Reagents, kits and methods for molecular barcoding

Abstract

Multimeric barcoding reagents for labelling a target nucleic acid comprise: first and second barcode molecules linked together, wherein each of the barcode molecules comprises a nucleic acid sequence comprising a barcode region; and first and second barcoded oligonucleotides. The multimeric barcoding reagents enable spatial sequencing. A single multimeric barcoding reagent can be used to label sub-sequences of an intact nucleic acid molecule or co-localised fragments of a nucleic acid molecule. The labelled sub-sequences can be sequenced and the sequencing data processed to determine the sequence of sub-sequences from a single intact nucleic acid molecule or from co-localised fragments of a nucleic acid molecule. Corresponding libraries, kits, methods and uses are provided.

1 .- 46 . (canceled) 
     
     
         47 . A library of multimeric barcoding reagents comprising at least 10 multimeric barcoding reagents for labelling a target nucleic acid for sequencing, wherein each multimeric barcoding reagent comprises:
 a. first and second barcode molecules comprised within a nucleic acid molecule, wherein each of the barcode molecules comprises a nucleic acid sequence comprising a barcode region; and   b. first and second barcoded oligonucleotides, wherein the first barcoded oligonucleotide comprises, optionally in the 5′ to 3′ direction, a barcode region complementary and annealed to the barcode region of the first barcode molecule and a target region capable of annealing or ligating to a first sub-sequence of the target nucleic acid, and wherein the second barcoded oligonucleotide comprises, optionally in the 5′ to 3′ direction, a barcode region complementary and annealed to the barcode region of the second barcode molecule and a target region capable of annealing or ligating to a second sub-sequence of the target nucleic acid;   and wherein the barcode regions of the first and second barcode molecules of each multimeric barcoding reagent are different to the barcode regions of at least 9 other multimeric barcoding reagents in the library.   
     
     
         48 . The library of multimeric barcoding reagents of  claim 47 , wherein the barcode regions uniquely identify each of the barcode molecules. 
     
     
         49 . The library of multimeric barcoding reagents of  claim 47 , wherein:
 a. each of the barcode molecules comprises a nucleic acid sequence comprising in the 5′ to 3′ direction an adapter region and a barcode region;   b. each first barcoded oligonucleotide comprises in the 5′ to 3′ direction a barcode region annealed to the barcode region of the first barcode molecule, an adapter region annealed to the adapter region of the first barcode molecule and a target region capable of annealing to a first sub-sequence of the target nucleic acid; and   c. each second barcoded oligonucleotide comprises in the 5′ to 3′ direction a barcode region annealed to the barcode region of the second barcode molecule, an adapter region annealed to the adapter region of the second barcode molecule and a target region capable of annealing to a second sub-sequence of the target nucleic acid.   
     
     
         50 . The library of multimeric barcoding reagents of  claim 47 , wherein each multimeric barcoding reagent comprises:
 a. at least 5, at least 10, at least 20, at least 25, at least 50, at least 75 or at least 100 barcode molecules linked together, wherein each barcode molecule is as defined in  claim 47 ; and   b. a barcoded oligonucleotide annealed to each barcode molecule, wherein each barcoded oligonucleotide is as defined in  claim 47 .   
     
     
         51 . The library of multimeric barcoding reagents of  claim 47 , wherein each target region comprises at least 5, at least 10, at least 15, at least 20, at least 25 or at least 50 nucleotides. 
     
     
         52 . The library of multimeric barcoding reagents of  claim 47 , wherein the library comprises at least 20, at least 25, at least 50, at least 75, at least 100, at least 250, at least 500, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7  or at least 10 8  multimeric barcoding reagents as defined in  claim 47 . 
     
     
         53 . A method of preparing a nucleic acid sample for sequencing, wherein the method comprises the steps of:
 a. contacting the nucleic acid sample with a library of multimeric barcoding reagents as defined in  claim 47 ;   b. annealing the target region of the first barcoded oligonucleotide to a first sub-sequence of a target nucleic acid, and annealing the target region of the second barcoded oligonucleotide to a second sub-sequence of the target nucleic acid; and   c. extending the first and second barcoded oligonucleotides to produce first and second different barcoded target nucleic acid molecules, wherein each of the barcoded target nucleic acid molecules comprises at least one nucleotide synthesized from the target nucleic acid as a template.   
     
