JP2018505688A - 標的化核酸配列包括度(coverage)のための方法 - Google Patents
標的化核酸配列包括度(coverage)のための方法 Download PDFInfo
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Abstract
Description
本出願は、2015年4月13日出願の米国特許仮出願第62/146,834号及び2015年2月24日出願の米国特許仮出願第62/119,996号の利益を主張し、これらは、その全体が全ての目的において参照として本明細書に組み込まれる。
本開示は、遺伝子材料の特徴決定に有用な方法、組成物及び系を提供する。特に、本明細書に記載されている方法、組成物及び系は、追加の多重複(redundant)配列情報がゲノムのこれらの選択部分から取得され得るように、ゲノムの選択部分の増加した多重複包括度を提供する。特定の場合において、この追加の配列情報は、ゲノムのこれらの選択部分の新規配列決定を可能にするのに十分な情報を提供する。
本開示に記載されている方法及び系は、分別された核酸が1つ以上の他の核酸から相対的に単離されて更なる処理及び/または反応を受けることができるように、異なる群または異なる領域への核酸の分別を提供する。そのような分別は、特定の例示的な場合において、個別の試料(例えば、核酸)を分離区分に沈着または区分化することを含むことができ、それぞれの区分は、それ自体の内容物を他の区分の内容物から分別した状態に維持している。本明細書において使用されるとき、区分とは、様々な異なる形態、例えば、ウエル、管、マイクロまたはナノウエル、中空などが含まれ得る容器または器を指す。しかし好ましい態様では、区分は流体流の中で流動可能である。これらの器は、例えば、内側流体中心またはコアを囲む外側隔壁を有するマイクロカプセルまたは微小胞から構成されていてもよく、あるいは、マトリックス内に材料を進入させる及び/または保持することができる多孔質マトリックスであってもよい。しかし好ましい態様では、これらの区分は、非水性連続相、例えば油相内の水性流体の液滴を構成し得る。様々な異なる器は、例えば、2013年8月13日出願の米国特許出願第13/966,150号に記載されている。同様に、非水性または油連続相中に安定した液滴を作り出すエマルション系は、例えば米国特許出願公開第2010−0105112号に詳細に記載されている。特定の場合において、マイクロ流体チャンネル網状組織は、本明細書に記載されている区分の生成に特に適している。そのようなマイクロ流体デバイスの例には、2015年4月9日出願の米国特許出願第14/682,952号において詳細に記載されたものが含まれ、この全開示は、全ての目的においてその全体が参照として本明細書に組み込まれる。細胞の水性混合物がその中を通って非水性流体中に抽出される多孔質膜を含む、代替的な機構を個別の細胞の区分化に用いることもできる。そのような系は、例えば、Nanomi,Incから一般に入手可能である。
本明細書に記載されている方法、組成物及び系は、核酸配列決定技術における使用に特に適応可能である。そのような配列決定技術には、短い読み込み及び長い読み込み配列決定技術を含む、当該技術に既知の任意の技術が含まれ得る。特定の態様において、本明細書に記載されている方法、組成物及び系は、短い読み込みで高い精度の配列決定技術に使用される。
理解されるように、本明細書において考察される方法及び系を使用して、任意の種類のゲノム材料から標的化配列情報を得ることができる。そのようなゲノム材料は、患者から採取した試料によって得てもよい。本明細書において考察される方法及び系に使用されるゲノム材料の例示的な試料及び種類には、ポリヌクレオチド、核酸、オリゴヌクレオチド、循環無細胞核酸、循環腫瘍細胞(CTC)、核酸断片、ヌクレオチド、DNA、RNA、ペプチドポリヌクレオチド、相補的DNA(cDNA)、二本鎖DNA(dsDNA)、一本鎖DNA(ssDNA)、プラスミドDNA、コスミドDNA、染色体DNA、ゲノムDNA(gDNA)、ウイルスDNA、細菌DNA、mtDNA(ミトコンドリアDNA)、リボソームRNA、無細胞DNA、無細胞胎児DNA(cffDNA)、mRNA、rRNA、tRNA、nRNA、siRNA、snRNA、snoRNA、scaRNA、マイクロRNA、dsRNA、ウイルスRNAなどが限定されることなく含まれる。まとめると、使用される試料は、特定の処理の必要性に応じて変わり得る。
