Methods and compositions for targeted nucleic acid sequencing

Abstract

The present invention is directed to methods, compositions and systems for capturing and analyzing sequence information contained in targeted regions of a genome. Such targeted regions may include exomes, partial exomes, introns, combinations of exonic and intronic regions, genes, panels of genes, and any other subsets of a whole genome that may be of interest.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 62 / 072,164, filed Oct. 29, 2014, which is expressly incorporated herein by reference in its entirety for all purposes.BACKGROUND OF THE INVENTION[0002]The ability to sequence genomes accurately and rapidly is revolutionizing biology and medicine. The study of complex genomes, and in particular, the search for the genetic basis of disease in humans, involves genetic analysis on a massive scale. Such genetic analysis on a whole genome level is costly not only monetarily but also in time and labor. These costs increase with protocols involving analyses of separate individual DNA samples. Sequencing (and re-sequencing) of polymorphic areas in the genome that are linked to disease development will contribute greatly to the understanding of diseases, such as cancer, and therapeutic development and will help meet the pharmacogenomics challenge to identify the genes and funct...

AI Technical Summary

Benefits of technology

[0012]In further aspects, the present disclosure provides a method of obtaining sequence information from one or more targeted portions of a genomic sample. Such a method includes without limitation the steps of: (a) providing individual first nucleic acid fragment molecules of the genomic sample in discrete partitions; (b) fragmenting the individual first nucleic acid fragment molecules within the discrete partitions to create a plurality of second fragments from each of the individual first nucleic acid fragment molecules; (c) attaching a common barcode sequence to the plurality of the second fragments within a discrete partition, such that each of the plurality of second fragments are attributable to the discrete partition in which they are contained; (d) applying a library of probes directed to the one or more targeted portions of the genomic sample to the second fragments; (e) conducting a sequencing reaction to identify sequences of the plurality of second fragments that hybridized to the library of probes, thereby obtaining sequence information from the one or more targeted portions of the genomic sample. In further embodiments, the library of probes are attached to binding moieties, and before the conducting step (e), the second fragments are captured on a surface comprising capture moieties through a reaction between the binding moieties and the capture moieties. In still further embodiments and prior to the conducting step (e), the second fragments are amplified before or after the second fragments are captured on the surface. In yet further embodiments, the binding moieties comprise biotin and the capture moieties comprise streptavidin. In still further embodiments, the sequencing reaction is a short read, high accuracy sequencing reaction. In still further embodiments, the second fragments are amplified such that the resultant amplification products are capable of forming partial or complete hairpin structures.

[0013]In further aspects and in accordance with any of the above, the present disclosure provides methods for obtaining sequence information from one or more targeted portions of a genomic sample while retaining molecular context. Such methods include the steps of: (a) providing starting genomic material; (b) distributing individual nucleic acid molecules from the starting genomic material into discrete partitions such that each discrete partition contains a first individual nucleic acid molecule; (c) fragmenting the first individual nucleic acid molecules in the discrete partitions to form a plurality of fragments; (d) providing a population enriched for fragments that include at least a portion of the one or more selected portions of the genome; (e) obtaining sequence information from the population, thereby sequencing one or more targeted portions of the genomic sample while retaining molecular context. In further embodiments, prior to the obtaining step (e), the plurality of fragments are tagged with a barcode to associate each fragment with the discrete partition in which it was formed. In still further embodiments, the individual nucleic acid molecules in step (b) are distributed such that molecular context of each first individual nucleic acid molecule is maintained.

[0014]In some aspects, the present disclosure provides methods of obtaining sequence information from one or more targeted portions of a genomic sample. Such methods include without limitation steps of (a) providing individual nucleic acid molecules of the genomic sample; (b) fragmenting the individual nucleic acid molecules to form a plurality of fragments, where each of the fragments further includes a barcode, and where fragments from the same individual nucleic molecule have a common barcode, thereby associating each fragment with the individual nucleic acid molecule from which it is derived; (c) enriching the plurality of fragments for fragments containing the one or more targeted portions of the genomic sample; and (d) conducting a sequencing reaction to identify sequences of the enriched plurality of fragments, thereby obtaining sequence information from the one or more targeted portions of the genomic sample. In further embodiments, the enriching step including applying a library of probes directed to the one or more targeted portions of the genomic sample. In yet further embodiments, the library of probes are attached to binding moieties, and prior to the conducting step, the fragments are captured through a reaction between the binding moieties and the capture moieties. In exemplary embodiments, the reaction between the binding moieties and the capture moieties immobilizes the fragments on a surface.

Problems solved by technology

Such genetic analysis on a whole genome level is costly not only monetarily but also in time and labor.

These costs increase with protocols involving analyses of separate individual DNA samples.

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