Reagents, kits and methods for molecular barcoding

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 using a multimeric barcode molecule, wherein the nucleic acid sample comprises a target nucleic acid, wherein the multimeric barcode molecule comprises first, second, and third barcode regions, and wherein preparing the nucleic acid sample for sequencing comprises producing first, second, and third barcoded target nucleic acid molecules, wherein the first barcoded target nucleic acid molecule comprises a first sub-sequence of the target nucleic acid and the nucleic acid sequence or complement thereof of the first barcode region, wherein the second barcoded target nucleic acid molecule comprises a second sub-sequence of the target nucleic acid and the nucleic acid sequence or complement thereof of the second barcode region, and wherein the third barcoded target nucleic acid molecule comprises a third sub-sequence of the target nucleic acid and the nucleic acid sequence or complement thereof of the third barcode region;
• b) sequencing the sample, wherein the step of sequencing comprises sequencing at least part of the first barcoded target nucleic acid molecule to generate a first sequence read, sequencing at least part of the second barcoded target nucleic acid molecule to generate a second sequence read, and sequencing at least part of the third barcoded target nucleic acid molecule to generate a third sequence read;
• and
• c) processing the sequence data obtained by step (b) to generate a synthetic long read from the target nucleic acid, wherein the processing comprises:
• 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 (i) to determine a group of sequences from the target nucleic acid that were labelled with barcode regions from the same multimeric barcode molecule;
• and wherein the synthetic long read comprises at least one nucleotide corresponding to the target nucleic acid from each of the first, second, and third sequence reads.