References

vavilov

Вавиловский журнал генетики и селекции

Vavilov Journal of Genetics and Breeding

2500-3259

Institute of Cytology and Genetics of Siberian Branch of the RAS

10.18699/VJ19.520

vavilov-2200

Research Article

АКТУАЛЬНЫЕ ТЕХНОЛОГИИ

MAINSTREAM TECHNOLOGIES

Новейшие технологии высокопроизводительного секвенирования транскриптома отдельных клеток

The new technologies of high-throughput single-cell RNA sequencing

https://orcid.org/0000-0003-3886-2880

Водясова

Е. А.

Vodiasova

E. A.

eavodiasova@gmail.com

https://orcid.org/0000-0002-7662-2573

Челебиева

Э. С.

Chelebieva

E. S.

https://orcid.org/0000-0003-3745-7066

Кулешова

О. Н.

Kuleshova

O. N.

Институт биологии южных морей имени А.О. Ковалевского Российской академии наукРоссияA.O. Kovalevsky Institute of Biology of the Southern Seas, RASRussian Federation

2019

22082019

235508518

2019

Водясова Е.А., Челебиева Э.С., Кулешова О.Н.

Vodiasova E.A., Chelebieva E.S., Kuleshova O.N.

This work is licensed under a Creative Commons Attribution 4.0 License.

https://vavilov.elpub.ru/jour/article/view/2200

Огромное количество полногеномных и транскриптомных данных, полученных с помощью современных технологий секвенирования нового поколения для целых организмов, не смогло дать ответы на многие вопросы в онкологии, иммунологии, физиологии, нейробиологии, зоологии и других областях науки и медицины. Так как основой всех одноклеточных и многоклеточных организмов является клетка, то необходимо изучение биологических процессов на ее уровне. Это понимание дало толчок развитию нового направления и появлению технологий, позволяющих работать с единичными клетками (технологии single-cell). Быстрое развитие не только приборной базы, но и различных усовершенствованных протоколов для работы с единичными клетками обусловлено актуальностью этих исследований во многих областях науки и медицины. Изучение особенностей различных этапов онтогенеза, определение закономерностей дифференциации клеток и последующего развития тканей, проведение геномного и транскриптомного анализов в различных областях медицины (особенно востребовано в иммунологии, онкологии), классификация типов и состояний клеток, закономерностей биохимических и физиологических процессов с применением технологий single-cell позволяют проводить комплексные исследования на новом уровне. Разработанные первые платформы для осуществления секвенирования транскриптомов отдельных клеток (scRNA-seq) проводили изоляцию не более ста клеток единовременно, что оказалось недостаточным в связи с выявленной высокой гетерогенностью клеток, обнаруженными минорными типами клеток, которые не детектировались по морфологическим признакам, и сложными регуляторными путями в организме. В настоящее время появились методики изоляции, захвата и секвенирования транскриптомов (scRNA-seq) десятков тысяч клеток единовременно. Однако новые технологии имеют определенные отличия как на этапе пробоподготовки, так и во время проведения биоинформатического анализа. В работе рассмотрены наиболее эффективные методы множественного параллельного scRNA-seq на примере современной платформы для изоляции и баркодирования клеток 10ХGenomics, а также особенности проведения такого эксперимента, дальнейший биоинформатический анализ полученных данных, перспективы использования и области применения новых высокопроизводительных технологий.

A wealth of genome and transcriptome data obtained using new generation sequencing (NGS) technologies for whole organisms could not answer many questions in oncology, immunology, physiology, neurobiology, zoology and other fields of science and medicine. Since the cell is the basis for the living of all unicellular and multicellular organisms, it is necessary to study the biological processes at its level. This understanding gave impetus to the development of a new direction – the creation of technologies that allow working with individual cells (single-cell technology). The rapid development of not only instruments, but also various advanced protocols for working with single cells is due to the relevance of these studies in many fields of science and medicine. Studying the features of various stages of ontogenesis, identifying patterns of cell differentiation and subsequent tissue development, conducting genomic and transcriptome analyses in various areas of medicine (especially in demand in immunology and oncology), identifying cell types and states, patterns of biochemical and physiological processes using single cell technologies, allows the comprehensive research to be conducted at a new level. The first RNA-sequencing technologies of individual cell transcriptomes (scRNA-seq) captured no more than one hundred cells at a time, which was insufficient due to the detection of high cell heterogeneity, existence of the minor cell types (which were not detected by morphology) and complex regulatory pathways. The unique techniques for isolating, capturing and sequencing transcripts of tens of thousands of cells at a time are evolving now. However, new technologies have certain differences both at the sample preparation stage and during the bioinformatics analysis. In the paper we consider the most effective methods of multiple parallel scRNA-seq using the example of 10XGenomics, as well as the specifics of such an experiment, further bioinformatics analysis of the data, future outlook and applications of new high-performance technologies.

scRNA-seqтранскриптомикаChromium 10XGenomicsсеквенированиеединичные клетки

scRNA-seqtranscriptomicsChromium 10XGenomicssequencingsingle cell

The research were supported by the Government of the Russian Federation, grant No. 14.W03.31.0015.

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The authors declare that there are no conflicts of interest present.