JP2020513741A - 連結型ライゲーション - Google Patents
連結型ライゲーション Download PDFInfo
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- JP2020513741A JP2020513741A JP2019531129A JP2019531129A JP2020513741A JP 2020513741 A JP2020513741 A JP 2020513741A JP 2019531129 A JP2019531129 A JP 2019531129A JP 2019531129 A JP2019531129 A JP 2019531129A JP 2020513741 A JP2020513741 A JP 2020513741A
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Abstract
Description
本出願は、2016年12月9日に出願された米国仮出願62/432,277号および2017年10月9日に出願された米国仮出願62/569,824号の利益およびこれらの仮出願に対する優先権を主張し、両仮出願は本明細書に参照により援用される。
本発明は、概して、核酸の捕捉、増幅およびシーケンシングに関する。
ハイスループットゲノムシーケンシングプラットフォームは、大量のデータを手頃な価格で生じるが、それらは十分に正確ではない。最良のシーケンシング技法であってもエラー率が約1パーセントである。これは単一のヒトゲノムの配列における何十万ものエラーに変換される。不正確な塩基呼び出し(base calling)により、配列の誤アラインメントおよび突然変異の誤識別が導かれる。塩基呼び出しおよびアラインメントアルゴリズムが利用可能であるが、増幅およびシーケンシングのエラーによって品質が負の影響を受ける。
二重にシードされたクラスターを使用したKRASアンプリコンのシーケンシングのエラーの減少
フローセルクラスターに単一の鋳型分子をシードした。単一の鋳型コピーは、図17に示されている通り、連結鋳型分子を1つのみフローセルに結合させた連結鋳型のライブラリーに由来するものであった。次いで、KRASアンプリコンにアラインメントされた最初の3000の単独でシードされたクラスターを、35よりも大きいという質の閾値を適用することでシーケンシングのエラーについて分析した。図16に示されている通り、単独でシードされたクラスターでは、約3000の平均深度で0.13%の平均エラーが生じた。単独でシードされたフローセルでは連結鋳型ライブラリーを使用したので、結果は、非連結鋳型分子を用いた標準の単一のシーディング方法を使用して生じるものよりも低いエラー率を表す可能性がある。
参照による組み込み
等価物
Claims (25)
- 標的核酸にアダプターを選択的にライゲートする方法であって、
標的核酸の第1の部分に相補的なプローブを含む第1の連結型ライゲーションアダプターを提供するステップであって、前記プローブは、第1のユニバーサルプライミング部位を含む第1のアダプターに連結されている、ステップと、
前記標的核酸を含む試料を、前記第1の連結型ライゲーションアダプターに曝露するステップと、
前記第1の連結型アダプターに前記標的核酸をライゲートするステップと、
前記第1のユニバーサルプライミング部位に相補的な第1のユニバーサルプライマーを使用するPCRによって、ライゲートされた標的核酸を増幅するステップと
を含む、方法。 - 前記標的核酸をシーケンシングすることをさらに含み、前記アダプターが、シーケンシングアダプターをさらに含む、請求項1に記載の方法。
- 前記標的核酸の第2の部分に相補的なプローブを含む第2の連結型ライゲーションアダプターを提供するステップであって、前記プローブは、第2のユニバーサルプライミング部位を含む第2のアダプターに連結されている、ステップと、
前記試料を、前記第2の連結型ライゲーションアダプターに曝露するステップと、
前記第2の連結型アダプターに前記標的核酸をライゲートするステップと
をさらに含み、
ライゲートされた標的核酸は、前記第1のユニバーサルプライマーおよび前記第2のユニバーサルプライミング部位に相補的な第2のユニバーサルプライマーを使用して増幅される、請求項1に記載の方法。 - 前記試料が、前記第1および第2の連結型ライゲーションアダプターに同時に曝露される、請求項3に記載の方法。
- 前記標的核酸の前記第1および前記第2の部分が同一である、請求項3に記載の方法。