     
         54 . A kit for labelling a target nucleic acid for sequencing, wherein the kit comprises:
 a. a library of multimeric barcoding reagents comprising at least 10 multimeric barcoding reagents, wherein each multimeric barcoding reagent comprises:
 i. first and second barcode molecules comprised within a nucleic acid molecule, wherein each of the barcode molecules comprises a nucleic acid sequence comprising, optionally in the 5′ to 3′ direction, an adapter region and a barcode region, and 
 ii. first and second barcode oligonucleotides, wherein the first barcode oligonucleotide comprises a barcode region complementary and annealed to the barcode region of the first barcode molecule, and wherein the second barcode oligonucleotide comprises a barcode region complementary and annealed to the barcode region of the second barcode molecule; 
 and wherein the barcode regions of the first and second barcode molecules of each multimeric barcoding reagent are different to the barcode regions of at least 9 other multimeric barcoding reagents in the library, and 
   b. first and second adapter oligonucleotides for each of the multimeric barcoding reagents, wherein the first adapter oligonucleotide comprises, optionally in the 5′ to 3′ direction, an adaptor region capable of annealing to the adapter region of the first barcode molecule and a target region capable of annealing or ligating to a first sub-sequence of the target nucleic acid, and wherein the second adapter oligonucleotide comprises, optionally in the 5′ to 3′ direction, an adaptor region capable of annealing to the adapter region of the second barcode molecule and a target region capable of annealing or ligating to a second sub-sequence of the target nucleic acid.   
     
     
         55 . The kit of  claim 54 , wherein the barcode regions uniquely identify each of the barcode molecules. 
     
     
         56 . The kit of  claim 54 , wherein the adapter regions of the adapter oligonucleotides comprise an identical constant region. 
     
     
         57 . The kit of  claim 54 , wherein each multimeric barcoding reagent comprises at least 5, at least 10, at least 20, at least 25, at least 50, at least 75 or at least 100 barcode molecules linked together, wherein each barcode molecule is as defined in  claim 54 ; and wherein the kit comprises an adaptor oligonucleotide capable of annealing to each barcode molecule, wherein each adaptor oligonucleotide is as defined in  claim 54 . 
     
     
         58 . The kit of  claim 54 , wherein each target region comprises at least 5, at least 10, at least 15, at least 20, at least 25 or at least 50 nucleotides. 
     
     
         59 . The kit of  claim 54 , wherein the kit comprises a library of at least 20, at least 25, at least 50, at least 75, at least 100, at least 250, at least 500, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108 or at least 109 multimeric barcoding reagents, wherein each multimeric barcoding reagent is as defined in  claim 54 , and adaptor oligonucleotides for each of the multimeric barcoding reagents, wherein each adaptor oligonucleotide is as defined in  claim 54 . 
     
     
         60 . A method of preparing a nucleic acid sample for sequencing, wherein the method comprises the steps of:
 a. contacting the nucleic acid sample with first and second adapter oligonucleotides as defined in  claim 54 ;   b. annealing the target region of the first adapter oligonucleotide to a first sub-sequence of a target nucleic acid, and annealing the target region of the second adapter oligonucleotide to a second sub-sequence of the target oligonucleotide;   c. contacting the nucleic acid sample with a library of multimeric barcoding reagents as defined in  claim 54 ;   d. annealing the adapter region of the first adapter oligonucleotide to the adapter region of the first barcode molecule, and annealing the adapter region of the second adapter oligonucleotide to the adapter region of the second barcode molecule; and   e. ligating the 3′ end of the first barcode oligonucleotide to the 5′ end of the first adapter oligonucleotide to produce a first barcoded oligonucleotide and ligating the free 3′ end of the second barcode oligonucleotide to the 5′ end of the second adapter oligonucleotide to produce a second barcoded oligonucleotide;
 and wherein the first and second adapter oligonucleotides or the first and second barcoded oligonucleotides are extended to produce first and second different barcoded target nucleic acid molecules each of which comprises at least one nucleotide synthesized from the target nucleic acid as a template. 
   
     
     
         61 . A kit for labelling a target nucleic acid for sequencing, wherein the kit comprises:
 a. a library of multimeric barcode molecules comprising at least 10 multimeric barcode molecules, each multimeric barcode molecule comprising first and second barcode molecules comprised within a nucleic acid molecule, wherein each of the barcode molecules comprises a nucleic acid sequence comprising, optionally in the 5′ to 3′ direction, an adapter region, a barcode region, and a priming region, and wherein the barcode regions of the first and second barcode molecules of each multimeric barcode molecule are different to the barcode regions of at least 9 other multimeric barcode molecules in the library;   b. first and second extension primers for each of the multimeric barcode molecules, wherein the first extension primer comprises a sequence capable of annealing to the priming region of the first barcode molecule, and wherein the second extension primer comprises a sequence capable of annealing to the priming region of the second barcode molecule; and   c. first and second adapter oligonucleotides for each of the multimeric barcode molecules, wherein the first adapter oligonucleotide comprises, optionally in the 5′ to 3′ direction, an adaptor region capable of annealing to the adapter region of the first barcode molecule and a target region capable of annealing or ligating to a first sub-sequence of the target nucleic acid, and wherein the second adapter oligonucleotide comprises, optionally in the 5′ to 3′ direction, an adaptor region capable of annealing to the adapter region of the second barcode molecule and a target region capable of annealing or ligating to a second sub-sequence of the target nucleic acid.   
     
