**5.3. Nanopore technology**

stranded DNA fragment anneals (by hybridization) to a complementary oligonucleotide on the flow-cell surface (resulting in an ∩-shaped bridge-like structure); then, a DNA polymer‐ ase enzyme synthesizes a complementary DNA strand by incorporation of unlabeled nucleotides. The resulting double-stranded DNA template is denatured, which releases the two single-stranded DNA fragments both from one another as well as from the flow-cell surface at one end: if the initial ssDNA fragment had its 5′ end free, then it is restored and the new copy remains bound at its 3′ end (which is complementary to the free 5′ end). Thus, the templates generated by bridge amplification form clusters of single-stranded fragments bound at one end and the number of DNA templates grows. (Then, the reverse templates are removed and only the forward strands remain – clones identical to the original tem‐

**•** Sequencing – The samples are ready for the sequencing step when the clusters are complete. A proprietary reversible terminator-based method is used: a sequencing primer is annealed (it is complementary to the part of the adapter that is adjacent to the DNA fragment of interest), followed by cycles of incorporation of terminator-bound dNTPs. In each cycle, there is a mixture of the four different dNTPs and they naturally compete for incorporation based on complementarity. As a result, only one complementary dNTP per cycle is incor‐ porated in the growing strand. In each cycle, a labeled terminator-bound dNTP is incorpo‐ rated in each chain in each cluster and is detected based on emission wavelength and intensity. The signal emitted by each nucleotide incorporated in each strand at the end of a cycle is recorded in real time in each cluster. The cycle is repeated *n* times to obtain an *n*base long sequence. When the new strand is synthesized and its sequence is recorded, it is removed. This approach of base-by-base sequencing achieves very high precision, as errors

**•** Data processing and analysis – The fluorescence emissions from different clusters are digitally processed in parallel and are visualized as nucleotide sequences for each individual cluster. Following sequence alignment, the data are compared against referent sequences to perform phylogenetic analysis, single-nucleotide polymorphism analysis, distance deter‐

This technique allows multiplex analysis to be carried out by creating distinct libraries based on the so-called index sequences (short nucleotide sequences specific for each library that can be used like a barcode), which are attached during library preparation step. Different libraries are first individually prepared and are then combined together and loaded in one and the same flow cell lane. The labeled libraries are sequenced simultaneously, in a single run of the equipment, and at the end of the process, the sequences are exported in a single file. Next, a demultiplexing algorithm is used to separate the sequences in different files based on their

The platform is compatible with different library preparation methods depending on the purpose of the sequencing analysis (whole genome sequencing, target sequencing, mRNA sequencing, 16s RNA gene sequencing, etc.). This is possible because the sequencing steps that come after the library preparation step are fundamental and do not depend on the library preparation method. There are ready-to-use, standardized library preparation kits designed

are eliminated even in repetitive sequence regions and homopolymers.

barcode. This is followed by alignment with referent sequences of interest.

mination, and a range of other analyses.

plates)

20 Nucleic Acids - From Basic Aspects to Laboratory Tools

Nanopore technology is an innovative NGS platform that also includes sequence library preparation. The library preparation step requires maximal purity of the target nucleic acid (DNA, PCR products, cDNA). This is achieved by magnetic particle purification (It is proba‐ bly possible to use other methods as well, e.g., column silica-based ones, if the manufacturers are reliable and guarantee high purification ofthe nucleic acid samples). The target nucleic acid samples are ligated between the 5′ end of one of the strands and the 3′ end of the other strand of the double-stranded DNA (in the shape of a hairpin). Bayley sequencing technology is used. Inthismethod,theDNAmoleculeisbasicallypassedthroughasystemconsistingofaprocessive exonuclease enzyme bound to a protein nanopore (Figure 6A). The enzyme unzips the DNA doublehelixandpushes/cleavesoneofthe strands throughthe apertureoftheproteinnanopore in a base-by-base manner (The aperture is only a few nanometers in diameter.). This contin‐ ues until the hairpin loop at the end of the first strand is reached and then the enzyme simply proceeds on to push the ligated reverse strand through the nanopore as well. The system is designed so that a constant ion current flows through the aperture of the free protein nano‐ pore. This current is specifically disrupted when each base enters the aperture. These specific disruptions inthe ioncurrent are recordedbyanelectronicdevice andare interpretedtoidentify each base. The disruption that each of the four bases causes in the ion current is different due todifferences intheir chemical structureandchemical characteristics.Recordings frommultiple channels in parallel allows high-throughput sequencing of DNA.

Protein nanopores are inserted into an electrically resistant polymer membrane so that a membrane potential can be created driving the ion current through the pore aperture. This ion current is disrupted in a specific way when a nucleotide passes through the aperture of the protein nanopore, which causes a change in the membrane potential.

