Reagents and methods for the analysis of linked nucleic acids
First Claim
1. A method of analysing a sample comprising a cell-free circulating microparticle, wherein the cell-free circulating microparticle contains at least two fragments of genomic DNA, and wherein the method comprises:
- (a) preparing the sample for sequencing comprising linking at least two of the at least two fragments of genomic DNA to produce a set of at least two linked fragments of genomic DNA; and
(b) sequencing each of the linked fragments in the set to produce at least two linked sequence reads.
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Abstract
Reagents and methods for the analysis of nucleic acids (e.g. genomic DNA) of circulating microparticles (i.e. microparticles originating from blood) are provided. The methods comprise linking at least two fragments of a target nucleic acid of a circulating microparticle to produce a set of at least two linked fragments of the target nucleic acid. In the methods, fragments of a target nucleic acid may be linked by techniques such as barcoding, partitioning, ligation and/or separate sequencing. The sequencing of a set of linked fragments provides a set of informatically linked sequence reads corresponding to the sequences of fragments from a single microparticle.
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Citations
27 Claims
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1. A method of analysing a sample comprising a cell-free circulating microparticle, wherein the cell-free circulating microparticle contains at least two fragments of genomic DNA, and wherein the method comprises:
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(a) preparing the sample for sequencing comprising linking at least two of the at least two fragments of genomic DNA to produce a set of at least two linked fragments of genomic DNA; and (b) sequencing each of the linked fragments in the set to produce at least two linked sequence reads. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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- 18. A method of preparing a sample for sequencing, wherein the sample comprises a cell-free circulating microparticle, wherein the cell-free circulating microparticle contains at least two fragments of genomic DNA, and wherein the method comprises appending the at least two fragments of genomic DNA of the cell-free circulating microparticle to a barcode sequence, or to different barcode sequences of a set of barcode sequences, to produce a set of linked fragments of genomic DNA.
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23. A method of preparing a sample for sequencing, wherein the sample comprises first and second cell-free circulating microparticles, and wherein each cell-free circulating microparticle contains at least two fragments of a target nucleic acid, and wherein the method comprises the steps of:
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(a) contacting the sample with a library comprising at least two multimeric barcoding reagents, wherein each multimeric barcoding reagent comprises first and second barcode regions linked together, wherein each barcode region comprises a nucleic acid sequence and wherein the first and second barcode regions of a first multimeric barcoding reagent are different to the first and second barcode regions of a second multimeric barcoding reagent of the library; and (b) appending barcode sequences to each of first and second fragments of the target nucleic acid of the first cell-free circulating microparticle to produce first and second barcoded target nucleic acid molecules for the first cell-free circulating microparticle, wherein the first barcoded target nucleic acid molecule comprises the nucleic acid sequence of the first barcode region of the first multimeric barcoding reagent and the second barcoded target nucleic acid molecule comprises the nucleic acid sequence of the second barcode region of the first multimeric barcoding reagent, and appending barcode sequences to each of first and second fragments of the target nucleic acid of the second cell-free circulating microparticle to produce first and second barcoded target nucleic acid molecules for the second cell-free circulating microparticle, wherein the first barcoded target nucleic acid molecule comprises the nucleic acid sequence of the first barcode region of the second multimeric barcoding reagent and the second barcoded target nucleic acid molecule comprises the nucleic acid sequence of the second barcode region of the second multimeric barcoding reagent. - View Dependent Claims (24, 25, 26, 27)
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Specification