Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics

Abstract

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio's single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.

Figure 1.

Overview of SMRT Sequencing Technology. Sequencing starts with preparing a library from double stranded DNA (A) to which hairpin adapters are ligated (B). This library is thereafter loaded onto a SMRT Cell made up of nanoscale observation chambers (Zero Mode Waveguides (ZMWs)). The DNA molecules in the library will be pulled to the bottom of the ZMW where the polymerase will incorporate fluorescently labelled nucleotides (C). Note that not all ZMWs will contain a DNA molecule because the library is loaded by diffusion. The fluorescence emitted by the nucleotides is recorded by a camera in real-time. Hence, not only the fluorescence color can be registered, but also the time between nucleotide incorporation which is called the interpulse duration (IPD) (D, right panel). When a sequencing polymerase encounters nucleotides on the DNA strand containing an (epigenetic) modification, like for example a 6-methyl adenosine modification (E, left panel), then the IPD will be delayed (E, right panel) compared to non-methylated DNA (D, right panel). Due to the circular structure of the library, a short insert will be covered multiple times by the continuous long read (CLR). Each pass of the original DNA molecule is termed a subread, which can be combined into one highly accurate consensus sequence termed a circular consensus sequence (CCS) or reads-of-insert (ROI) (F–H, left panel). Though SMRT sequencing always uses a circular template, long insert libraries typically only have a single pass and hence generate a linear sequence with single pass error rates (black nucleotides) (FG, right panel). Afterwards, overlapping single passes can be combined into one consensus sequence of high quality (H, right panel). Overall, CCS reads have the advantage of being very accurate while single passes stand out for their long read lengths (>20 kb).