TP53遺伝子を標的にする増幅反応を実施した。腫瘍タンパク質p53は、p53、細胞腫瘍抗原p53(UniProt名称)、リンタンパク質p53、腫瘍抑制因子p53、抗原NY−CO−13、または形質転換関連タンパク質53(TRP53)としても知られており、ヒトにおいてTP53遺伝子によりコードされるタンパク質である。p53タンパク質は、多細胞生物体において重要であり、細胞周期を調節し、したがって腫瘍抑制因子として機能して、癌を予防する。このように、p53は、ゲノム突然変異を防止することによって安定性を保存する役割のため、「ゲノムの守護者(the guardian of the genome)」と記載されてきた。したがって、TP53は、腫瘍抑制遺伝子と分類される。
Claims (93)
- ゲノムの1つ以上の選択部分を配列決定する方法であって、
(a)出発ゲノム材料を準備することと、
(b)前記出発ゲノム材料の個別の核酸分子を、それぞれの分離区分が個別の核酸分子を含有するように、分離区分の中に分配することと、
(c)前記分離区分中の前記個別の核酸分子の少なくともいくつかの選択部分を増幅して、アンプリコンの個体群を形成することと、
(d)前記アンプリコンの個体群にバーコードを付けて、前記アンプリコンの複数のバーコード付き断片を形成し、所定の分離区分内の断片が、それぞれ共通のバーコードを含み、それによって、それぞれの断片を、それが誘導された前記個別の核酸分子と関連づけることと、
(e)前記複数の断片から配列情報を取得し、それによってゲノムの1つ以上の選択部分を配列決定することと
を含む、前記方法。 - 前記ゲノムの前記1つ以上の選択部分が、前記ゲノムの高度に多形性の領域を含む、請求項1に記載の方法。
- 前記ゲノムの前記1つ以上の選択部分の前記配列決定が、新規の配列決定である、請求項1〜2に記載の方法。
- 前記増幅が、少なくとも3.5メガベース対(Mb)の範囲にわたるPCR増幅を含む、請求項1〜3に記載の方法。
- 前記増幅が、少なくとも3.0Mbの領域にわたってねじれている複数のプライマー対を利用するPCR増幅を含む、請求項1〜4の記載の方法。
- 前記複数のプライマー対が、前記プライマー配列の増幅を防止するためにウラシルを含有する、請求項5に記載の方法。
- 前記取得ステップ(e)が、短い読み込み長さ(read−length)配列決定反応及び長い読み込み長さ配列決定反応からなる群から選択される配列決定反応を含む、請求項1〜6に記載の方法。
- 前記配列決定反応が、短い読み込みで高い精度の配列決定反応である、請求項7に記載の方法。
- 取得ステップ(e)において生成される前記配列情報が、由来する個別の核酸の分子構成を保持する、請求項1〜8に記載の方法。
- 前記取得ステップ(e)の前に、前記複数の断片は、
(i)前記ゲノムの前記1つ以上の選択部分の中の、または近くの領域に相補的なプローブを、前記断片とハイブリッド形成させて、プローブ断片複合体を形成すること、
(ii)プローブ断片複合体を固体支持体の表面に捕捉すること
によって、前記ゲノムの前記1つ以上の選択部分の少なくとも一部を含む断片がさらに豊富化される、請求項1〜9に記載の方法。 - 前記固体支持体がビーズを含む、請求項10に記載の方法。
- 前記方法が、前記複数の断片の重複配列に基づいて、推定コンティグ中において2つ以上の前記個別の核酸分子を連結することをさらに含み、前記推定コンティグが、少なくとも10kbのN50長さを含む、請求項1〜11に記載の方法。
- 前記推定コンティグが、少なくとも20kbのN50長さを含む、請求項12に記載の方法。
- 前記推定コンティグが、少なくとも40kbのN50長さを含む、請求項12に記載の方法。
- 前記推定コンティグが、少なくとも50kbのN50長さを含む、請求項12に記載の方法。
- 前記推定コンティグが、少なくとも100kbのN50長さを含む、請求項12に記載の方法。
- 前記推定コンティグが、少なくとも200kbのN50長さを含む、請求項12に記載の方法。
- 前記バーコード付き断片の前記バーコードが、追加の配列セグメントをさらに含む、請求項1〜17に記載の方法。