     
         62 . The kit of  claim 61 , wherein the first and second extension primers are identical in sequence. 
     
     
         63 . A kit for labelling a target nucleic acid for sequencing, wherein the kit comprises:
 a. a library of multimeric barcode molecules comprising at least 10 multimeric barcode molecules, each multimeric barcode molecule comprising first and second barcode molecules comprised within a nucleic acid molecule, wherein each of the barcode molecules comprises a nucleic acid sequence comprising, optionally in the 5′ to 3′ direction, an adapter region and a barcode region, and wherein the barcode regions of the first and second barcode molecules of each multimeric barcode molecule are different to the barcode regions of at least 9 other multimeric barcode molecules in the library; and   b. first and second adapter oligonucleotides for each of the multimeric barcode molecules, wherein the first adapter oligonucleotide comprises, optionally in the 5′ to 3′ direction, an adaptor region capable of annealing to the adapter region of the first barcode molecule and a target region capable of annealing or ligating to a first sub-sequence of the target nucleic acid, and wherein the second adapter oligonucleotide comprises, optionally in the 5′ to 3′ direction, an adaptor region capable of annealing to the adapter region of the second barcode molecule and a target region capable of annealing or ligating to a second sub-sequence of the target nucleic acid.   
     
     
         64 . The kit of  claim 61 , wherein the barcode regions uniquely identify each of the barcode molecules. 
     
     
         65 . The kit of  claim 61 , wherein the adapter regions of the adapter oligonucleotides comprise an identical constant region. 
     
     
         66 . The kit of  claim 61 , wherein each multimeric barcode molecule comprises at least 5, at least 10, at least 20, at least 25, at least 50, at least 75 or at least 100 barcode molecules linked together, wherein each barcode molecule is as defined in  claim 61 ; and wherein the kit comprises an adaptor oligonucleotide capable of annealing to each barcode molecule, wherein each adaptor oligonucleotide is as defined in  claim 61 . 
     
     
         67 . The kit of  claim 61 , wherein each target region comprises at least 5, at least 10, at least 15, at least 20, at least 25 or at least 50 nucleotides. 
     
     
         68 . The kit of  claim 61 , wherein the kit comprises a library of at least 20, at least 25, at least 50, at least 75, at least 100, at least 250, at least 500, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108 or at least 109 multimeric barcode molecules, wherein each multimeric barcode molecule is as defined in  claim 61 , and adaptor oligonucleotides for each of the multimeric barcode molecules, wherein each adaptor oligonucleotide is as defined in  claim 61 . 
     
     
         69 . A method of preparing a nucleic acid sample for sequencing, wherein the method comprises the steps of:
 a. contacting the nucleic acid sample with first and second adapter oligonucleotides as defined in  claim 61 ;   b. annealing the target region of the first adapter oligonucleotide to a first sub-sequence of a target nucleic acid, and annealing the target region of the second adapter oligonucleotide to a second sub-sequence of the target oligonucleotide;   c. contacting the nucleic acid sample with a library of multimeric barcode molecules as defined in  claim 61  and first and second extension primers as defined in  claim 61 ;   d. annealing the adapter region of the first adapter oligonucleotide to the adapter region of the first barcode molecule, and annealing the adapter region of the second adapter oligonucleotide to the adapter region of the second barcode molecule;   e. extending the first extension primer using the barcode region of the first barcode molecule as a template to produce a first barcode oligonucleotide, and extending the second extension primer using the barcode region of the second barcode molecule as a template to produce a second barcode oligonucleotide, wherein the first barcode oligonucleotide comprises a sequence complementary to the barcode region of the first barcode molecule and the second barcode oligonucleotide comprises a sequence complementary to the barcode region of the second barcode molecule; and   f. ligating the 3′ end of the first barcode oligonucleotide to the 5′ end of the first adapter oligonucleotide to produce a first barcoded oligonucleotide and ligating the 3′ end of the second barcode oligonucleotide to the 5′ end of the second adapter oligonucleotide to produce a second barcoded oligonucleotide;
 and wherein the first and second adapter oligonucleotides, or the first and second barcoded oligonucleotides, are extended to produce first and second different barcoded target nucleic acid molecules each of which comprises at least one nucleotide synthesized from the target nucleic acid as a template. 
   
