Methods and reagents for molecular barcoding

A method of preparing a nucleic acid sample for sequencing, wherein the nucleic acid sample comprises a formalin-fixed paraffin-embedded (FFPE) sample, and wherein the method comprises the steps of:

• (a) contacting the nucleic acid sample with a library of multimeric barcoding reagents, wherein the library comprises first and second multimeric barcoding reagents, wherein each multimeric barcoding reagent comprises first and second different barcode regions linked together, wherein each barcode region comprises a nucleic acid sequence, and wherein the first and second barcode regions of the first multimeric barcoding reagent are different to the first and second barcode regions of the second multimeric barcoding reagent, and
• (b) appending barcode sequences from the first multimeric barcoding reagent to each of the nucleic acid sequences of first and second fragments of a first target nucleic acid molecule to produce first and second different barcoded target nucleic acid molecules, 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 from the second multimeric barcoding reagent to each of the nucleic acid sequences of first and second fragments of a second target nucleic acid molecule to produce third and fourth different barcoded target nucleic acid molecules, wherein the third barcoded target nucleic acid molecule comprises the nucleic acid sequence of the first barcode region of the second multimeric barcoding reagent and the fourth barcoded target nucleic acid molecule comprises the nucleic acid sequence of the second barcode region of the second multimeric barcoding reagent,
• wherein prior to and/or during step (a) and/or prior to and/or during step (b), the method further comprises the step of removing or depleting all or a fraction of the paraffin from the nucleic acid sample.