Biomedical Chemistry: Research and Methods, 2018, 1(4), e00086

Prospects for the Use of Third Generation Sequencers for Quantitative Profiling of Transcriptome

S.P. Radko1*, L.K. Kurbatov1, K.G. Ptitsyn2, Y.Y. Kiseleva3, E.A. Ponomarenko1, A.V. Lisitsa1, A.I. Archakov1

1Institute of Biomedical Chemistry 10 Pogodinskaya str., Moscow, 119121 Russia; *e-mail:
2Adzhinomoto-Genetika 1 Dorozhny 1-st Road, Moscow, 117545 Russia
2Russian Scientific Center of Roentgenoradiology, 86 Profsoyuznaya str., Moscow, 117997 Russia

Keywords:third generation sequencing; transcriptome; quantitative profiling


The whole version of this paper is available in Russian.

Transcriptome profiling is widely employed to analyze transcriptome dynamics when studying various biological processes at the cell and tissue levels. Unlike the second generation sequencers, which sequence relatively short fragments of nucleic acids, the third generation DNA/RNA sequencers developed by biotechnology companies “PacBio” and “Oxford Nanopore Technologies” allow one to sequence transcripts as single molecules and may be considered as potential molecular counters capable to measure the number of copies of each transcript with high throughput, sensitivity, and specificity. In the present review, the features of single molecule sequencing technologies offered by “PacBio” and “Oxford Nanopore Technologies” are considered alongside with their utility for transcriptome analysis, including the analysis of transcript isoforms. The prospects and limitations of the single molecule sequencing technology in application to quantitative transcriptome profiling are also discussed.

Figure 1. Schematic illustration of single-molecule real-time (SMRT) DNA sequencing. DNA polymerase with bound DNA template are immobilized to the bottom of zero-mode waveguide. The sequencing of the template is observed in real time by detecting enzymatic processing of fluorescent phospholinked nucleotides during the synthesis of the complementary DNA.
Figure 2. Schematic representation of SMRTbell template and the stepwise treatments of raw reads during SMRT-sequencing.
Figure 3. Illustration of different activities of φ29 DNA polymerase as a motor protein during the nanopore sequencing of DNA.
I. Helicase activity.
II. 3'-5'-exonuclease activity.
III. Polymerase activity.
Figure 4. Scheme of the design of double-stranded DNA fragment comprised of alternating «template» and «complement» sequences.y.


This work has been performed within the framework of the Fundamental Scientific Research Program of the State Academies of Sciences for 2013-2020.


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