     
     
         70 . A method of sequencing a sample prepared according the method of  claim 53  comprising the steps of:
 a. isolating the barcoded target nucleic acid molecules, and 
 b. producing a sequence read from each barcoded target nucleic acid molecule that comprises the barcode region, the target region and at least one additional nucleotide from the target nucleic acid. 
 
     
     
         71 . A method for processing sequencing data obtained by the method of  claim 70  comprising the steps of:
 a. identifying for each sequence read the sequence of the barcode region and the sequence from the target nucleic acid; and 
 b. using the information from step (a) to determine a group of sequences from the target nucleic acid that were labelled with barcode regions from the same multimeric barcoding reagent. 
 
     
     
         72 . A method of generating a synthetic long read from a target nucleic acid comprising the steps of:
 a. preparing a nucleic acid sample for sequencing according to the method of  claim 53 ;   b. sequencing the sample according to a method comprising the steps of
 i. isolating the barcoded target nucleic acid molecules, and 
 ii. producing a sequence read from each barcoded target nucleic acid molecule that comprises the barcode region, the target region and at least one additional nucleotide from the target nucleic acid; and 
   c. processing the sequence data obtained by step (b) according to a method comprising the steps of
 i. identifying for each sequence read the sequence of the barcode region and the sequence from the target nucleic acid, and 
 ii. using the information from step (c)(i) to determine a group of sequences from the target nucleic acid that were labelled with barcode regions from the same multimeric barcoding reagent; 
   wherein step (c) generates a synthetic long read comprising at least one nucleotide from each of the at least two sequence reads.   
     
     
         73 . A method of sequencing two or more co-localized target nucleic acids comprising the steps of:
 a. preparing a nucleic acid sample for sequencing according to the method of  claim 53 ;   b. sequencing the sample according to a method comprising the steps of
 i. isolating the barcoded target nucleic acid molecules, and 
 ii. producing a sequence read from each barcoded target nucleic acid molecule that comprises the barcode region, the target region and at least one additional nucleotide from the target nucleic acid; and 
   c. processing the sequence data obtained by step (b) according to a method comprising the steps of
 i. identifying for each sequence read the sequence of the barcode region and the sequence from a target nucleic acid of the co-localized target nucleic acids, and 
 ii. using the information from step (c)(i) to determine a group of sequences from the co-localized target nucleic acids that were labelled with barcode regions from the same multimeric barcoding reagent; 
   wherein step (c) identifies at least two sequence reads comprising nucleotides from at least two target nucleic acids co-localized in the sample.   
     
     
         74 . A method of sequencing target nucleic acids from an individual cell comprising the steps of:
 a. preparing a nucleic acid sample for sequencing according to the method of  claim 53 , wherein the multimeric barcoding reagents are introduced into the cell;   b. sequencing the sample according to a method comprising the steps of
 i. isolating the barcoded target nucleic acid molecules, and 
 ii. producing a sequence read from each barcoded target nucleic acid molecule that comprises the barcode region, the target region and at least one additional nucleotide from the target nucleic acid; and 
   c. processing the sequence data obtained by step (b) according to a method comprising the steps of
 i. identifying for each sequence read the sequence of the barcode region and the sequence from a target nucleic acid of the target nucleic acids from the cell, and 
 ii. using the information from step (c)(i) to determine a group of sequences from the target nucleic acids from the cell that were labelled with barcode regions from the same multimeric barcoding reagent; 
   wherein step (c) identifies at least two sequence reads comprising nucleotides from at least two target nucleic acids from the cell.   
     
     
         75 . Use of a library of multimeric barcoding reagents as defined in  claim 47  to:
 a. produce two or more sequence reads from a target nucleic acid, wherein two or more sequence reads are identified as derived from the same target nucleic acid and combined to produce a synthetic long read; 
 b. label a formalin-fixed paraffin-embedded nucleic acid sample, wherein the multimeric barcoding reagent or the components of the kit is/are introduced into the sample and used to label a set of two or more co-localized target nucleic acids for sequencing; 
 c. label target nucleic acids in an individual cell, wherein the multimeric barcoding reagent or the components of the kit is/are introduced into a cell and used to label a set of two or more target nucleic acids in the cell for sequencing; or 
 d. label target nucleic acids in a sample of human plasma or serum, wherein the multimeric barcoding reagent or the components of the kit is/are used to label a set of two or more target nucleic acids in the plasma or serum for sequencing.