vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJGB-23-11vavilov-3635Research ArticleАКТУАЛЬНЫЕ ТЕХНОЛОГИИMAINSTREAM TECHNOLOGIESВлияние предварительной обработки образцов периферической крови человека на качество Hi-C библиотекInfluence of human peripheral blood samples preprocessing on the quality of Hi-C librarieshttps://orcid.org/0000-0002-7972-5949ГридинаМ. М.GridinaM. M.

Новосибирск

Novosibirsk

gridinam@gmail.comhttps://orcid.org/0000-0003-3480-3963ВеснаЭ.VesnaE.

Новосибирск

Novosibirsk

https://orcid.org/0000-0001-5458-0408МиньженковаМ. Е.MinzhenkovaM. E.

Москва

Moscow

https://orcid.org/0000-0002-0641-1084ШиловаН. В.ShilovaN. V.

Москва

Moscow

РыжковаО. П.RyzhkovaO. P.

Москва

Moscow

НазаренкоЛ. П.NazarenkoL. P.

Томск

Tomsk

БеляеваЕ. О.BelyaevaE. O.

Томск

Tomsk

ЛебедевИ. Н.LebedevI. N.

Томск

Tomsk

https://orcid.org/0000-0002-5573-3100ФишманВ. С.FishmanV. S.

Новосибирск

Novosibirsk

Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesRussian FederationМедико-генетический научный центр им. академика Н.П. БочковаРоссияResearch Centre for Medical GeneticsRussian FederationТомский национальный исследовательский медицинский центр Российской академии наукРоссияTomsk National Research Medical Center of the Russian Academy of SciencesRussian Federation2023070320232718387Copyright © Гридина М.М., Весна Э., Миньженкова М.Е., Шилова Н.В., Рыжкова О.П., Назаренко Л.П., Беляева Е.О., Лебедев И.Н., Фишман В.С., 20232023Гридина М.М., Весна Э., Миньженкова М.Е., Шилова Н.В., Рыжкова О.П., Назаренко Л.П., Беляева Е.О., Лебедев И.Н., Фишман В.С.Gridina M.M., Vesna E., Minzhenkova M.E., Shilova N.V., Ryzhkova O.P., Nazarenko L.P., Belyaeva E.O., Lebedev I.N., Fishman V.S.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/3635

Метод захвата конформации хроматина в его полногеномном варианте (Hi-C) – мощный инструмент не только для выявления закономерностей пространственной организации генома, но и для понимания влияния их нарушения на развитие заболеваний. Кроме того, метод может быть использован для детекции хромосомных перестроек, в том числе сбалансированных транслокаций и инверсий. Применение метода Hi-C для поиска хромосомных перестроек получает все более широкое распространение. Это связано с тем, что современные высокопроизводительные методы анализа генома позволяют эффективно детектировать точечные мутации и несбалансированные хромосомные перестройки. Однако чувствительность этих методов для определения сбалансированных транслокаций и инверсий остается достаточно низкой. Хранение образцов цельной крови может влиять на количество и целостность выделяемой из них геномной ДНК, а кроме того, приводить к искажению результатов последующих анализов в том случае, если хранение осуществлялось в ненадлежащих условиях. Метод Hi-C крайне требователен к исходному материалу, так как необходимым условием для его успешного применения и получения качественных данных является сохранение пространственной укладки хроматина внутри ядра. Цель нашего исследования состояла в том, чтобы определить оптимальные условия хранения крови для проведения последующего анализа Hi-C. Были выбраны 10 различных условий хранения образцов крови и пробоподготовки. Для каждого условия приготовлены Hi-C библиотеки и отсеквенированы, после чего оценивалось качество полученных библиотек. В результате сформулированы требования к хранению и подготовке образцов, необходимые для получения качественных Hi-C данных. Нами установлен минимальный объем образца крови, достаточный для проведения Hi-C анализа. Помимо этого, мы определили способы выделения ядерных элементов крови и их долгосрочного хранения, наиболее подходящие для последующего проведения Hi-C анализа. Основное требование, сформулированное нами, – не замораживать цельную кровь.

The genome-wide variant of the chromatin conformation capture technique (Hi-C) is a powerful tool for revealing patterns of genome spatial organization, as well as for understanding the effects of their disturbance on disease development. In addition, Hi-C can be used to detect chromosomal rearrangements, including balanced translocations and inversions. The use of the Hi-C method for the detection of chromosomal rearrangements is becoming more widespread. Modern high-throughput methods of genome analysis can effectively reveal point mutations and unbalanced chromosomal rearrangements. However, their sensitivity for determining translocations and inversions remains rather low. The storage of whole blood samples can affect the amount and integrity of genomic DNA, and it can distort the results of subsequent analyses if the storage was not under proper conditions. The Hi-C method is extremely demanding on the input material. The necessary condition for successfully applying Hi-C and obtaining high-quality data is the preservation of the spatial chromatin organization within the nucleus. The purpose of this study was to determine the optimal storage conditions of blood samples for subsequent Hi-C analysis. We selected 10 different conditions for blood storage and sample processing. For each condition, we prepared and sequenced Hi-C libraries. The quality of the obtained data was compared. As a result of the work, we formulated the requirements for the storage and processing of samples to obtain high-quality Hi-C data. We have established the minimum volume of blood sufficient for conducting Hi-C analysis. In addition, we have identified the most suitable methods for isolation of peripheral blood mononuclear cells and their long-term storage. The main requirement we have formulated is not to freeze whole blood.

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