An array of microscaffolds holds the membrane in which the nanopores are embedded, giving stability to the structure during operation. Each microscaffold on the sensor array chip contains anindividualelectrode,allowingformultiplenanoporeexperimentstobeperformedinparallel. Each nanopore channel is controlled and measured by an individual channel on a correspond‐ ing, bespoke application specific integrated circuit (ASIC) (Figure 6B) [59].

Another NGS platform is the Ion Torrent sequencing technology. In this method, following library preparation, the sequencing step is actually performed in wells that contain the DNA template, an underlying sensor, and electronics. It works as follows: when a new nucleotide is incorporated in the growing DNA strand, a proton (H+ ) is released, which causes a change in the pH in the well. This leads to changes in the surface potential of a metal-oxide sensing layer and to changes in the potential of the source terminal of the underlying field-effect transistor. This is the signal that a complementary nucleotide has been incorporated at the end of the growing DNA strand. To determine the DNA sequence, the equipment needs to be able to differentiate between the four bases. This is done by flushing each well with one type of nucleotide at a time. If the base is complementary, the nucleotide will be incorporated and a

corresponding, bespoke application specific integrated circuit (ASIC) (Figure 6B) [59].

data). 1 – DNA library is prepared; 2 –the dsDNA strands are separated and one of the strands passes through 3 – a processive exonuclease enzyme, and 4 – a protein nanopore, in a base-bybase manner, causing acharacteristic disruption in the electrical current flowing through the nanopore, which is in fact the signal that is detected in real time. When the end of the first strand is reached, the 3- to 5-end hairpin ligation of the two DNA strands allows the processive enzyme to simply continue to push the second strand through the nanopore; 5 – Representative image of DNA sequencing data. (B) 1 – A nanopore protein; 2 – Array of Microscaffold; 3 – Array Chip; 4 – Application Specific Integrated Circuit. **Figure 6.** (А) Underlying principle used in nanopore technology sequencing (illustrative data). 1 – DNA library is pre‐ pared; 2 – the dsDNA strands are separated and one of the strands passes through 3 – a processive exonuclease en‐ zyme, and 4 – a protein nanopore, in a base-by-base manner, causing a characteristic disruption in the electrical current flowing through the nanopore, which is in fact the signal that is detected in real time. When the end of the first strand is reached, the 3′- to 5′-end hairpin ligation of the two DNA strands allows the processive enzyme to simply continue to push the second strand through the nanopore; 5 – Representative image of DNA sequencing data. (B) 1 – A nano‐ pore protein; 2 – Array of Microscaffold; 3 – Array Chip; 4 – Application Specific Integrated Circuit.

**Figure 6**. (А) Underlying principle used in nanopore technology sequencing (illustrative

change in the pH will be recorded; and if the base is not complementary, there will be no pH change [60]. Another NGS platform is theIon Torrent sequencing technology. In this method, following library preparation, the sequencing step is actually performed in wells that containthe DNA template, anunderlying sensor, and electronics. It works as follows: when a new nucleotide

NGS technologies are a new trend and yet more approaches and applications in nucleic acid analysis are being developed. is incorporated in the growing DNA strand, a proton (H<sup>+</sup> ) is released, which causes a change in the pH in the well. This leads to changes in the surface potential of a metal-oxide sensing layerand to changes in the potential of the source terminal of the underlying field-effect transistor.This is the signal that a complementary nucleotide has been incorporated at the end of

the growing DNA strand. To determine the DNA sequence, the equipment needs to be able to differentiate between the four bases. This is done by flushing each well with one type of nucleotide at a time. If the base is complementary, the nucleotide will be incorporated and a

#### **Author details** change in the pH will be recorded; and if the base is not complementary, there will be no pH change [60].

Ivo Nikolaev Sirakov\*

Address all correspondence to: insirakov@gmail.com

Medical University – Sofia, Department of Microbiology, Sofia, Bulgaria

### **References**

[1] Fabre AL, Colotte M, Luis A, Tuffet S, Bonnet J. An efficient method for long-term room temperature storage of RNA. Euro J Human Genetics 2014;22:379–85; doi: 10.1038/ejhg.2013.145


change in the pH will be recorded; and if the base is not complementary, there will be no pH

Another NGS platform is theIon Torrent sequencing technology. In this method, following library preparation, the sequencing step is actually performed in wells that containthe DNA template, anunderlying sensor, and electronics. It works as follows: when a new nucleotide

**Figure 6**. (А) Underlying principle used in nanopore technology sequencing (illustrative data). 1 – DNA library is prepared; 2 –the dsDNA strands are separated and one of the strands passes through 3 – a processive exonuclease enzyme, and 4 – a protein nanopore, in a base-bybase manner, causing acharacteristic disruption in the electrical current flowing through the nanopore, which is in fact the signal that is detected in real time. When the end of the first strand is reached, the 3- to 5-end hairpin ligation of the two DNA strands allows the processive enzyme to simply continue to push the second strand through the nanopore; 5 – Representative image of DNA sequencing data. (B) 1 – A nanopore protein; 2 – Array of Microscaffold; 3 –