- 前記試料が、前記第1の連結型ライゲーションアダプターに曝露された後に、前記第2の連結型ライゲーションアダプターに曝露される、請求項5に記載の方法。
- 前記標的核酸が融合核酸である、請求項1に記載の方法。
- 前記融合核酸の一部のみが公知である、請求項7に記載の方法。
- 前記標的核酸の前記第1の部分に相補的な前記プローブが、前記第1のアダプターに近接した固体支持体に結合されており、前記第1のアダプターも、前記固体支持体に結合されている、請求項1に記載の方法。
- 前記標的核酸の第2の部分に相補的なプローブを含む第2の連結型ライゲーションアダプターを提供するステップであって、前記プローブは、第2のユニバーサルプライミング部位を含む第2のアダプターに連結されている、ステップと、
前記試料を、前記第2の連結型ライゲーションアダプターに曝露するステップと、
前記第2の連結型アダプターに前記標的核酸をライゲートするステップと
をさらに含み、
前記試料は、前記第1のユニバーサルプライマーおよび前記第2のユニバーサルプライミング部位に相補的な第2のユニバーサルプライマーを使用して増幅される、請求項9に記載の方法。 - 増幅の前に、前記固体支持体を洗浄して、前記試料中に存在する未結合核酸を除去するステップをさらに含む、請求項10に記載の方法。
- 前記固体支持体が、フローセルである、請求項9に記載の方法。
- 前記標的核酸の前記第1の部分に相補的な前記プローブが、ポリエチレングリコール誘導体、オリゴ糖、脂質、炭化水素、ポリマー、反転塩基およびタンパク質からなる群から選択されるリンカーによって前記第1のアダプターに連結されている、請求項1に記載の方法。
- 前記リンカーが切断可能である、請求項13に記載の方法。
- 前記標的核酸が、二本鎖DNA(dsDNA)であり、
前記第1の連結型ライゲーションアダプターが、前記プローブと複合体化したリコンビナーゼをさらに含み、前記第1のアダプターが、二本鎖アダプターであり、
前記標的核酸を含む前記試料が、一本鎖結合タンパク質の存在下で前記第1の連結型ライゲーションアダプターに曝露される、請求項1に記載の方法。 - 前記アダプターが、ランダムヌクレオチドの配列を含む、請求項1に記載の方法。
- 前記アダプターが、ユニバーサルプライミング部位を含まない、請求項1に記載の方法。
- 前記標的核酸がDNAまたはRNAである、請求項1に記載の方法。
- 前記曝露、ライゲーションおよび増幅ステップが、ドロップレット中で実施される、請求項1に記載の方法。
- 鋳型DNA分子に二本鎖アダプターをライゲートする方法であって、
リンカーによって第2の二本鎖アダプターに連結された第1の二本鎖アダプターを含む連結型アダプターを提供するステップと、
前記連結型アダプターを、鋳型DNA分子に曝露するステップと、
前記鋳型DNA分子の一方の末端に、前記第1のアダプターをライゲートするステップと、
前記鋳型DNA分子のもう一方の末端に、前記第2のアダプターをライゲートするステップと
を含む、方法。 - 前記リンカーが、ポリエチレングリコール誘導体、オリゴ糖、脂質、炭化水素、ポリマー、反転塩基およびタンパク質からなる群から選択される、請求項20に記載の方法。
- 前記リンカーが切断可能である、請求項20に記載の方法。
- 前記第1のアダプターが、Y−アダプターである、請求項20に記載の方法。
- 前記第1のアダプターが、ヘアピンアダプターである、請求項20に記載の方法。
- 前記鋳型DNA分子および連結型アダプターが、一本鎖DNA(ssDNA)を含む、請求項20に記載の方法。
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US20220186298A1 (en) | 2022-06-16 |
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WO2018104908A2 (en) | 2018-06-14 |
JP7048609B2 (ja) | 2022-04-05 |
EP3551769A4 (en) | 2020-10-28 |
US20190300942A1 (en) | 2019-10-03 |
JP2022081635A (ja) | 2022-05-31 |
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WO2018104908A3 (en) | 2018-08-16 |
US11268137B2 (en) | 2022-03-08 |
US20240052403A1 (en) | 2024-02-15 |
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