**4.1. Single-molecule real-time (SMRT) sequencing by pacific biosciences**

Pacific Biosciences (http://www.pacificbiosciences.com) markets the PacBio RS II sequencer and the SMRT real-time sequencing system [74, 75]. SMRT sequencing is performed in SMRT cells that contain 150,000 ultra-microwells at a zeptoliter scale where one molecule of DNA polymerase is immobilized at the bottom of each well using the biotin-streptavidin system in nanostructures known as zero-mode waveguides (ZMWs). Once the template single-strand DNA is coupled with immobilized DNA polymerase, fluorescently labeled dNTP analogs are added and detected when the nucleotide is incorporated into the growing strand. CCD cameras continuously monitor the 150,000 ZMWs as a series of observed pulses that are converted into single molecular traces representing the template sequences. Since all four nucleotides are added simultaneously and measured in real time, the speed of sequencing is much increased compared to technologies where individual nucleotides are flushed sequen‐ tially. Although the reported accuracy was 99.3% initially with read lengths of about 900 bp [4], circularizing the template and sequencing it several times using a technology called SMRTbell templates provided longer reads and improved the accuracy to >99.999% [76, 77]. Once sequencing is initiated, the system's computational Blade Center performs real-time signal processing, base calling, and quality assessment. Primary analysis data, including trace and pulse data, read-length, distribution, polymerase speed, and quality measurement, is streamed directly to the secondary analysis software called SMRT Analysis that is capable of processing sequencing data in real time. The secondary analysis tools also include a full suite of tools to analyze single-molecule sequencing data for a broad range of applications.

#### **4.2. Helicos sequencing by the genetic analysis system**

The Helicos sequencing system was the first commercial implementation of single-molecule fluorescent sequencing [66, 78], marketed by the now bankrupt Helicos Biosciences. Today, the sequencing provider Seqll (http://seqll.com) sequences genomic DNA and RNA using the Helicos sequencing system and HeliScope single-molecule sequencers. DNA is sheared, tailed with polyA, and hybridized to a flow cell surface containing oligo-dT for sequencing-bysynthesis of billions of molecules in parallel. The polyA-tailed fragments of DNA molecules are hybridized directly to the oligo-dT50 bound on the surface of disposable glass flow cells. The addition of fluorescent nucleotides with a terminating nucleotide pauses the cyclical process until an image of one nucleotide for each DNA sequence has been captured, and then the process is repeated until the fragments have been completely sequenced [75, 76]. This sequencing system is a combination of sequencing by hybridization and sequencing by synthesis using a DNA polymerase [79]. Sample preparation does not require ligation or PCR amplification and, therefore, largely avoids the GC content and size biases observed in other technologies [56]. The HeliScope sequencing read lengths range from 25 to over 60 bases, with 35 bases being the average. This method has successfully sequenced the human genome [80] to provide disease signatures in a clinical evaluation [81] and sequenced RNA to produce quantitative transcriptomes of tissues and cells [82].
