Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
AssigneeTecan Genomics, Inc.
InventorsBenjamin G. Schroeder, Doug Amorese
Life of this patent
Estimating the term…
Abstract
Provided herein are methods, compositions and kits for the generation of bisulfite-converted next generation sequencing (NGS) libraries. The methods, compositions and kits provided herein can be useful, for example, for the production of libraries from genomic DNA that allow for determination of the methylation status across the genome, i.e. the methylome. The methods, compositions and kits provided herein can also be utilized to query methylation status at a particular genomic locus or loci. Moreover, the methods provided herein can be employed for high-throughput sequencing of bisulfite-converted DNA while maintaining the directional (strandedness) information of the original nucleic acid sample.
Claims20
A method for generating a nucleic acid library, the method comprising:
- ligating first strands of oligonucleotide adapters of similar construct to 5′ ends of double-stranded DNA fragments in a sample, wherein the first strand of each adapter comprises one or more guanine;
- extending 3′ ends of the double-stranded DNA fragments with a DNA polymerase in the presence of a dCTP analog resistant to bisulfite treatment, thereby creating extension products comprising one or more cytosines resistant to bisulfite treatment and complementary to the guanines;
- treating the extension products with bisulfite, thereby converting unmodified cytosines bases in the DNA fragments and adapter sequences to uracil;
- and
- amplifying the bisulfite-treated extension products.
Dependencies are inferred from the claim text.