microbeПроблемы особо опасных инфекцийProblems of Particularly Dangerous Infections0370-10692658-719XRussian Research Anti-Plague Institute “Microbe”10.21055/0370-1069-2024-1-176-181microbe-1959Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESГибридная сборка полных геномов штаммов Yersinia pestisHybrid Assembly of Complete Genomes of Yersinia pestis Strainshttps://orcid.org/0000-0001-7190-4427ФедоровА. В.FedorovA. V.

46, Universitetskaya St., Saratov, 410005

https://orcid.org/0000-0002-4909-2394КрасновЯ. М.KrasnovYa. M.

Краснов Ярослав Михайлович, 

410005, Саратов, ул. Университетская, 46

Yaroslav M. Krasnov, 

46, Universitetskaya St., Saratov, 410005

rusrapi@microbe.ruhttps://orcid.org/0000-0002-9190-099XНарышкинаЕ. А.NaryshkinaE. A.

410005, Саратов, ул. Университетская, 46

46, Universitetskaya St., Saratov, 410005

rusrapi@microbe.ruСоседоваЕ. А.SosedovaE. A.

410005, Саратов, ул. Университетская, 46

46, Universitetskaya St., Saratov, 410005

rusrapi@microbe.ruhttps://orcid.org/0000-0002-8260-4670КатышевА. Д.KatyshevA. D.

410005, Саратов, ул. Университетская, 46

46, Universitetskaya St., Saratov, 410005

rusrapi@microbe.ruhttps://orcid.org/0000-0001-5403-989XЕрошенкоГ. А.EroshenkoG. A.

410005, Саратов, ул. Университетская, 46

46, Universitetskaya St., Saratov, 410005

rusrapi@microbe.ruhttps://orcid.org/0000-0003-2438-8364КуклеваЛ. М.KuklevaL. M.

410005, Саратов, ул. Университетская, 46

46, Universitetskaya St., Saratov, 410005

rusrapi@microbe.ruhttps://orcid.org/0000-0003-0954-5683ОсинаН. А.OsinaN. A.

410005, Саратов, ул. Университетская, 46

46, Universitetskaya St., Saratov, 410005

rusrapi@microbe.ruhttps://orcid.org/0000-0003-3788-3452КутыревВ. В.KutyrevV. V.

410005, Саратов, ул. Университетская, 46

46, Universitetskaya St., Saratov, 410005

rusrapi@microbe.ruФКУН «Российский научно-исследовательский противочумный институт «Микроб»РоссияRussian Research Anti-Plague Institute “Microbe”Russian Federation20240404202401176181Copyright © Федоров А.В., Краснов Я.М., Нарышкина Е.А., Соседова Е.А., Катышев А.Д., Ерошенко Г.А., Куклева Л.М., Осина Н.А., Кутырев В.В., 20242024Федоров А.В., Краснов Я.М., Нарышкина Е.А., Соседова Е.А., Катышев А.Д., Ерошенко Г.А., Куклева Л.М., Осина Н.А., Кутырев В.В.Fedorov A.V., Krasnov Y.M., Naryshkina E.A., Sosedova E.A., Katyshev A.D., Eroshenko G.A., Kukleva L.M., Osina N.A., Kutyrev V.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://journal.microbe.ru/jour/article/view/1959

Цель исследования – сборка полноразмерных нуклеотидных последовательностей хромосомы и плазмид для 13 штаммов Yersinia pestis из 11 природных очагов чумы, находящихся на территории Российской Федерации, используя данные двух технологий секвенирования.

Материалы и методы

Материалы и методы. Штаммы Y. pestis выращивали на агаре Хоттингера (рН 7,2) при 37 °С. Выделение ДНК проводили методом фенол-хлороформной экстракции. Для генетического анализатора MinIon (Oxford Nanopore) подготовку ДНК-фрагметов проводили методом лигирования по модифицированному протоколу. Для генетического анализатора Ion S5 (IonTorrent) подготовку образцов проводили по стандартному протоколу получения библиотеки с размером фрагментов ДНК 400 пар нуклеотидов (п.н.). Полученные единичные прочтения отфильтровывались по среднему качеству Q30 для IonTorrent и Q7 для Oxford Nanopore.

Результаты и обсуждение

Результаты и обсуждение. Проведена подготовка фрагментов ДНК, содержащих 50000 и более пар нуклеотидов, для последующего секвенирования с использованием технологии секвенирования через нанопоры (Oxford Nanopore). Использован алгоритм Trycycler для гибридной сборки генома штаммов Y. pestis и коррекции возникающих при этом процессе ошибок, позволяющий собрать полноразмерные нуклеотидные последовательности хромосомы и плазмид для каждого генома штамма. В международную генетическую базу данных NCBI GenBank депонированы нуклеотидные последовательности хромосом геномов 13 штаммов Y. pestis из 11 природных очагов чумы, находящихся на территории Российской Федерации. Установлено, что для сборки полноразмерных геномов штаммов Y. pestis необходимо значительное количество прочтений размером 50000 п.н. и более, а использование алгоритма Trycycler позволяет получить более точную сборку полных геномов бактерий.

The aim of the study was to assemble full-length nucleotide sequences of the chromosome and plasmids for 13 Yersinia pestis strains from 11 natural plague foci located in the Russian Federation, using data from two sequencing technologies.

Materials and methods

Materials and methods. Y. pestis strains were grown on Hottinger’s agar (pH 7.2) at 37 °C. DNA was isolated using phenol-chloroform extraction. For the MinIon genetic analyzer (Oxford Nanopore), DNA fragments were prepared by ligation according to a modified protocol. For the Ion S5 genetic analyzer (IonTorrent), sample preparation was carried out according to the standard protocol for obtaining a library with a DNA fragment size of 400 nucleotide pairs (bp). The resulting single reads were filtered by average quality Q30 for IonTorrent and Q7 for Oxford Nanopore.

Results and discussion

Results and discussion. DNA fragments containing 50 000 or more nucleotide pairs were prepared for subsequent sequencing using nanopore sequencing technology (Oxford Nanopore). The Trycycler algorithm was applied for hybrid assembly of the genome of Y. pestis strains and correction of errors arising during this process, allowing the obtainment of full-length nucleotide sequences of the chromosome and plasmids for each genome of the strain. The nucleotide sequences of the chromosomes of 13 Y. pestis strains from 11 natural foci of plague located in the Russian Federation have been deposited in the international genetic database NCBI GenBank. It has been established that to assemble full-length genomes of Y. pestis strains, a significant number of reads with a size of 50 000 bp or more is required, and the use of the Trycycler algorithm allows one to generate a more accurate assembly of complete bacterial genomes.

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