Method for low frequency somatic cell mutation identification and quantification

A method of detecting genomic location of a transposon in a subject, comprising

• a) providing isolated DNA sample from a test sample of the subject;
• b) fragmenting the DNA sample to obtain DNA fragments;
• c) ligating adaptors to both ends of the DNA fragments to obtain a transposon-adaptor library;
• d) amplifying the transposon-adaptor library with primers, wherein a first primer comprises a transposon specific sequence capable of hybridizing to an internal sequence of the transposon, a second primer comprises an adapting sequence capable of hybridizing to the adaptor, thereby amplifying the transposon and flanking sequences thereof;
• e) sequencing the obtained product from amplification to obtain the flanking sequences;
• and
• f) aligning the obtained flanking sequences with a reference genomic sequence to obtain genomic location and orientation of the transposon;
• or
• a method of identifying a tissue or cell specific transposon insertion event, comprising
• a) providing isolated DNA sample from a test sample of an individual;
• b) fragmenting the DNA sample to obtain DNA fragments;
• c) ligating adaptors to both ends of the DNA fragments to obtain a transposon-adaptor library;
• d) amplifying the transposon-adaptor library with primers, wherein a first primer comprises a transposon specific sequence capable of hybridizing to an internal sequence of the transposon, a second primer comprises an adapting sequence capable of hybridizing to the adaptor, thereby amplifying the transposon and flanking sequences thereof;
• e) sequencing the obtained product from amplification to obtain the flanking sequences:
• f) aligning the obtain flanking sequences with a reference genomic sequence to obtain genomic location and orientation of the transposon;
• and
• g) comparing the location obtained in step f) with a reference location to identify the transposon which is present in step f) but absent in the reference location as the specific transposon insertion in the test tissue or cell;
• or
• a method of quantifying tissue or cell specific transposon insertion events in an individual, comprising
• a) providing isolated DNA sample from a test sample of the individual;
• b) fragmenting the DNA sample to obtain DNA fragments;
• c) ligating adaptors to both ends of the DNA fragments to obtain a transposon-adaptor library;
• d) amplifying the transposon-adaptor library with primers, wherein a first primer comprises a transposon specific sequence capable of hybridizing to an internal sequence of the transposon, a second primer comprises an adapting sequence capable of hybridizing to the adaptor, thereby amplifying the transposon and flanking sequence thereof;
• e) sequencing the obtained product from amplification to obtain the flanking sequences;
• f) aligning the obtain flanking sequences with a reference genomic sequence to obtain genomic location and orientation of the transposon;
• and
• g) comparing the location obtained in step f) with a reference location to identify the transposon which is present in step f) but absent in the reference location as the specific transposon insertion in the test tissue or cell;
• and
• h) counting tissue or cell specific transposon insertion events in the sequencing data of the library and transposon insertion events in the reference genome corresponding to the individual, and calculating the ratio of the two numbers to quantify the tissue or cell specific transposon insertion events.