3.4. Fourth-Generation Sequencing

The fourth-generation sequencing integrated nanopore technology into SMS. This technology performs real-time sequencing without amplification and repeated cycles by eliminating synthesis and therefore is called as 4G sequencing. The 4^thGS, also called in situ sequencing technology, has opened new horizons in DNA sequencing by making it possible to identify order of nucleotides in the fixed cells and tissues [21]. It differs from other sequencing generation approaches in two ways. Firstly, spatial distribution of the DNA reads over the sample can be observed which provide very useful information for highlighting tissue heterogeneity based upon the known markers. The second difference is that large number of cells can be analyzed simultaneously. For example, robust single cell RNA sequencing approaches were developed, which are cheap and are capable to sequence a number of cells with very few pictograms of the starting material [51]. Drawback of this technique is that tissue material is composed of several thousands of cells and sequencing single cells is not technically and computationally an easy job. However, it is predicted that in situ sequencing will be used to extract clinically important information from data produced by conventional NGS approaches. Targeted in situ sequencing method may be applied for filtering validated biomarkers directly on the samples whereas nontargeted technique may be useful for developing molecular profiles of the samples for classifying a disease on the molecular level or to satisfy the patients. Integrating in situ sequencing in the conventional NGS methods would expedite the development of these methods and these will eventually become essential tools for personalized medicine. Nanopore sequencing, the most popular 4^thGS platform, has ability to identify molecules (proteins, DNA, RNA, etc.) while they are passed through nanoscale holes entrenched in a thin membrane [52]. In this approach, an electric field forces individual molecules to pass through a nanopore having 2 nm diameter. Due to very thin pore, single-stranded molecules are passed through the pore in a firm linear order. Distinguished electric signals are generated as DNA molecule passes through the pore. The most famous nanopore technology is the Oxford nanopore Technology. It is one of the most robust sequence technologies and can sequence whole genome with 1 million base pairs long reads and diagnose diseases very efficiently and with very low cost [53]. The MinION, which was released in 2014, is the first application of nanopore technology. Other higher throughput nanopore devices from Oxford Nanopore Technologies are GridION Mk1 and PromethION 24/48. GridION Mk1has 1-5 flow cells with the ability of generating 250 GB data. PromethION 24/48 has 1-48 flow cells and can produce data up to 15 TB [54]. Nanopore sequencing is classified into three categories. In case of 1D, single-stranded DNA is sequenced. In 2D, two strands of the DNA were bounded by a hairpin-like structure. The first sequence of one strand of DNA is obtained, and then, the second strand DNA is sequenced. In this way, sequencing is repeated twice to raise base calling quality. 1D² is very close to 2D, but hairpin structure is not needed for keeping connected two strands of DNA.