- 前記追加の配列セグメントが、プライマー、結合配列、ランダムn−merオリゴヌクレオチド、ウラシル核酸塩基を含むオリゴヌクレオチドからなる群から選択される1つ以上のメンバーを含む、請求項18に記載の方法。
- 前記バーコード付けが、少なくとも700,000個のバーコードのライブラリーから選択されるバーコードを結合することを含む、請求項1〜19に記載の方法。
- 前記分離区分内の前記アンプリコンの前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の約100×〜5000×の包括度を表す、請求項1〜20に記載の方法。
- 前記分離区分内の前記アンプリコンの前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の約200×〜1000×の包括度を表す、請求項1〜20に記載の方法。
- 前記分離区分内の前記アンプリコンの前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の少なくとも1000×の包括度を表す、請求項1〜20に記載の方法。
- 前記分離区分内の前記アンプリコンの前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の少なくとも2000×の包括度を表す、請求項1〜20に記載の方法。
- 前記分離区分内の前記アンプリコンの前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の少なくとも5000×の包括度を表す、請求項1〜20に記載の方法。
- ゲノム試料の1つ以上の不十分に特徴決定された部分から配列情報を取得する方法であって、
(a)前記ゲノム試料の個別の第1の核酸断片分子を分離区分の中に提供することと、
(b)前記分離区分内の前記個別の第1核酸断片分子を断片化して、前記個別の第1の核酸断片分子のそれぞれから複数の第2の断片を作り出すことと、
(c)不十分に特徴決定された前記複数の第2の断片の選択領域を増幅して、アンプリコンの個体群を形成することと、
(d)共通のバーコード配列を、前記アンプリコンのそれぞれが、それらが含有されている前記分離区分に属するように、それぞれの分離区分内の前記アンプリコンに結合することと、
(e)前記アンプリコンの配列を特定し、それによって、前記ゲノム試料の1つ以上の不十分に特徴決定された部分から配列情報を取得することと
を含む、前記方法。 - 前記増幅が、少なくとも3.5メガベース対(Mb)の範囲にわたるPCR増幅を含む、請求項26に記載の方法。
- 前記増幅が、少なくとも3.0Mbの領域にわたってねじれている複数のプライマー対を利用するPCR増幅を含む、請求項26〜27の記載の方法。
- 前記複数のプライマー対が、前記プライマー配列の増幅を防止するためにウラシルを含有する、請求項28に記載の方法。
- 前記特定ステップ(e)が、短い読み込み長さ配列決定反応及び長い読み込み長さ配列決定反応からなる群から選択される配列決定反応を含む、請求項26〜29に記載の方法。
- 前記配列決定反応が、短い読み込みで高い精度の配列決定反応である、請求項30に記載の方法。
- 前記特定ステップ(e)が、前記特定が、同じ個別の第1の核酸断片分子から誘導されるアンプリコンを特定することをさらに含むように、前記アンプリコン配列の前記分子構成を保存する、請求項26〜31に記載の方法。
- 前記方法が、前記複数の第の2断片の重複配列に基づいて、推定コンティグ中において2つ以上の前記個別の第1の断片分子を連結することをさらに含み、前記推定コンティグが、少なくとも10kbのN50長さを含む、請求項26〜32に記載の方法。
- 前記推定コンティグが、少なくとも20kbのN50長さを含む、請求項33に記載の方法。
- 前記推定コンティグが、少なくとも40kbのN50長さを含む、請求項33に記載の方法。
- 前記推定コンティグが、少なくとも50kbのN50長さを含む、請求項33に記載の方法。
- 前記推定コンティグが、少なくとも100kbのN50長さを含む、請求項33に記載の方法。
- 前記推定コンティグが、少なくとも200kbのN50長さを含む、請求項33に記載の方法。
- 前記バーコード配列が、追加の配列セグメントをさらに含む、請求項26〜38に記載の方法。
