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US 10,648,103 B2Granted
Universal blocking oligo system and improved hybridization capture methods for multiplexed capture reactions
AssigneeInvitae Corporation
InventorEric Olivares
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Abstract
Provided herein, in some embodiments, are novel compositions and improved methods for nucleic acid manipulation and analysis that can be applied to multiplex nucleic acid sequencing. In certain embodiments, the novel compositions and methods presented herein are more cost effective, more conducive to automation, and faster than traditional approaches. Also provided herein are novel blocking nucleic acids.
Claims19
A composition for use in massive parallel nucleic acid sequencing comprising:
- a) a library of nucleic acids comprising a plurality of library inserts wherein each nucleic acid of the library comprises (i) at least one library insert obtained from one of four or more samples, (ii) a first non-native nucleic acid, and (iii) a second non-native nucleic acid, wherein the first non-native nucleic acid and the second non-native nucleic acid are located on opposing sides of the at least one library insert, and the first non-native nucleic acid comprises a first distinguishable nucleic acid barcode and the second non-native nucleic acid comprises a second distinguishable nucleic acid barcode, wherein the first and second distinguishable nucleic acid barcodes are unique to the one of the four or more samples;
- and
- b) four universal blocking (U-block) nucleic acids, wherein (i) a first U-block nucleic acid and a second U-block nucleic acid are configured to hybridize to the first non-native nucleic acid on opposing sides of the first distinguishable nucleic acid barcode, (ii) a third U-block nucleic acid and a fourth U-block nucleic acid are configured to hybridize to the second non-native nucleic acid on opposing sides of the second distinguishable nucleic acid barcode, and (iii) each of the U-block nucleic acids does not substantially hybridize to a portion of the first or second distinguishable nucleic acid barcode.
A method of analyzing a nucleic acid library comprising:
- a) obtaining a library of nucleic acids comprising a plurality of library inserts wherein each nucleic acid of the library comprises (i) at least one library insert obtained from one of four or more samples, (ii) a first non-native nucleic acid, and (iii) a second non-native nucleic acid, wherein the first non-native nucleic acid and the second non-native nucleic acid are located on opposing sides of the at least one library insert, and the first non-native nucleic acid comprises a first distinguishable nucleic acid barcode and the second non-native nucleic acid comprises a second distinguishable nucleic acid barcode, wherein the first and second distinguishable nucleic acid barcodes are unique to the one of the four or more samples;
- b) contacting the library of nucleic acids with four universal blocking (U-block) nucleic acids, wherein (i) a first U-block nucleic acid and a second U-block nucleic acid are configured to hybridize to the first non-native nucleic acid on opposing sides of the first distinguishable nucleic acid barcode, (ii) a third U-block nucleic acid and a fourth U-block nucleic acid are configured to hybridize to the second non-native nucleic acid on opposing sides of the second distinguishable nucleic acid barcode, and (iii) each of the U-block nucleic acids does not substantially hybridize to a portion of the first or second distinguishable nucleic acid barcode;
- c) contacting the library of nucleic acids with one or more capture nucleic acids, each comprising a first member of a binding pair, wherein the one or more capture nucleic acids are configured to specifically hybridize to a subset of the nucleic acids of the library;
- d) capturing the capture nucleic acids, thereby providing captured nucleic acids comprising the subset of nucleic acids of the library;
- e) contacting the captured nucleic acids with a set of primers under amplification condition, thereby providing amplicons;
- and
- f) analyzing the amplicons.
4 more elements
Dependencies are inferred from the claim text.