RU2016107196A - Библиотеки для секвенирования нового поколения - Google Patents
Библиотеки для секвенирования нового поколения Download PDFInfo
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Claims (33)
1. Способ определения нуклеотидной последовательности-мишени, включающий:
a) создание библиотеки для секвенирования нового поколения, что включает
1) амплификацию нуклеотидной последовательности-мишени с использованием праймера, содержащего специфичную в отношении мишени последовательность и универсальную последовательность А, с получением ампликона, где указанный ампликон может быть одноцепочечным или двуцепочечным;
2) лигирование первого адаптерного олигонуклеотида, содержащего универсальную последовательность В, с указанным ампликоном для образования адаптера-ампликона; и
3) создание библиотеки, представляющей собой «лестницу» фрагментов, содержащей множество фрагментов для применения в качестве библиотеки для секвенирования нового поколения; и
b) определение нуклеотидной последовательности фрагмента указанной библиотеки, представляющей собой «лестницу» фрагментов, где указанная нуклеотидная последовательность содержит нуклеотидную субпоследовательность нуклеотидной последовательности-мишени.
2. Способ по п. 1, отличающийся тем, что указанный праймер дополнительно содержит нуклеотидную последовательность-штрихкод, связанную с указанной нуклеиновой кислотой-мишенью, и указанный способ дополнительно включает определение нуклеотидной последовательности-штрихкода фрагмента указанной библиотеки, представляющей собой «лестницу» фрагментов.
3. Способ по п. 1, дополнительно включающий лигирование второго адаптерного олигонуклеотида, содержащего универсальную последовательность С, с 3' концами указанных фрагментов библиотеки, представляющей собой «лестницу» фрагментов, для создания библиотеки для секвенирования нового поколения, при этом определение нуклеотидной последовательности фрагмента указанной библиотеки, представляющей собой «лестницу» фрагментов, включает использование
олигонуклеотидного праймера, комплементарного универсальной последовательности С.
4. Способ по п. 2, отличающийся тем, что определение указанной нуклеотидной последовательности-штрихкода фрагмента указанной библиотеки, представляющей собой «лестницу» фрагментов, включает использование олигонуклеотидного праймера, комплементарного универсальной последовательности В.
5. Способ по п. 1, отличающийся тем, что указанная нуклеотидная последовательность фрагмента библиотеки, представляющей собой «лестницу» фрагментов, содержит от 15 до 1000 нуклеотидов.
6. Способ по п. 2, включающий объединение указанной нуклеотидной последовательности-штрихкода с источником указанной нуклеотидной последовательности-мишени.
7. Способ по п. 2, дополнительно включающий группировку нуклеотидных последовательностей фрагментов указанной библиотеки, представляющей собой «лестницу» фрагментов, имеющих одинаковую нуклеотидную последовательность-штрихкод.
8. Способ по п. 1, дополнительно включающий сборку множества нуклеотидных последовательностей фрагментов указанной библиотеки, представляющей собой «лестницу» фрагментов, с обеспечением консенсусной последовательности.
9. Способ по п. 8, дополнительно включающий картирование указанной консенсусной последовательности на референсной последовательности.
10. Способ по п. 8, отличающийся тем, что указанная консенсусная последовательность сохраняет информацию о фазировании и/или сцеплении указанной нуклеиновой кислоты-мишени.
11. Способ по п. 1, отличающийся тем, что указанная библиотека, представляющая собой «лестницу» фрагментов, содержит нуклеотидную последовательность указанной нуклеиновой кислоты-мишени и:
1) 5' конец нуклеотидной субпоследовательности m находится на нуклеотиде xm указанной нуклеотидной последовательности-мишени, и 3' конец указанной нуклеотидной субпоследовательности находится на нуклеотиде ym указанной нуклеотидной последовательности-мишени;
2) 5' конец нуклеотидной субпоследовательности (m+1) находится на нуклеотиде xm+1 указанной нуклеотидной последовательности-мишени, и 3' конец указанной нуклеотидной субпоследовательности находится на нуклеотиде ym+1 указанной нуклеотидной последовательности-мишени;
3) m варьирует от 1 до n;
4) xm=xm+1; и
5) (ym+1-ym)<20.
12. Композиция для применения в качестве библиотеки для секвенирования нового поколения для получения последовательности нуклеиновой кислоты-мишени, при этом указанная композиция содержит n нуклеиновых кислот, при этом каждая нуклеиновая кислота содержит нуклеотидную субпоследовательность указанной нуклеиновой кислоты-мишени, и:
1) 5' конец нуклеотидной субпоследовательности m находится на нуклеотиде xm указанной нуклеотидной последовательности-мишени, и 3' конец указанной нуклеотидной субпоследовательности находится на нуклеотиде ym указанной нуклеотидной последовательности-мишени; и
2) 5' конец нуклеотидной субпоследовательности (m+1) находится на нуклеотиде xm+1 указанной нуклеотидной последовательности-мишени, и 3' конец указанной субпоследовательности находится на нуклеотиде ym+1 указанной нуклеотидной последовательности-мишени;
3) m варьирует от 1 до n;
4) xm=xm+1; и
5) (ym+1-ym)<20.
13. Композиция по п. 12, отличающаяся тем, что нуклеиновая кислота содержит: 3'-О-модифицированный аналог нуклеотида, 3'-О-алкинил аналог нуклеотида, 3'-О-пропаргил аналог нуклеотида или аналог нуклеотида, содержащий обратимый терминатор.
14. Композиция по п. 12, отличающаяся тем, что нуклеиновая кислота содержит адаптер, присоединенный к аналогу нуклеотида с помощью связи, образованной с помощью «клик-химии».
15. Набор для создания библиотеки секвенирования, содержащий:
a) 3'-О-модифицированный аналог нуклеотида; и
b) адаптерный олигонуклеотид.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201361867224P | 2013-08-19 | 2013-08-19 | |
| US61/867,224 | 2013-08-19 | ||
| PCT/US2014/051739 WO2015026853A2 (en) | 2013-08-19 | 2014-08-19 | Next-generation sequencing libraries |
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| Publication Number | Publication Date |
|---|---|
| RU2016107196A true RU2016107196A (ru) | 2017-09-26 |
| RU2016107196A3 RU2016107196A3 (ru) | 2018-07-27 |
| RU2698125C2 RU2698125C2 (ru) | 2019-08-22 |
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| RU2016107196A RU2698125C2 (ru) | 2013-08-19 | 2014-08-19 | Библиотеки для секвенирования нового поколения |
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|---|---|
| US (4) | US10036013B2 (ru) |
| EP (3) | EP3626866B1 (ru) |
| CN (1) | CN105917036B (ru) |
| CA (1) | CA2921620C (ru) |
| ES (2) | ES2873850T3 (ru) |
| RU (1) | RU2698125C2 (ru) |
| WO (1) | WO2015026853A2 (ru) |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2790295C2 (ru) * | 2019-01-11 | 2023-02-16 | Иллумина Кембридж Лимитед | Сложные комплексы связанной на поверхности транспосомы |
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