targeted sequencing of cancer related gene mutations at the SM level [31]. In this paper, we describe the performance of our new GenoCare platform for SM sequencing without preliminary PCR amplification.…”

Resequencing theEscherichia coligenome by GenoCare single molecule sequencing platform

“…The GenoCare Platform: Our sequencing tests have all been performed on the GenoCare platform designed and fabricated by the Direct Genomics company. The GenoCare sequencer has been developed on the basis of a single molecule sequencing-by-synthesis process described previously [37] [38]. The platform is highly automated and user-friendly.…”

“…Our approach is devised to provide simple operation and high-throughput, unbiased data. Recently, we demonstrated a direct targeted sequencing of cancer related gene mutations [37] and M13 virus genome sequencing [38] at the single molecule level. In this study, we resequenced the E. coli genome using two different platforms: the Illumina MiSeq system and the new Direct Genomics GenoCare platform.…”

Single Molecule Sequencing of Cell-free DNA from Maternal Plasma for Noninvasive Trisomy Detection

“…Helicos technology, a PCR-free singlemolecule-sequencing platform, has been applied for high sensitivity and specificity noninvasive fetal aneuploidy detection without obvious GC bias [15,16]. In our previous study, we demonstrated a direct targeted sequencing of cancer related gene mutations by single molecule sequencing method [17]. Recently, we reported single molecule sequencing of the M13 virus genome and Escherichia coli genome by GenoCare platform developed by Direct Genomics.…”