Abstract
Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.
Highlights
The sequencing of the human genome [1, 2] and the ensuing development of next-generation sequencing technologies (NGS) has revolutionized the life sciences and brought new approaches to applications as diverse as pathogen detection [3, 4], forensics [5, 6], and clinical diagnosis [7,8]
M13 genomic DNA was sheared into fragments of ~200bp, poly-A tailed with tail length of 50100nt, and blocked by ddATP-Cy3
The cloning vector M13mp18 was sequenced on this new GenoCare platform
Summary
The sequencing of the human genome [1, 2] and the ensuing development of next-generation sequencing technologies (NGS) has revolutionized the life sciences and brought new approaches to applications as diverse as pathogen detection [3, 4], forensics [5, 6], and clinical diagnosis [7,8]. The advent of precision medicine [9] promises profound advances in the clinic, leveraging sequencing results for diagnosis of cancer [10, 11] and inherited disease [12, 13]. Despite the advantages of NGS platforms, the costly, time-intensive process of NGS sample library preparation and the use of polymerase chain reaction (PCR) amplification limit the efficiency and practicality of NGS in the clinic. The preparation of DNA libraries in NGS generally requires a preliminary step based on PCR amplification. This process introduces bias and can result in incorrect interpretation of raw data [14, 15]. The popular Illumina sequencing platform produces data sets with uneven coverage and serious defects in GC-poor or GC-rich regions.
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