- 前記追加の配列セグメントが、プライマー、結合配列、ランダムn−merオリゴヌクレオチド、ウラシル核酸塩基を含むオリゴヌクレオチドからなる群から選択される1つ以上のメンバーを含む、請求項39に記載の方法。
- 前記結合ステップ(d)が、少なくとも700,000個のバーコードのライブラリーから選択されるバーコードを結合することを含む、請求項26〜40に記載の方法。
- それぞれの分離区分中の前記ゲノム試料が、単一細胞のゲノムDNAを含む、請求項26〜41に記載の方法。
- それぞれの分離区分が、異なる染色体のゲノムDNAを含む、請求項26〜41に記載の方法。
- 前記分離区分が、エマルションの液滴を含む、請求項26〜43に記載の方法。
- 前記特定ステップ(e)の前に、前記アンプリコンが、得られた増幅産物が部分的または完全なヘアピン構造を形成する能力があるようにさらに増幅される、請求項26〜44に記載の方法。
- 前記分離区分内の前記バーコード付きアンプリコンが、前記ゲノムの前記1つ以上の不十分に特徴決定された部分の約100×〜5000×の包括度を表す、請求項26〜45に記載の方法。
- 前記分離区分内の前記バーコード付きアンプリコンが、前記ゲノムの前記1つ以上の不十分に特徴決定された部分の約200×〜1000×の包括度を表す、請求項26〜45に記載の方法。
- 前記分離区分内の前記バーコード付きアンプリコンが、前記ゲノムの前記1つ以上の不十分に特徴決定された部分の少なくとも1000×の包括度を表す、請求項26〜45に記載の方法。
- 前記分離区分内の前記バーコード付きアンプリコンが、前記ゲノムの前記1つ以上の不十分に特徴決定された部分の少なくとも2000×の包括度を表す、請求項26〜45に記載の方法。
- 前記分離区分内の前記バーコード付きアンプリコンが、前記ゲノムの前記1つ以上の不十分に特徴決定された部分の少なくとも5000×の包括度を表す、請求項26〜45に記載の方法。
- 分子構成を保持しながら、ゲノム試料の1つ以上の部分から配列情報を取得する方法であって、
(a)出発ゲノム材料を準備することと、
(b)前記出発ゲノム材料の個別の核酸分子を、分離区分がそれぞれ第1の個別核酸分子を含有するように、分離区分の中に分配することと、
(c)前記ゲノムの前記1つ以上の選択部分の少なくとも一部を含む断片が豊富化された個体群を提供することと、
(d)共通のバーコード配列を、前記断片のそれぞれが、それらが含有されていた前記分離区分に属するように、それぞれの分離区分内の前記断片に結合することと、
(e)前記断片から配列情報を取得し、それによって、分子構成を保持しながら、前記ゲノム試料の1つ以上の標的化部分を配列決定することと
を含む、前記方法。 - 前記提供ステップ(c)が、前記ゲノムの前記1つ以上の選択部分の配列を含有する前記断片の少なくとも一部のPCR増幅を含む、請求項51に記載の方法。
- 前記ゲノムの前記1つ以上の選択部分が、前記ゲノムの少なくとも3.0Mbの長さの近接領域を含む、請求項51〜52に記載の方法。
- 前記取得ステップ(e)が、短い読み込み長さ配列決定反応及び長い読み込み長さ配列決定反応からなる群から選択される配列決定反応を含む、請求項51〜53に記載の方法。
- 前記配列決定反応が、短い読み込みで高い精度の配列決定反応である、請求項54に記載の方法。
- 前記特定ステップ(e)が、前記特定が、同じ個別の第1の核酸断片分子から誘導される断片を特定することをさらに含むように、前記断片配列の前記分子構成を保存する、請求項51〜55に記載の方法。
- 前記方法が、前記複数の第の2の断片の重複配列に基づいて、推定コンティグ中において2つ以上の前記個別の第1の断片分子を連結することをさらに含み、前記推定コンティグが、少なくとも10kbのN50長さを含む、請求項51〜56に記載の方法。
- 前記推定コンティグが、少なくとも20kbのN50長さを含む、請求項57に記載の方法。
- 前記推定コンティグが、少なくとも40kbのN50長さを含む、請求項57に記載の方法。
- 前記推定コンティグが、少なくとも50kbのN50長さを含む、請求項57に記載の方法。
- 前記推定コンティグが、少なくとも100kbのN50長さを含む、請求項57に記載の方法。
- 前記推定コンティグが、少なくとも200kbのN50長さを含む、請求項57に記載の方法。
- 前記バーコード配列が、追加の配列セグメントをさらに含む、請求項51〜62に記載の方法。