**Figure 6.** (А) Underlying principle used in nanopore technology sequencing (illustrative data). 1 – DNA library is pre‐ pared; 2 – the dsDNA strands are separated and one of the strands passes through 3 – a processive exonuclease en‐ zyme, and 4 – a protein nanopore, in a base-by-base manner, causing a characteristic disruption in the electrical current flowing through the nanopore, which is in fact the signal that is detected in real time. When the end of the first strand is reached, the 3′- to 5′-end hairpin ligation of the two DNA strands allows the processive enzyme to simply continue to push the second strand through the nanopore; 5 – Representative image of DNA sequencing data. (B) 1 – A nano‐

parallel. Each nanopore channel is controlled and measured by an individual channel on a corresponding, bespoke application specific integrated circuit (ASIC) (Figure 6B) [59].

NGS technologies are a new trend and yet more approaches and applications in nucleic acid

the pH in the well. This leads to changes in the surface potential of a metal-oxide sensing layerand to changes in the potential of the source terminal of the underlying field-effect transistor.This is the signal that a complementary nucleotide has been incorporated at the end of the growing DNA strand. To determine the DNA sequence, the equipment needs to be able to differentiate between the four bases. This is done by flushing each well with one type of nucleotide at a time. If the base is complementary, the nucleotide will be incorporated and a change in the pH will be recorded; and if the base is not complementary, there will be no pH

pore protein; 2 – Array of Microscaffold; 3 – Array Chip; 4 – Application Specific Integrated Circuit.

) is released, which causes a change in

[1] Fabre AL, Colotte M, Luis A, Tuffet S, Bonnet J. An efficient method for long-term room temperature storage of RNA. Euro J Human Genetics 2014;22:379–85; doi:

change [60].

**Author details**

**References**

10.1038/ejhg.2013.145

Ivo Nikolaev Sirakov\*

change [60].

analysis are being developed.

22 Nucleic Acids - From Basic Aspects to Laboratory Tools

Address all correspondence to: insirakov@gmail.com

Array Chip; 4 – Application Specific Integrated Circuit.

is incorporated in the growing DNA strand, a proton (H<sup>+</sup>

Medical University – Sofia, Department of Microbiology, Sofia, Bulgaria


[16] Brawerman G, Mendecki J, Lee SY. A procedure for the isolation of mammalian mes‐

[17] Puissant C, Houdebine LM. An improvement of the single-step method of RNA iso‐ lation by acid guanidinium thiocyanate – phenol – chloroform extraction. BioTechni‐

[18] Birnboim HC1. Extraction of high molecular weight RNA and DNA from cultured

[19] Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc 2010 Jun;2010(6):pdb.prot5439. doi: 10.1101/

[20] Wyatt GR. The purine and pyrimidine composition of deoxypentose nucleic acids.

[21] Clark LB, Tinoco I Jr. Correlations in the ultraviolet spectra of the purine and pyrimi‐

[22] Barbara R, Karin F, Claus V, Martin W, Peter R. Impact of long-term storage on sta‐ bility of standard DNA for nucleic acid-based methods. J Clin Microbiol Nov

[23] Bonner G, Klibanov AM. Structural stability of DNA in nonaqueous solvents. Bio‐

[24] Haralambiev H. Animal Viruses. Diagnostic of virus infection. Publisher – Pandora,

[25] Watson JD, Crick FHC. A structure for deoxyribose nucleic acid. Nature

[26] Langer PR, Waldrop AA, Ward DC. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239:487. doi: 10.1126/

[27] Saiki RK,Gelfand DH,Stoffel S, Scharf SJ, Higuchi R,Horn, GT,Mullis KB, Erlich H. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymer‐

[28] Linz U, Delling U, Rubsamem W. Systematic studies on parameters influencing the performance of the polymerase chain reaction. J Clin Chem Clin Biochem 1990;28:5–

[29] Sarkar G, Kapelner S, Sommer SS. Formamide can dramatically improve the specific‐

[30] He Q, Marjamäki M, Soini H, Mertsola J, Viljanen MK. Primers are decisive for sensi‐

dine bases. J Am Chem Soc 1965;87(1):11–5.doi: 10.1021/ja01079a003

senger ribonucleic acid. Biochemistry 1972;11(4):1972.

mammalian cells. Methods Enzymol 1992;216:154–60.

ques 1990;8:148–9.

24 Nucleic Acids - From Basic Aspects to Laboratory Tools

pdb.prot5439

Biochem J 1951;48(5):584–90.

2010;4260–2. doi:10.1128/JCM.01230-10

technol Bioeng 2000;68:339–44.

ase. Science 1988;239(4839):487–91.

ity of PCR. Nucleic Acids Res 1990;18:7465–8.

tivity of PCR. Biotechniques 1994;17(1):82, 84, 86–7.

Haskovo, 2002, pp. 58–59.

1953;171:737–8.

science.2448875

13.

