Method for identification and enumeration of nucleic acid sequence, expression, copy, or dna methylation changes using combined nuclease, ligase, polymerase, and sequencing reactions

Abstract

The present invention relates to a method for the highly specific, targeted capture of regions of human genomes and transcriptomes from the blood, i.e. from cell free circulating DNA, exosomes, microRNA, circulating tumor cells, or total blood cells, to allow for the highly sensitive detection of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using combined nuclease, ligation, polymerase, and massively parallel sequencing reactions. The method generates a collection of different circular chimeric single-stranded nucleic acid constructs, suitable for sequencing on multiple platforms. In some embodiments, each construct of the collection comprised a first single stranded segment of original genomic DNA from a host organism and a second single stranded synthetic nucleic acid segment that is linked to the first single stranded segment and comprises a nucleotide sequence that is exogenous to the host organism. These chimeric constructs are suitable for identifying and enumerating mutations, copy changes, translocations, and methylation changes. In other embodiments, input mRNA, lncRNA, or miRNA is used to generate circular DNA products that reflect the presence and copy number of specific mRNA's, lncRNA's splice-site variants, translocations, and miRNA.

1 .- 82 . (canceled) 
     
     
         83 . A method for sequencing a plurality of target nucleic acid molecules, said method comprising:
 providing a sample containing a plurality of target nucleic acid molecules, wherein said target nucleic acid molecules have 3′ and 5′ ends;   appending a 3′ linker sequence and a 5′ linker sequence to the 3′ and 5′ ends of the plurality of target nucleic acid molecules, respectively, to form 3′ and 5′ linker-appended target nucleic acid molecules, wherein said 3′ and 5′ linker sequences collectively comprise (i) one or more patient identifier sequences, (ii) a first solid support primer-specific portion, (iii) a second solid support primer-specific portion, (iv) one or more sequencing primer binding sequences, or any combination of (i), (ii), (iii), and (iv);   providing one or more first oligonucleotide probes, each first oligonucleotide probe comprising a portion complementary to the 3′ linker-appended target nucleic acid molecules, and a portion complementary to the 5′ linker-appended target nucleic acid molecules;   hybridizing the 3′ and 5′ linker-appended target nucleic acid molecules and the one or more first oligonucleotide probes, wherein the first oligonucleotide probes hybridize in a base specific manner to complementary sequences of the 3′ and 5′ linker-appended target nucleic acid molecules to form one or more ligation-competent junctions suitable for coupling 3′ and 5′ ends of the 3′ and 5′ linker-appended target nucleic acid molecules;   ligating the 3′ and 5′ linker-appended target nucleic acid molecules at the one or more ligation-competent junctions to form a plurality of circular chimeric single-stranded nucleic acid constructs;   providing a solid support on which a plurality of first oligonucleotide primers is immobilized, each first oligonucleotide primer comprising a nucleotide sequence that is complementary to the first solid support primer-specific portion of the plurality of circular chimeric single-stranded nucleic acid constructs;   hybridizing the plurality of circular chimeric single-stranded nucleic acid constructs to the plurality of first oligonucleotide primers immobilized on the solid support;   blending the plurality of circular chimeric single-stranded nucleic acid constructs hybridized to the plurality of first oligonucleotide primers immobilized on the solid support with a polymerase to form a rolling circle amplification reaction mixture;   subjecting the rolling circle amplification reaction mixture to an extension treatment where the polymerase extends the one or more hybridized oligonucleotide primers to produce a plurality of primary extension products, each primary extension product comprising one or more tandem linear sequences, wherein each tandem linear sequence is complementary to one of the circular chimeric single-stranded nucleic acid constructs in the collection; and   sequencing the circular chimeric single-stranded nucleic acid constructs directly or said plurality of primary extension products thereof.   
     
     
         84 . The method of  claim 83  further comprising:
 removing unligated target nucleic acid molecules, first oligonucleotide probes, and other non-circularized nucleic acid molecules from the sample after said ligating. 
 
     
     
         85 . The method of  claim 83 , wherein the polymerase is a strand-displacing polymerase. 
     
     
         86 . The method of  claim 83  further comprising:
 providing a collection of crosslinking oligonucleotides, each crosslinking oligonucleotide comprising two or more repeats of a nucleotide sequence, wherein said nucleotide sequence is the same as at least a portion of the nucleotide sequence of the circular chimeric single-stranded nucleic acid constructs of the collection; and 
 capturing one or more tandem linear sequences of a primary extension product on a crosslinking oligonucleotide of the collection, thereby condensing the primary extension product into a compact structure prior to said sequencing. 
 
     
     
         87 . The method of  claim 83 , wherein said sequencing comprises:
 hybridizing a sequencing primer to each tandem linear sequence of the primary extension product on the solid support;   extending the sequencing primer; and   sequencing each tandem linear sequence of the primary extension product on the solid support based on said extending.   
     