- 前記追加の配列セグメントが、プライマー、結合配列、ランダムn−merオリゴヌクレオチド、ウラシル核酸塩基を含むオリゴヌクレオチドからなる群から選択される1つ以上のメンバーを含む、請求項63に記載の方法。
- 前記結合ステップ(d)が、少なくとも700,000個のバーコードのライブラリーから選択されるバーコードを結合することを含む、請求項51〜64に記載の方法。
- それぞれの分離区分中の前記ゲノム材料が、単一細胞のゲノムDNAを含む、請求項51〜65に記載の方法。
- それぞれの分離区分が、異なる染色体のゲノムDNAを含む、請求項51〜65に記載の方法。
- 前記分離区分が、エマルションの液滴を含む、請求項51〜67に記載の方法。
- 前記取得ステップ(e)の前に、前記断片が、得られた増幅産物が部分的または完全なヘアピン構造を形成する能力があるようにさらに増幅される、請求項51〜68に記載の方法。
- 前記提供ステップ(c)が、前記ゲノムの前記1つ以上の選択部分の少なくとも一部を含む前記断片のPCR増幅を含む、請求項51〜69に記載の方法。
- 前記分離区分内の前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の約100×〜5000×の包括度を表す、請求項51〜70に記載の方法。
- 前記分離区分内の前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の約200×〜1000×の包括度を表す、請求項51〜70に記載の方法。
- 前記分離区分内の前記アンプリコンの前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の少なくとも1×の包括度を表す、請求項51〜70に記載の方法。
- 前記分離区分内の前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の少なくとも2000×の包括度を表す、請求項51〜70に記載の方法。
- 前記分離区分内の前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の少なくとも5000×の包括度を表す、請求項51〜70に記載の方法。
- 分子構成を保持しながら、ゲノム試料の1つ以上の部分から配列情報を取得する方法であって、
(a)出発ゲノム材料を準備することと、
(b)前記出発ゲノム材料の個別の核酸分子を、分離区分がそれぞれ第1の個別核酸分子を含有するように、分離区分の中に分配することと、
(c)前記ゲノムの前記1つ以上の選択部分の少なくとも一部を含む断片の配列が豊富化されている少なくともいくつかの前記分離区分内に、個体群を提供することと、
(d)共通のバーコード配列を、前記断片のそれぞれが、それらが含有されていた前記分離区分に属するように、それぞれの分離区分内の前記断片に結合することと、
(e)前記ゲノムの前記1つ以上の選択部分の少なくとも一部を含む断片を含有する分離区分を、前記ゲノムの前記1つ以上の選択部分を含む断片を含有しない分離区分から分別することと、
(f)前記ゲノムの前記1つ以上の選択部分の少なくとも一部を含む前記断片から配列情報を取得し、それによって、分子構成を保持しながら、前記ゲノム試料の1つ以上の標的化部分を配列決定することと
を含む、前記方法。 - 前記提供ステップ(c)が、前記ゲノムの前記1つ以上の選択部分の少なくとも一部を含むアンプリコンの個体群を産生する、前記ゲノムの前記1つ以上の選択部分の少なくとも一部を含む前記断片の定方向PCR増幅を含む、請求項76に記載の方法。
- 前記提供ステップ(c)が、検出可能標識を前記アンプリコンに結合することをさらに含む、請求項77に記載の方法。
- 前記分別ステップ(e)が、前記検出可能標識のシグナルを放出する前記区分を、そのような信号を有さない前記区分から選別することを含む、請求項78に記載の方法。
- 前記検出可能標識が蛍光分子を含む、請求項78〜79に記載の方法。
- 前記取得ステップ(f)の前に、前記分離区分が合わされ、前記断片が一緒にプールされる、請求項76〜80に記載の方法。
- 前記特定ステップ(f)が、前記特定が、同じ個別の第1の核酸断片分子から誘導される断片を特定することをさらに含むように、前記断片配列の前記分子構成を保存する、請求項76〜81に記載の方法。
