Method for quantitating nucleic acid library

A method for quantitating a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments, the method comprising:

• contacting a plurality of extension primers and a plurality of nucleic acid binding dye molecules with the plurality of nucleic acid molecules;
• producing an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase;
• subsequent to producing the extension product of each of the plurality of target nucleic acid fragments, measuring a signal produced by the plurality of nucleic acid binding dye molecules;
• and
• estimating an average size of the plurality of target nucleic acid fragments based on a number of the plurality of target nucleic acid fragments and the signal produced by the plurality of nucleic acid binding dye molecules.

A method of sequencing a nucleic acid sample, comprising:

• generating a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments using a nucleic acid sample;
• contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules, wherein each of the plurality of detectably-labeled probes comprises a first labeled nucleic acid domain comprising a first label;
• producing an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase;
• hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the target nucleic acid fragments during extending the respective one of the plurality of extension primers with the polymerase;
• detecting a first signal produced as a result of hydrolyzing the plurality of detectably-labeled probes;
• calculating a number of the plurality of target nucleic acid fragments based on signals detected upon a single cycle of extension reactions;
• diluting the plurality of target nucleic acid fragments to a predetermined concentration;
• and
• sequencing at least one portion of the plurality of target nucleic acid fragments.

A method for quantitating a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments and a plurality of adapter molecules, the method comprising:

• contacting a plurality of detectably-labeled probes, a plurality of extension primers, and a plurality of hybridizing oligonucleotides with the plurality of nucleic acid molecules, wherein each of the a plurality of detectably-labeled probes comprises a first labeled nucleic acid domain comprising a first label, and each of the a plurality of hybridizing oligonucleotides comprises a blocker nucleic acid domain;
• producing an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase;
• hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the target nucleic acid fragments during extending the respective one of the plurality of extension primers with the polymerase;
• and
• detecting a first signal produced as a result of hydrolyzing the plurality of detectably-labeled probes.

A mixture, comprising:

• a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments;
• a plurality of detectably-labeled probes;
• and
• a plurality of hybridized adapter molecules;
• wherein each of the plurality of detectably-labeled probes comprises a first labeled nucleic acid domain comprising a first label;
• each of the plurality of target nucleic acid fragments comprises a first adapter sequence, a target insert sequence, and a second adapter sequence;
• the first adapter sequence is linked to the second adapter sequence through the target insert sequence;
• at least a portion of each of the plurality of detectably-labeled probes is complementary to at least a portion of the first adapter sequence;
• wherein each of the plurality of hybridized adapter molecules comprises:
• the first adapter sequence;
• the second adapter sequence;
• and
• a partially hydrolyzed hybridizing oligonucleotide, at least a portion of which is hybridized to a domain of a respective one of a plurality of adapter molecules;
• wherein the first adapter sequence is directly linked to the second adapter sequence;
• and
• the domain comprises a portion of the first adapter sequence and a portion of the second adapter sequence directly adjacent to each other.

A kit for quantitating a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments, wherein each of the plurality of target nucleic acid fragments comprises a first adapter sequence, a target insert sequence, and a second adapter sequence;

• the first adapter sequence is linked to the second adapter sequence through the target insert sequence;
• wherein the kit comprises:
• a plurality of detectably-labeled probes;
• a plurality of extension primers;
• and
• instructions directing a user to (1) producing an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase;
• and (2) calculating a number of the plurality of target nucleic acid fragments based on signals detected upon a single cycle of extension reactions;
• wherein each of the plurality of detectably-labeled probes comprises a first labeled nucleic acid domain comprising a first label;
• at least a portion of each of plurality of the detectably-labeled probes is complementary to at least a portion of the first adapter sequence;
• and
• at least a portion of each of the plurality of extension primers is complementary to at least a portion of the second adapter sequence.