Method for measuring mutation rate

A method of identifying mutation,

• the method comprising the following steps (1) to (6):
• (1) preparing a library of genome fragments for next generation sequencing (NGS), comprising the following steps (a) to (c):
• (a) preparing DNA-adaptor ligates for a plurality of genomic DNA by:
• (i) cleaving each genomic DNA with a restriction enzyme;
• and
• (ii) joining adaptors to both ends of each cleaved genomic DNA, each adaptor comprising at least a random sequence that distinguishes each adaptor molecule from another by genomic DNA origin, wherein each end of a cleavage site of the genomic DNA is joined by at least one adaptor;
• (b) obtaining amplification products by using the DNA-adaptor ligates prepared in step (a) as templates and performing a polymerase chain reaction (PCR) using a primer that binds to the 3′ end of a nucleotide sequence of interest at a specific region of genomic DNA and a primer that binds to the 5′ end of the adaptors;
• (b-2) using the amplification products of step (b) as a template and performing nested PCR using:
• (i) a forward primer that binds to all or part of a nucleotide sequence of the specific region of genomic DNA;
• and
• (ii) a reverse primer that binds to all or part of the adaptor except for the random sequences,
• to produce nested amplification products, and
• (c) performing a second PCR by using the nested amplification products prepared in step (b-2) as templates and a primer pair which binds to both ends of the templates to form genome fragments;
• (2) determining the sequence of each genome fragment included in the library through NGS;
• (3) grouping the amplification products into a number of n by aligning the amplification products prepared in step (c) based on n number of landmarks on a reference genome sequence;
• (4) selecting mi number of genome fragments for each group, wherein mi is the number of genome fragments selected in the ith landmark, by sub-grouping the amplification products constituting the group according to the random sequences, then selecting one genome fragment for each mi number of sub-groups;
• (5) determining one representative nucleotide sequence without mutations for each group by comparing the nucleotide sequences of mi number of genome fragments;
• and
• (6) determining a mutation when there is a nucleotide sequence different from the representative nucleotide sequence of each group among the mi genomic fragments of n groups.