公开(公告)号：EP3907299A1

公开(公告)日：2021-11-10

申请号：EP21173115.3

申请日：2012-04-12

申请人： The Johns Hopkins University

发明人： VOGELSTEIN, Bert ,  KINZLER, Kenneth ,  PAPADOPOULOS, Nickolas ,  KINDE, Isaac

IPC分类号： C12Q1/6874 ,  C12N15/11 ,  C12Q1/6806

摘要： The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called "Safe-SeqS" for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant ("super-mutants") if =95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

公开(公告)号：EP2697397B1

公开(公告)日：2017-04-05

申请号：EP12772013.4

申请日：2012-04-12

申请人： The Johns Hopkins University

发明人： VOGELSTEIN, Bert ,  KINZLER, Kenneth W. ,  PAPADOPOULOS, Nickolas ,  KINDE, Isaac

IPC分类号： C12Q1/68 ,  C12N15/11

CPC分类号： C12Q1/6874 ,  C12Q1/6806 ,  C12Q1/6869 ,  C12Q1/6876 ,  C12Q2563/179 ,  C12Q2600/158 ,  C12Q2535/122 ,  C12Q2565/514

摘要： The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called "Safe-SeqS" for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant ("super-mutants") if =95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

公开(公告)号：EP2912468A1

公开(公告)日：2015-09-02

申请号：EP13851273.6

申请日：2013-10-17

申请人： The Johns Hopkins University

发明人： KINDE, Isaac ,  KINZLER, Kenneth W. ,  VOGELSTEIN, Bert ,  PAPADOPOULOS, Nickolas ,  DIAZ, Luis ,  BETTEGOWDA, Chetan ,  WANG, Yuxuan

IPC分类号： G01N33/574 ,  G01N33/68 ,  C12Q1/68

CPC分类号： C12Q1/6886 ,  C12Q2600/154 ,  C12Q2600/156 ,  C12Q2600/158 ,  C12Q2600/16 ,  G01N33/57442 ,  G01N33/57449

摘要： The recently developed liquid-based Papanicolaou (Pap) smear allows not only cytologic evaluation but also collection of DNA for detection of HPV, the causative agent of cervical cancer. We tested these samples to detect somatic mutations present in rare tumor cells that might accumulate in the cervix once shed from endometrial and ovarian cancers. A panel of commonly mutated genes in endometrial and ovarian cancers was assembled and used to identify mutations in all 46 endometrial or cervical cancer tissue samples. We were able also able to identify the same mutations in the DNA from liquid Pap smears in 100% of endometrial cancers (24 of 24) and in 41% of ovarian cancers (9 of 22). We developed a sequence-based method to query mutations in 12 genes in a single liquid Pap smear without prior knowledge of the tumor's genotype.

公开(公告)号：EP4239081A3

公开(公告)日：2023-11-08

申请号：EP23170939.5

申请日：2013-03-22

申请人： The Johns Hopkins University

发明人： VOGELSTEIN, Bert ,  KINZLER, Kenneth W. ,  PAPADOPOULOS, Nickolas ,  KINDE, Isaac

IPC分类号： C12Q1/6883 ,  C12N15/11 ,  G01N33/53 ,  G01N33/48 ,  C12Q1/6827

摘要： Massively parallel sequencing of cell-free, maternal plasma DNA was recently demonstrated to be a safe and effective screening method for fetal chromosomal aneuploidies. Here, we report an improved sequencing method achieving significantly increased throughput and decreased cost by replacing laborious sequencing library preparation steps with PCR employing a single primer pair. Using this approach, samples containing as little as 4% trisomy 21 DNA could be readily distinguished from euploid samples.

公开(公告)号：EP4239081A2

公开(公告)日：2023-09-06

申请号：EP23170939.5

申请日：2013-03-22

申请人： The Johns Hopkins University

发明人： VOGELSTEIN, Bert ,  KINZLER, Kenneth W. ,  PAPADOPOULOS, Nickolas ,  KINDE, Isaac

IPC分类号： C12Q1/6827 ,  C12Q1/6883

摘要： Massively parallel sequencing of cell-free, maternal plasma DNA was recently demonstrated to be a safe and effective screening method for fetal chromosomal aneuploidies. Here, we report an improved sequencing method achieving significantly increased throughput and decreased cost by replacing laborious sequencing library preparation steps with PCR employing a single primer pair. Using this approach, samples containing as little as 4% trisomy 21 DNA could be readily distinguished from euploid samples.