- 前記特定ステップ(f)が、短い読み込み長さ配列決定反応及び長い読み込み長さ配列決定反応からなる群から選択される配列決定反応を含む、請求項76〜82に記載の方法。
- 前記配列決定反応が、短い読み込みで高い精度の配列決定反応である、請求項83に記載の方法。
- 前記分離区分が、エマルションの液滴を含む、請求項76〜84に記載の方法。
- 前記分離区分内の前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の約100×〜5000×の包括度を表す、請求項76〜85に記載の方法。
- 前記分離区分内の前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の約200×〜1000×の包括度を表す、請求項76〜85に記載の方法。
- 前記分離区分内の前記アンプリコンの前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の少なくとも1000×の包括度を表す、請求項76〜85に記載の方法。
- 前記分離区分内の前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の少なくとも2000×の包括度を表す、請求項76〜85に記載の方法。
- 前記分離区分内の前記バーコード付き断片が、前記ゲノムの前記1つ以上の選択部分の少なくとも5000×の包括度を表す、請求項76〜85に記載の方法。
- 分子構成を保持しながら、ゲノム試料の1つ以上の部分から配列情報を取得する方法であって、
(a)ゲノム材料を準備することと、
(b)個別の核酸分子を前記ゲノム材料から分別して、分別された個別の核酸分子を形成することと、
(c)前記分別された個別の核酸分子からの前記ゲノムの前記1つ以上の選択部分の少なくとも一部を含む断片が豊富化された個体群を提供し、少なくとも複数の前記断片が、それらが誘導される前記個別の核酸分子に属することと、
(d)前記断片から配列情報を取得し、それによって、分子構成を保持しながら、前記ゲノム試料の1つ以上の標的化部分を配列決定することと
を含む、前記方法。 - 前記分別ステップ(b)が、1つ以上の個別の核酸分子を分離区分に分配することを含む、請求項91に記載の方法。
- 前記提供ステップ(c)が、前記ゲノムの前記1つ以上の選択部分の少なくとも一部を含むアンプリコンの個体群を産生する、前記ゲノムの前記1つ以上の選択部分の少なくとも一部を含む前記核酸分子の定方向増幅を含む、請求項91〜92に記載の方法。
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WO2014210353A2 (en) * | 2013-06-27 | 2014-12-31 | 10X Technologies, Inc. | Compositions and methods for sample processing |
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WO2016138148A1 (en) | 2016-09-01 |
EP3262188A1 (en) | 2018-01-03 |
CN107532202A (zh) | 2018-01-02 |
IL254120A0 (en) | 2017-10-31 |
EP3936619A1 (en) | 2022-01-12 |
CA2975958A1 (en) | 2016-09-01 |
EP3262188B1 (en) | 2021-05-05 |
MX2017010857A (es) | 2017-12-11 |
US20220403464A1 (en) | 2022-12-22 |
CN115651972A (zh) | 2023-01-31 |
US20160281160A1 (en) | 2016-09-29 |
AU2016222719A1 (en) | 2017-09-07 |
KR20170119710A (ko) | 2017-10-27 |
BR112017018054A2 (pt) | 2018-07-24 |
US11274343B2 (en) | 2022-03-15 |
AU2016222719B2 (en) | 2022-03-31 |
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