Quantifying dna sequences

A method for quantifying target deoxyribonucleic acid (DNA) sequences, said method comprising the steps of:

• contacting, in each container of N separate containers, N being an integer equal to or larger than two, target DNA sequences, under hybridization conditions, with ML left ligation oligonucleotides comprising, from a 5′ end to a 3′ end, a sequencing read-1 primer site, a respective first unique molecular identifier (UMI) and a respective sequence complementary to a respective first segment of a target DNA sequence and MR right ligation oligonucleotides comprising, from a 5′ end to a 3′ end, a respective sequence complementary to a respective second segment of a target DNA sequence, a respective second UMI and a sequencing read-2 primer site, wherein ML and MR are each an integer equal to or larger than two, said respective first UMI being different for each of said ML left ligation oligonucleotides and said respective second UMI being different for each of said MR right ligation oligonucleotides;
• adding, in each container of said N separate containers, a ligating agent capable of ligating together said 3′ end of a left ligation oligonucleotide and said 5′ end of a right ligation oligonucleotide while hybridized to a target DNA sequence to form a ligated product comprising two UMIs and hybridized to said target DNA sequence;
• immobilizing, in each container of said N separate containers, said ligated product in complex with said target DNA sequence onto a solid phase having preference for DNA sequences of a length in terms of number of deoxyribonucleotides equal to or larger than a minimum length and removing a supernatant;
• amplifying, in each container of said N separate containers, said ligated product in presence of a left amplification primer comprising, from a 5′ end to a 3′ end, a first common sequence and said sequencing read-1 primer site and a right amplification primer comprising, from a 5′ end to a 3′ end, a second common sequence, a barcode sequence and a ligation oligonucleotide binding site complementary to said sequencing read-2 primer site to form an amplified product comprising two UMIs and one barcode sequence;
• mixing together the content of said N separate containers;
• sequencing at least a respective portion of said amplified products by addition of a sequencing read-1 primer comprising said sequencing read-1 primer site and a sequencing read-2 primer comprising said read-2 primer site to form respective sequence reads comprising at least nucleotide sequences of two UMIs, one barcode sequence and a target DNA sequence;
• demultiplexing said sequence reads based on nucleotide sequences of said barcode sequences;
• mapping said demultiplexed sequence reads to known DNA sequences based on nucleotide sequences of said target DNA sequence;
• and
• quantifying unique target DNA sequences in said N containers based on said demultiplexed and mapped sequence reads and based on nucleotide sequences of said UMIs.

A kit for quantifying target deoxyribonucleic acid (DNA) sequences, said kit comprises:

• ML left ligation oligonucleotides comprising, from a 5′ end to a 3′ end, a sequencing read-1 primer site, a respective first unique molecular identifier (UMI) and a respective sequence complementary to a respective first segment of a target DNA sequence;
• MR right ligation oligonucleotides comprising, from a 5′ end to a 3′ end, a respective sequence complementary to a respective second segment of a target DNA sequence, a respective second UMI and a sequencing read-2 primer site, wherein ML and MR are each an integer equal to or larger than two, said respective first UMI being different for each of said ML left ligation oligonucleotides and said respective second UMI being different for each of said MR right ligation oligonucleotides;
• a left amplification primer comprising, from a 5′ end to a 3′ end, a first common sequence and said sequencing read-1 primer site;
• a right amplification primer comprising, from a 5′ end to a 3′ end, a second common sequence, a barcode sequence and a ligation oligonucleotide binding site complementary to said sequencing read-2 primer site;
• a sequencing read-1 primer comprising said sequencing read-1 primer site;
• and
• a sequencing read-2 primer comprising said read-2 primer site.