     
         88 . The method of  claim 83 , wherein said sequencing is carried out using a method selected from the group consisting of fluorescent primer hybridization, molecular beacon hybridization, primer extension, exonuclease-based sequencing, ligase detection reaction, ligase chain reaction, pyrosequencing, fluorescence-based sequencing-by-synthesis, fluorescence-based sequencing-by-ligation, nanopore based sequencing, ion-based sequencing-by-synthesis, and ion-based sequencing-by-ligation. 
     
     
         89 . The method of  claim 83 , wherein the solid support further comprises:
 a plurality of second oligonucleotide primers immobilized on the solid support, each second oligonucleotide primer comprising a nucleotide sequence that is the same as the second solid support primer-specific portion of the chimeric single stranded nucleic acid constructs of the collection.   
     
     
         90 . The method of  claim 89 , wherein two or more second oligonucleotide primers immobilized on the solid support hybridize to two or more tandem linear sequences of a primary extension product, said method further comprising:
 extending the two or more hybridized second oligonucleotide primers using a polymerase to form a plurality of immobilized secondary extension products, each secondary extension product comprising a nucleotide sequence that is complementary to a portion of the primary extension product; and   removing the primary extension products from the solid support, whereby said sequencing involves sequencing the plurality of immobilized secondary extension products.   
     
     
         91 . A method for sequencing a plurality of target nucleic acid molecules, said method comprising:
 providing a sample containing a plurality of target nucleic acid molecules, wherein said target nucleic acid molecules have 3′ and 5′ ends;   appending a 3′ linker sequence and a 5′ linker sequence to the 3′ and 5′ ends of the plurality of target nucleic acid molecules, respectively, to form one or more 3′ and 5′ linker-appended target nucleic acid molecules, wherein said 3′ and 5′ linker sequences collectively comprise (i) one or more patient identifier sequences, (ii) one or more sequencing primer binding sequences, or both (i) and (ii);   providing (a) one or more first oligonucleotide probes, wherein the first oligonucleotide probes comprise a portion complementary to the 3′ linker-appended target nucleic acid molecules, a portion complementary to the 5′ linker-appended target nucleic acid molecules, and a further portion comprising a first solid support primer-specific portion and optionally a second solid support primer-specific portion and (b) one or more second oligonucleotide probes comprising a nucleotide sequence complementary to the further portion of the first oligonucleotide probe(s);   hybridizing the 3′ and 5′ linker-appended target nucleic acid molecules, the one or more first oligonucleotide probes, and the one or more second oligonucleotide probes, under conditions effective for the one or more first oligonucleotide probes to hybridize in a base specific manner to complementary sequences of the 3′ and 5′ linker-appended target nucleic acid molecules and to hybridize to the second oligonucleotide probe to form one or more ligation competent junctions suitable for coupling 3′ and 5′ ends of the 3′ and 5′ linker-appended target nucleic acid molecules with the second oligonucleotide probe;   ligating the 3′ and 5′ linker-appended target nucleic acid molecules at the one or more ligation junctions to form a plurality of circular chimeric single-stranded nucleic acid constructs;   providing a solid support on which a plurality of first oligonucleotide primers is immobilized, each first oligonucleotide primer comprising a nucleotide sequence that is complementary to the first solid support primer-specific portion of the plurality of circular chimeric single-stranded nucleic acid constructs;   hybridizing the plurality of circular chimeric single-stranded nucleic acid constructs to the plurality of first oligonucleotide primers immobilized on the solid support;   blending the plurality of circular chimeric single-stranded nucleic acid constructs hybridized to the plurality of first oligonucleotide primers immobilized on the solid support with a polymerase to form a rolling circle amplification reaction mixture;   subjecting the rolling circle amplification reaction mixture to an extension treatment where the polymerase extends the one or more hybridized oligonucleotide primers to produce a plurality of primary extension products, each primary extension product comprising one or more tandem linear sequences, wherein each tandem linear sequence is complementary to one of the circular chimeric single-stranded nucleic acid constructs in the collection; and   sequencing the circular chimeric single-stranded nucleic acid constructs directly or said plurality of primary extension products thereof.   
     
     
         92 . The method of  claim 91  further comprising:
 removing unligated target nucleic acid molecules, first oligonucleotide probes, and other non-circularized nucleic acid molecules from the sample after said ligating. 
 
     
     
         93 . The method of  claim 91 , wherein the polymerase is a strand-displacing polymerase. 
     
     
         94 . The method of  claim 91  further comprising:
 providing a collection of crosslinking oligonucleotides, each crosslinking oligonucleotide comprising two or more repeats of a nucleotide sequence, wherein said nucleotide sequence is the same as at least a portion of the nucleotide sequence of the circular chimeric single-stranded nucleic acid constructs of the collection; and 
 capturing one or more tandem linear sequences of a primary extension product on a crosslinking oligonucleotide of the collection, thereby condensing the primary extension product into a compact structure prior to said sequencing. 
 