公开(公告)号：EP3907297A1

公开(公告)日：2021-11-10

申请号：EP21173117.9

申请日：2012-04-12

申请人： The Johns Hopkins University

发明人： VOGELSTEIN, Bert ,  KINZLER, Kenneth ,  PAPADOPOULOS, Nickolas ,  KINDE, Isaac

IPC分类号： C12Q1/6806 ,  C12N15/11 ,  C12Q1/6874

摘要： The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called "Safe-SeqS" for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant ("super-mutants") if =95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

公开(公告)号：EP2912468B1

公开(公告)日：2018-09-12

申请号：EP13851273.6

申请日：2013-10-17

申请人： The Johns Hopkins University

发明人： KINDE, Isaac ,  KINZLER, Kenneth W. ,  VOGELSTEIN, Bert ,  PAPADOPOULOS, Nickolas ,  DIAZ, Luis ,  BETTEGOWDA, Chetan ,  WANG, Yuxuan

IPC分类号： C12Q1/6886

CPC分类号： C12Q1/6886 ,  C12Q2600/154 ,  C12Q2600/156 ,  C12Q2600/158 ,  C12Q2600/16 ,  G01N33/57442 ,  G01N33/57449

摘要： The recently developed liquid-based Papanicolaou (Pap) smear allows not only cytologic evaluation but also collection of DNA for detection of HPV, the causative agent of cervical cancer. We tested these samples to detect somatic mutations present in rare tumor cells that might accumulate in the cervix once shed from endometrial and ovarian cancers. A panel of commonly mutated genes in endometrial and ovarian cancers was assembled and used to identify mutations in all 46 endometrial or cervical cancer tissue samples. We were able also able to identify the same mutations in the DNA from liquid Pap smears in 100% of endometrial cancers (24 of 24) and in 41% of ovarian cancers (9 of 22). We developed a sequence-based method to query mutations in 12 genes in a single liquid Pap smear without prior knowledge of the tumor's genotype.

公开(公告)号：EP3246416A1

公开(公告)日：2017-11-22

申请号：EP17154750.8

申请日：2012-04-12

申请人： The Johns Hopkins University

发明人： VOGELSTEIN, Bert ,  KINZLER, Kenneth ,  PAPADOPOULOS, Nickolas ,  KINDE, Isaac

IPC分类号： C12Q1/68 ,  C12N15/11

CPC分类号： C12Q1/6874 ,  C12Q1/6806 ,  C12Q1/6869 ,  C12Q1/6876 ,  C12Q2563/179 ,  C12Q2600/158 ,  C12Q2535/122 ,  C12Q2565/514

摘要： The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called "Safe-SeqS" for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant ("super-mutants") if =95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

摘要翻译： 存在于一小部分DNA模板中的突变的鉴定对于生物医学研究的若干领域的进展是必不可少的。 尽管大规模并行测序仪器原则上非常适合于此任务，但这些仪器的错误率通常太高，以至于无法确定罕见变体。 我们在这里描述了一种可以大幅度提高大规模并行测序仪器的灵敏度的方法。 这种方法的一个例子叫做安全测序系统（Safe-Sequencing System）的“Safe-SeqS”，包括（i）为每个模板分子分配一个唯一标识符（UID） （ii）扩增每个独特标记的模板分子以产生UID家族; 和（iii）扩增产物的冗余测序。 具有相同UID的PCR片段确实是突变体（“超级突变体”），如果其中95％含有相同的突变。 我们说明了这种方法用于确定聚合酶保真度的效用，体外合成的寡核苷酸的准确性以及正常细胞核和线粒体基因组突变的发生率。

公开(公告)号：EP2697397A2

公开(公告)日：2014-02-19

申请号：EP12772013.4

申请日：2012-04-12

申请人： The Johns Hopkins University

发明人： VOGELSTEIN, Bert ,  KINZLER, Kenneth W. ,  PAPADOPOULOS, Nickolas ,  KINDE, Isaac

IPC分类号： C12Q1/68 ,  C12N15/11

CPC分类号： C12Q1/6874 ,  C12Q1/6806 ,  C12Q1/6869 ,  C12Q1/6876 ,  C12Q2563/179 ,  C12Q2600/158 ,  C12Q2535/122 ,  C12Q2565/514

摘要： The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called "Safe-SeqS" for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant ("super-mutants") if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized
in vitro , and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

摘要翻译： 存在于一小部分DNA模板中的突变的鉴定对于生物医学研究的若干领域的进展是必不可少的。 尽管大规模并行测序仪器原则上非常适合于此任务，但这些仪器的错误率通常太高，以至于无法确定罕见变体。 我们在这里描述了一种可以大幅度提高大规模并行测序仪器的灵敏度的方法。 这种方法的一个例子叫做安全测序系统（Safe-Sequencing System）的“Safe-SeqS”，包括（i）为每个模板分子分配一个唯一标识符（UID） （ii）扩增每个独特标记的模板分子以产生UID家族; 和（iii）扩增产物的冗余测序。 具有相同UID的PCR片段如果≥95％含有相同的突变，则是真正的突变体（“超级突变体”）。 我们说明了这种方法用于确定聚合酶保真度的效用，体外合成的寡核苷酸的准确性以及正常细胞核和线粒体基因组突变的发生率。