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Preparation of templates for methylation analysis

  • US 9,868,982 B2
  • Filed: 02/07/2008
  • Issued: 01/16/2018
  • Est. Priority Date: 02/07/2007
  • Status: Active Grant
First Claim
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1. A method of genome-wide analysis of the methylation status of cytosine bases in a whole genome sample, comprising:

  • a. providing a whole genome sample and fragmenting the whole genome sample to produce fragmented double stranded nucleic acid target fragments, wherein the fragments are obtained from said whole genome, and wherein the fragments span across said whole genome;

    b. ligating forked universal adaptors to the fragmented double stranded nucleic acid target fragments to produce adaptor-ligated double stranded nucleic acid target fragments comprising identical nucleic acid bases at each termini, wherein after said ligation step, said adaptor-ligated double stranded nucleic acid target fragments comprise a region of double stranded nucleic acids and at least one region of single stranded nucleic acids and wherein all cytosine bases in said forked universal adaptors are methylated, and wherein said forked universal adaptors are phosphorylated at the 5′

    end and said at least one region of single stranded nucleic acids is capable of hybridizing to SEQ ID NO;

    6;

    c. treating the adaptor-ligated double stranded nucleic acid target fragments with bisulfite to convert non-methylated cytosine bases to uracil thereby producing treated adaptor-ligated double stranded nucleic acid target fragments;

    d. optionally performing an amplification step to produce amplicons of the treated adapter-ligated double stranded nucleic acid target fragments;

    e. attaching the treated adaptor-ligated double stranded nucleic acid fragments or the amplicons thereof to a solid support;

    f. sequencing the treated adaptor-ligated double stranded nucleic acid target fragments or the amplicons thereof to generate sequences; and

    g. analyzing the sequences of step to determine which cytosine bases were converted to uracil bases, thereby determining the methylation status of the whole genome sample.

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