     
     
         95 . The method of  claim 91 , wherein said sequencing comprises:
 hybridizing a sequencing primer to each tandem linear sequence of the primary extension product on the solid support;   extending the sequencing primer; and   sequencing each tandem linear sequence of the primary extension product on the solid support based on said extending.   
     
     
         96 . The method of  claim 91 , wherein said sequencing is carried out using a method selected from the group consisting of fluorescent primer hybridization, molecular beacon hybridization, primer extension, exonuclease-based sequencing, ligase detection reaction, ligase chain reaction, pyrosequencing, fluorescence-based sequencing-by-synthesis, fluorescence-based sequencing-by-ligation, nanopore based sequencing, ion-based sequencing-by-synthesis, and ion-based sequencing-by-ligation. 
     
     
         97 . The method of  claim 91 , wherein the solid support further comprises:
 a plurality of second oligonucleotide primers immobilized on the solid support, each second oligonucleotide primer comprising a nucleotide sequence that is the same as the second solid support primer-specific portion of the chimeric single stranded nucleic acid constructs of the collection.   
     
     
         98 . The method of  claim 97 , wherein two or more second oligonucleotide primers immobilized on the solid support hybridize to two or more tandem linear sequences of a primary extension product, said method further comprising:
 extending the two or more hybridized second oligonucleotide primers using a polymerase to form a plurality of immobilized secondary extension products, each secondary extension product comprising a nucleotide sequence that is complementary to a portion of the primary extension product; and   removing the primary extension products from the solid support, whereby said sequencing involves sequencing the plurality of immobilized secondary extension products.   
     
     
         99 . A system comprising:
 a collection of different chimeric nucleic acid constructs, each construct comprising:
 one or more single-stranded linker-appended nucleic acid molecules comprising a target nucleic acid molecule from a host organism, wherein said single-stranded linker-appended nucleic acid molecule has 3′ and 5′ ends; wherein the 3′ and 5′ ends of said single-stranded linker-appended nucleic acid molecule have appended 3′ linker sequences and appended 5′ linker sequences on the 3′ and 5′ ends, respectively, which are exogenous to the host organism; and wherein said 3′ and 5′ linker sequences collectively comprise (i) one or more patient identifier sequences, (ii) a first solid support primer-specific portion, (iii) a second solid support primer-specific portion, (iv) one or more sequencing primer binding sequences, or any combination of (i), (ii), (iii), and (iv); 
 one or more first oligonucleotide probes hybridized to the single-stranded linker-appended nucleic acid molecule(s), wherein the one or more first oligonucleotide probes comprises a nucleotide sequence that is exogenous to the host organism, said nucleotide sequence comprising a portion complementary to the 3′ end of the linker-appended nucleic acid molecule(s), and a portion complementary to the 5′ end of the linker-appended nucleic acid molecule(s), wherein there is a ligation competent junction between the 3′ end of the linker-appended nucleic acid molecule(s) and the 5′ end of the linker-appended nucleic acid molecule(s). 
   
     
     
         100 . A system comprising:
 a collection of different chimeric nucleic acid constructs, each construct comprising:
 one or more single-stranded linker-appended nucleic acid molecules comprising a target nucleic acid molecule from a host organism, wherein said single-stranded linker-appended nucleic acid molecule has 3′ and 5′ ends; wherein the 3′ and 5′ ends of said single-stranded linker-appended nucleic acid molecule have appended 3′ linker sequences and appended 5′ linker sequences on the 3′ and 5′ ends, respectively, which are exogenous to the host organism; and wherein said 3′ and 5′ linker sequences collectively comprise (i) one or more patient identifier sequences, (ii) one or more sequencing primer binding sequences, or both; 
 one or more first oligonucleotide probes hybridized to the single-stranded linker-appended nucleic acid molecule(s), wherein the one or more first oligonucleotide probes comprise a nucleotide sequence that is exogenous to the host organism, said nucleotide sequence comprising a portion complementary to the 3′ sequence of the linker-appended nucleic acid molecule(s), a portion complementary to the 5′ sequence of the linker-appended nucleic acid molecule(s), and a further portion comprising a first solid support primer-specific portion and optionally a second solid support primer-specific portion; 
 one or more second oligonucleotide probes hybridized to the further portion of the first oligonucleotide probe, wherein the second oligonucleotide probe comprises a sequence complementary to the further portion of the first oligonucleotide probe, wherein the 3′ and 5′ ends of the second oligonucleotide probe are adjacent to the 5′ and 3′ ends of the single-stranded linker-appended nucleic acid molecule with a junction suitable for ligation between them.