**4.3. Nanopore sequencing by Oxford Nanopore Technologies (MinION and PromethION)**

Oxford Nanopore Technologies provides the latest single-molecule sequencing system [83, 84]. The MinION Mkl is a portable handheld device for DNA and RNA sequencing that attaches directly to a laptop/computer using a USB port, whereas the PromethION is a small bench-top system. Nanopore sequencing uses pores formed from proteins, such as haemoly‐ sin, a biological protein channel system in *Staphylococcus aureus* [85]. The idea behind DNA and RNA sequencing using nanopores is that the conductivity of ion currents in the pore changes when the strand of nucleic acid passes through it [83]. The flow of ion current depends on the shape of the molecule translocating through the pore. Since nucleotides have different shapes, each nucleotide is recognized by its effect on the change of the ionic current [86]. The key advantage of this approach is that sample preparation is minimal compared to secondgeneration sequencing methods, and long read lengths can be obtained in the kbp range. In addition, there are no amplification or ligation steps required before sequencing. The main problem with this technology is the requirement to optimize the speed of DNA translocation through the nanopore to ensure reliable measurement of the ionic current changes and reduce the high error rates of base calling [83–86]. At this time, Oxford Nanopore Technologies is in the beta testing phase, and users are required to join the MinION Access Programme and pay a fee of \$1000 [83] to access a MinION starter pack (3 MinION MkI flow cells, a Nanopore sequencing kit, and a wash kit). Laver et al. [87] have assessed the performance of an earlier version of the MinION sequencing device and concluded that "the MinION is an exciting prospect; however, the current error rate limits its ability to compete with existing sequencing technologies, though we do show that MinION sequence reads can enhance contiguity of *de novo* assembly when used in conjunction with Illumina MiSeq data." They resequenced three bacterial genomes and estimated the error rate to be 38.2%, with mean and median reads of 2 and 1 kb, respectively, and with the longest single read of 98 kb. The low depth of coverage provided by the present nanopore technology is a possible barrier to accurate eukaryotic genome sequencing at the moment. Nevertheless, these are not intangibles and nanopore nucleic acid sequencing is envisaged to include methylation and direct RNA sequencing in the near future [83].

#### **4.4. NGS by electron microscopy**

read lengths, flexible workflow, reduced turnaround time, and a cheaper price than those

Third-generation single-molecule sequencing technologies have emerged to reduce the price of sequencing and to simplify the preparatory procedures and sequencing methods [4, 74, 75].

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.

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

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

**4. Third-generation sequencing: Emerging technologies for single-**

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

provided by the other platforms [54, 73].

12 Next Generation Sequencing - Advances, Applications and Challenges

**molecule sequencing**

The sequence of long, intact DNA molecules can be visualized and identified by using electron microscopy. The first report on the successful application of electron microscopy for NGS was for the partial sequencing of DNA base pairs within intact DNA molecules using synthesized genomes of 3.3 and 7.2 kb length that were sequenced by enzymatically incorporating modified bases that contained atoms of increased atomic number and allowed for the direct visualization and identification of individually labeled bases [88]. In this sequencing process, the double strands of the DNA sample are separated into single strands using common enzymes and reactions. Then, the single-stranded DNA is labeled by PCR using dNTPs attached to heavyatom metal labels that can be separated into identifiable electron microscope-generated images showing large black dots, small black dots, and large gray dots along the DNA molecule linearized by ZSG threading. Standard image-based technologies perform the reads and analysis of the labeled DNA using image analysis software that provides sequence data in real time. The sequenced molecules are reads in the range of 5 to 50 kb in length that are useful for *de novo* genome assembly and for analysis of full haplotypes and copy number variants. The company ZS Genetics (http://www.zsgenetics.com) offers a service to provide accurate, longread, single-molecule DNA sequences using the NGS electron microscopy platform.
