Single-molecule real-time sequencing

To prepare the library, DNA fragments are put into a circular form using hairpin adapter ligations.

[4] The zero-mode waveguide (ZMW) is a nanophotonic confinement structure that consists of a circular hole in an aluminum cladding film deposited on a clear silica substrate.

Due to the behavior of light when it travels through a small aperture, the optical field decays exponentially inside the chamber.

[10] Pacific Biosciences (PacBio) commercialized SMRT sequencing in 2011,[11] after releasing a beta version of its RS instrument in late 2010.

The resulting P4 attributes provided higher-quality assemblies using fewer SMRT Cells and with improved variant calling.

[16] When coupled with input DNA size selection (using an electrophoresis instrument such as BluePippin) yields average read length over 7 kilobases.

The throughput with the new chemistry was estimated between 500 million to 1 billion bases per SMRT Cell, depending on the sample being sequenced.

Throughput per experiment for the technology is both influenced by the read length of DNA molecules sequenced as well as total multiplex of a SMRT Cell.

The prototype of the SMRT Cell contained about 3000 ZMW holes that allowed parallelized DNA sequencing.

[22] In April 2013, the company released a new version of the sequencer called the "PacBio RS II" that uses all 150,000 ZMW holes concurrently, doubling the throughput per experiment.

[23][24] The highest throughput mode in November 2013 used P5 binding, C3 chemistry, BluePippin size selection, and a PacBio RS II officially yielded 350 million bases per SMRT Cell though a human de novo data set released with the chemistry averaging 500 million bases per SMRT Cell.

In September 2015, the company announced the launch of a new sequencing instrument, the Sequel System, that increased capacity to 1 million ZMW holes.

[39] In 2013, scientists estimated that long-read sequencing could be used to fully assemble and finish the majority of bacterial and archaeal genomes.

[41] In August 2012, scientists from the Broad Institute published an evaluation of SMRT sequencing for SNP calling.

[49][50] SMRT sequencing has several applications in reproductive medical genetics research when investigating families with suspected parental gonadal mosaicism.

Deep sequencing enables determination of allele frequencies in sperm cells, of relevance for estimation of recurrence risk for future affected offspring.