Digital measurements from targeted sequencing

Abstract

Disclosed herein are methods, compositions and kits for quantitating one or more specific nucleic acids within a plurality of nucleic acids. In some embodiments, a sequencing library is constructed from enriched probe extension products specific for the specific nucleic acids and sequenced. In some embodiments, the resulting reads are used for removing duplicate reads. In some embodiments, counting of verified probes is used to quantitate or determine the number of specific nucleic acid molecules in the starting nucleic acid sample.

What is claimed is: 
     
         1 . A method for quantitating a plurality of specific nucleic acid molecules in a composition comprising: a. generating a plurality of probe extension products, wherein each probe extension product comprises a probe sequence that is complementary to a probe target region within a specific nucleic acid molecule; b. sequencing the plurality of probe extension products to generate a sequence for each of the plurality of probe extension products; c. aligning the sequence of each of the plurality of probe extension products to a reference sequence database, wherein the reference sequence database comprises probe sequences; and d. determining the number of alignments for the sequence of each probe extension product with a sequence in the reference sequence database, wherein the number of alignments indicates the quantity of each of the specific nucleic acid molecule that the probe of the probe extension product is complementary to. 
     
     
         2 . A method for quantitating a plurality of specific nucleic acid molecules comprising: a. generating a plurality of probe extension products, wherein each probe extension product comprises (i) a first adapter, and (ii) a probe sequence complementary to a probe target region within a specific nucleic acid molecule; b. sequencing the plurality of probe extension products to generate sequence data comprising a sequence for each of the plurality of probe extension products; c. identifying the presence of the probe sequence of each probe extension product within the sequence data; and d. determining the number of each of the probe sequences within the plurality of probe extension products, wherein the number of each of the probe sequences indicates the quantity of each of the plurality of specific nucleic acid molecules to which each of the probes sequences is complementary to. 
     
     
         3 . A method for quantitating a plurality of specific nucleic acid molecules within a plurality of nucleic acid molecules comprising: a. appending a first adaptor sequence to a 5′ end to each of a plurality of nucleic acid molecules; b. hybridizing a plurality of probes to the plurality of specific nucleic acid molecules, wherein each probe is complementary to a probe target region within a specific nucleic acid molecule; c. extending each probe into the appended first adaptor sequence to generate a plurality of probe extension products having the first adaptor sequence and a second adaptor sequence to produce a plurality of probe extension products; d. sequencing the plurality of probe extension products to generate sequence data for each of the plurality of probe extension products; e. aligning the sequence for each of the plurality of probe extension products to a pre-determined sequence within a reference copy of a probe database, wherein said pre-determined sequence is specific to each probe; and f. determining the number of each probe sequence aligned to its pre-determined sequence, wherein the number indicates the quantity of the specific nucleic acids molecule to which the probe is complementary to.