Method for detecting the mutation and methylation of tumor-specific genes in ctDNA

1 . A method for constructing a sequencing library, comprising the following steps sequentially:

(1) taking a DNA sample and digesting it with a methylation-sensitive restriction endonuclease;

(2) the DNA sample digested in step (1) is subjected to end repair and adding A treatment at the 3′ end sequentially;

(3) ligating the DNA sample processed in step (2) with an adapter in an adapter mixture, and obtaining a library after PCR amplification;

(4) amplifying the library obtained in step (3);

the adapter mixture consists of n adapters;

each adapter is formed by an upstream primer A and a downstream primer A to form a partial double-stranded structure; the upstream primer A includes a sequencing adapter A, a random tag, an anchor sequence A and a base T sequentially from the 5′ end; the downstream primer A includes an anchor sequence B and a sequencing adapter B sequentially from the 5′ end; the partial double-stranded structure is formed by the reverse complementation of the anchor sequence A and the anchor sequence B;

the sequencing adapter A and sequencing adapter B are corresponding sequencing adapters selected according to different sequencing platforms;

the random tag is 8-14 bases in length;

the anchor sequence A has a length of 12-20 bp, and has ≤3 consecutive repeating bases;

the n adapters use n different anchor sequences A(s), and the four bases in each anchor sequence A are balanced, and the number of mismatched bases ≥3;

n is any natural number ≥8;

a primer pair used for amplification in step (4) consists of two single-stranded DNA molecules shown in SEQ ID NO. 25 and SEQ ID NO. 26 in the sequence listing;

wherein, the number of mismatched bases ≥3 means that the adapter mixture contains n anchor sequences A(s), and there are at least 3 differences in the bases between each anchor sequence A; the difference is different positions or different sequences.

2 . The construction method according to claim 1 , wherein:

the DNA sample is a genomic DNA, cDNA, ct DNA or cf DNA sample.

3 . A DNA library constructed by the method according to claim 1 ; wherein, DNA molecules in the DNA library contain double-stranded adapters, which are Y-shaped adapters, each DNA molecule includes a random tag composed of 8-14 random bases and an anchor sequence with a length of 12-20 bp on one or both ends of the adapter; the DNA library contains an adapter mixture employing n distinct anchor sequences, where n≥8; each anchor sequence is balanced of the four nucleotide bases, and the number of mismatched bases between any two anchor sequences is ≥3.

4 . A kit for detecting tumor mutation and/or methylation in DNA samples, comprising the adapter mixture and primer combinations according to claim 1 ;

wherein the tumor is a liver malignant tumor;

the primer combinations include primer set I, primer set II, primer set III, primer set IV, primer set V, primer set VI, primer set VII and primer set VIII, wherein:

the primer set I includes 78 single-stranded DNA molecules, and the nucleotide sequences of the 78 single-stranded DNA molecules are shown in SEQ ID NO. 28 to SEQ ID NO. 105 in the sequence listing sequentially;

the primer set II includes 82 single-stranded DNA molecules, and the nucleotide sequences of the 82 single-stranded DNA molecules are shown in SEQ ID NO. 106 to SEQ ID NO. 187 in the sequence listing sequentially;

the primer set III includes 14 single-stranded DNA molecules, and the nucleotide sequences of the 14 single-stranded DNA molecules are shown in SEQ ID NO. 188 to SEQ ID NO. 201 in the sequence listing sequentially;

the primer set IV includes 15 single-stranded DNA molecules, and the nucleotide sequences of the 15 single-stranded DNA molecules are shown in SEQ ID NO. 202 to SEQ ID NO. 216 in the sequence listing sequentially;

the primer set V includes 75 single-stranded DNA molecules, and the 75 single-stranded DNA molecules sequentially include the nucleotide sequences shown in SEQ ID NO. 220 to SEQ ID NO. 294 of the sequence listing from the 16th position from the 5′ end to the 3′ end;

the primer set VI includes 79 single-stranded DNA molecules, and the 79 single-stranded DNA molecules sequentially include the nucleotide sequences shown in SEQ ID NO. 295 to SEQ ID NO. 373 of the sequence listing from the 16th position from the 5′ end to the 3′ end;

the primer set VII includes 14 single-stranded DNA molecules, and the 14 single-stranded DNA molecules sequentially include the nucleotide sequences shown in SEQ ID NO. 374 to SEQ ID NO. 387 of the sequence listing from the 16th position from the 5′ end to the 3′ end;

the primer set VIII includes 15 single-stranded DNA molecules, and the 15 single-stranded DNA molecules sequentially include the nucleotide sequences shown in SEQ ID NO. 388 to SEQ ID NO. 402 of the sequence listing from the 16th position from the 5′ end to the 3′ end.

5 . A method for detecting target mutation and/or methylation in a DNA sample, comprising the following steps:

(1) constructing a library;

(2) performing two rounds of nested PCR amplification to the library obtained in step (1), sequencing the product, and analyzing the occurrence of target mutation and/or methylation in the DNA sample according to the sequencing result;

in the step (2), primer combination A is used to carry out the first round of PCR amplification;

primer combination A consists of upstream primer A and downstream primer combination A;

the upstream primer A is a library amplification primer used for library amplification in step (1);

the downstream primer combination A is a combination of Y primers designed according to X target sites; X and Y are both natural numbers greater than 1, and X≤Y;

using the product of the first round of PCR as a template, carrying out the second round of PCR amplification with primer combination B;

primer combination B consists of upstream primer B, downstream primer combination B and index primer;

the upstream primer B is a library amplification primer and the 3′ end is the same as that of the upstream primer A, and is used for the amplification of the product of the first round of PCR;

the index primer includes a segment A for sequencing, an index sequence for distinguishing samples, and a segment B for sequencing from the 5′ end;

the primer in the downstream primer combination B has the segment B and form a nested relationship with the primer detecting the same target site in the downstream primer combination A;

wherein, the method for constructing a library in step (1) comprises the following steps sequentially:

1) taking a DNA sample and digesting it with a methylation-sensitive restriction endonuclease;

2) the DNA sample digested in step 1) is subjected to end repair and adding A treatment at the 3′ end sequentially;

3) ligating the DNA sample processed in step 2) with an adapter in an adapter mixture, and obtaining a library after PCR amplification;

4) amplifying the library obtained in step 3);

the adapter mixture consists of n adapters;

each adapter is formed by an upstream primer A and a downstream primer A to form a partial double-stranded structure; the upstream primer A includes a sequencing adapter A, a random tag, an anchor sequence A and a base T sequentially from the 5′ end; the downstream primer A includes an anchor sequence B and a sequencing adapter B sequentially from the 5′ end; the partial double-stranded structure is formed by the reverse complementation of the anchor sequence A and the anchor sequence B;

the sequencing adapter A and sequencing adapter B are corresponding sequencing adapters selected according to different sequencing platforms;

the random tag is 8-14 bases in length;

the anchor sequence A has a length of 12-20 bp, and has ≤3 consecutive repeating bases;

the n adapters use n different anchor sequences A(s), and the four bases in each anchor sequence A are balanced, and the number of mismatched bases ≥3;

n is any natural number ≥8;

a primer pair used for amplification in step 4) consists of two single-stranded DNA molecules shown in SEQ ID NO. 25 and SEQ ID NO. 26 in the sequence listing.

6 . The method according to claim 5 , wherein:

the method for analyzing the target mutation in the DNA sample is: DNA molecules whose sequencing data meet the criterion A are traced back to a molecular cluster; the molecular clusters which meet the criterion B are labeled as a pair of duplex molecular clusters; for a mutation, if the following (a1) or (a2) is satisfied, the mutation is a true mutation from the original DNA sample: (a1) supported by at least one pair of duplex molecular clusters; (a2) supported by at least 4 molecular clusters; criterion A means satisfying (I), (II) and (III) at the same time; (I) the length of the DNA inserts is the same and the sequences are the same except for the mutation sites; (II) the random tag sequences are the same; (III) the anchor sequences are the same; criterion B means satisfying both IV and (V); (IV) the length of the DNA inserts is the same and the sequences are the same except for the mutation sites; (V) the anchor sequences at both ends of the molecular cluster are the same but in opposite positions;

the method for analyzing methylation in the DNA sample is: the DNA molecules whose sequencing data meet the criterion C are labeled as a cluster, and the number of clusters whose ends are the restriction sites of interest is calculated respectively, and recorded as unmethylated fragments; the number of all the clusters whose amplified fragments reach or exceed the first restriction site is calculated, and recorded as the total number of fragments; the average methylation level of the corresponding region is calculated according to the number of two fragments; the methylation level of the region=(1−the number of unmethylated fragments/the total number of fragments)×100%; criterion C means satisfying (VI), (VII), and (VIII) at the same time; (VI) the random tag sequences are the same; (VII) the anchor sequences are the same; (VIII) the length of the DNA inserts is the same and the sequences are the same except for the mutation sites.

7 . A method for detecting multiple target mutations and/or methylation in a DNA sample, comprising the following steps:

(1) constructing a library according to the method described in claim 1 ;

(2) enriching and sequencing the target region of the library of step (1), and analyzing the occurrence of target mutation and/or methylation in the DNA sample according to the sequencing result.

8 . The method according to claim 7 , wherein:

the method for analyzing the target mutation in the DNA sample is: DNA molecules whose sequencing data meet the criterion A are traced back to a molecular cluster; the molecular clusters which meet the criterion B are labeled as a pair of duplex molecular clusters; for a mutation, if the following (a1) or (a2) is satisfied, the mutation is a true mutation from the original DNA sample: (a1) supported by at least one pair of duplex molecular clusters; (a2) supported by at least 4 molecular clusters; criterion A means satisfying (I), (II), and (III) at the same time; (I) the length of the DNA inserts is the same and the sequences are the same except for the mutation sites; (II) the random tag sequences are the same; (III) the anchor sequences are the same; criterion B means satisfying both (IV) and (V); (IV) the length of the DNA inserts is the same and the sequences are the same except for the mutation sites; (V) the anchor sequences at both ends of the molecular cluster are the same but in opposite positions; the method for analyzing methylation in the DNA sample is: the DNA molecules whose sequencing data meet the criterion C are labeled as a cluster, and the number of clusters whose ends are the restriction sites of interest is calculated respectively, and recorded as unmethylated fragments; the number of all the clusters whose amplified fragments reach or exceed the first restriction site is calculated, and recorded as the total number of fragments; the average methylation level of the corresponding region is calculated according to the number of two fragments; the methylation level of the region=(1−the number of unmethylated fragments/the total number of fragments)×100%; criterion C means satisfying (VI), (VII), and (VIII) at the same time; (VI) the random tag sequences are the same; (VII) the anchor sequences are the same; (VIII) the length of the DNA inserts is the same and the sequences are the same except for the mutation sites.

9 . A method for distinguishing blood samples from tumor patients and blood samples from non-tumor patients, comprising the following steps:

constructing a library according to the method described in claim 1 ;

enriching and sequencing the target region of the library, and analyzing the occurrence of target mutation and/or methylation in the DNA sample according to the sequencing result;

distinguishing blood samples from tumor patients and blood samples from non-tumor patients according to occurrence of target mutation and/or methylation in the DNA sample.