The Experts below are selected from a list of 130278 Experts worldwide ranked by ideXlab platform

Tony E Godfrey - One of the best experts on this subject based on the ideXlab platform.

  • simple multiplexed pcr based barcoding of dna for ultrasensitive mutation detection by next generation sequencing
    Nature Protocols, 2017
    Co-Authors: Anders Stahlberg, Paul M Krzyzanowski, Matthew Egyud, Lincoln Stein, Stefan Filges, Tony E Godfrey
    Abstract:

    Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) Library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex Library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) Library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the Library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of Library Content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.

  • simple multiplexed pcr based barcoding of dna enables sensitive mutation detection in liquid biopsies using sequencing
    Nucleic Acids Research, 2016
    Co-Authors: Anders Stahlberg, Paul M Krzyzanowski, Jennifer B Jackson, Matthew Egyud, Lincoln Stein, Tony E Godfrey
    Abstract:

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing Library construction with flexible Library Content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.

Anders Stahlberg - One of the best experts on this subject based on the ideXlab platform.

  • simple multiplexed pcr based barcoding of dna for ultrasensitive mutation detection by next generation sequencing
    Nature Protocols, 2017
    Co-Authors: Anders Stahlberg, Paul M Krzyzanowski, Matthew Egyud, Lincoln Stein, Stefan Filges, Tony E Godfrey
    Abstract:

    Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) Library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex Library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) Library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the Library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of Library Content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.

  • simple multiplexed pcr based barcoding of dna enables sensitive mutation detection in liquid biopsies using sequencing
    Nucleic Acids Research, 2016
    Co-Authors: Anders Stahlberg, Paul M Krzyzanowski, Jennifer B Jackson, Matthew Egyud, Lincoln Stein, Tony E Godfrey
    Abstract:

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing Library construction with flexible Library Content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.

Matthew Egyud - One of the best experts on this subject based on the ideXlab platform.

  • simple multiplexed pcr based barcoding of dna for ultrasensitive mutation detection by next generation sequencing
    Nature Protocols, 2017
    Co-Authors: Anders Stahlberg, Paul M Krzyzanowski, Matthew Egyud, Lincoln Stein, Stefan Filges, Tony E Godfrey
    Abstract:

    Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) Library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex Library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) Library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the Library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of Library Content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.

  • simple multiplexed pcr based barcoding of dna enables sensitive mutation detection in liquid biopsies using sequencing
    Nucleic Acids Research, 2016
    Co-Authors: Anders Stahlberg, Paul M Krzyzanowski, Jennifer B Jackson, Matthew Egyud, Lincoln Stein, Tony E Godfrey
    Abstract:

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing Library construction with flexible Library Content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.

Lincoln Stein - One of the best experts on this subject based on the ideXlab platform.

  • simple multiplexed pcr based barcoding of dna for ultrasensitive mutation detection by next generation sequencing
    Nature Protocols, 2017
    Co-Authors: Anders Stahlberg, Paul M Krzyzanowski, Matthew Egyud, Lincoln Stein, Stefan Filges, Tony E Godfrey
    Abstract:

    Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) Library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex Library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) Library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the Library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of Library Content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.

  • simple multiplexed pcr based barcoding of dna enables sensitive mutation detection in liquid biopsies using sequencing
    Nucleic Acids Research, 2016
    Co-Authors: Anders Stahlberg, Paul M Krzyzanowski, Jennifer B Jackson, Matthew Egyud, Lincoln Stein, Tony E Godfrey
    Abstract:

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing Library construction with flexible Library Content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.

Paul M Krzyzanowski - One of the best experts on this subject based on the ideXlab platform.

  • simple multiplexed pcr based barcoding of dna for ultrasensitive mutation detection by next generation sequencing
    Nature Protocols, 2017
    Co-Authors: Anders Stahlberg, Paul M Krzyzanowski, Matthew Egyud, Lincoln Stein, Stefan Filges, Tony E Godfrey
    Abstract:

    Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) Library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex Library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) Library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the Library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of Library Content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.

  • simple multiplexed pcr based barcoding of dna enables sensitive mutation detection in liquid biopsies using sequencing
    Nucleic Acids Research, 2016
    Co-Authors: Anders Stahlberg, Paul M Krzyzanowski, Jennifer B Jackson, Matthew Egyud, Lincoln Stein, Tony E Godfrey
    Abstract:

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing Library construction with flexible Library Content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.