Sets of experimentally validated gene specific primer pairs are provided. Embodiments of the sets include 10 or more gene specific primer pairs of forward and reverse primers. The forward and reverse primers of each primer pair include gene specific primers that are experimentally validated as suitable for use in a multiplex amplification assay. In some instances, each of the forward and reverse primers includes an anchor domain that includes a universal primer binding site. The sets find use in a variety of different applications, including high-throughput sequencing applications.

Provided are methods for preparing a library of target nucleic acid sequences, as well as compositions and uses therefor. Methods comprise contacting a nucleic acid sample with a plurality of adaptors capable of amplification of one or more target nucleic acid sequences under conditions wherein the target nucleic acid(s) undergo a first amplification; digesting the resulting first amplification products; repairing the digested target amplicons; and amplifying the repaired products in a second amplification, thereby producing a library of target nucleic acid sequence. Each of the plurality of adaptor compositions comprise a handle and a targeted nucleic acid sequence and optionally one or more tag sequences. Provided methods may be carried out in a single, addition only workflow reaction, allowing for rapid production of highly multiplexed targeted libraries, optionally including unique tag sequences. Resulting library compositions are useful for a variety of applications, including sequencing applications.

Provided are methods for preparing a library of target nucleic acid sequences, as well as compositions and uses therefor. Methods comprise contacting a nucleic acid sample with a plurality of adaptors capable of amplification of one or more target nucleic acid sequences under conditions wherein the target nucleic acid(s) undergo a first amplification; digesting the resulting first amplification products; repairing the digested target amplicons; and amplifying the repaired products in a second amplification, thereby producing a library of target nucleic acid sequence. Each of the plurality of adaptor compositions comprise a handle and a targeted nucleic acid sequence and optionally one or more tag sequences. Provided methods may be carried out in a single, addition only workflow reaction, allowing for rapid production of highly multiplexed targeted libraries, optionally including unique tag sequences. Resulting library compositions are useful for a variety of applications, including sequencing applications.

Provided herein are compositions and methods for determining the quality of nucleic acids in a sample that includes a first set of primers and a second set of primers. Additionally, provided herein are compositions and methods for determining nucleic the acid quality in a sample including sets of primers for amplification of repetitive nucleic acid sequences.

Provided herein are compositions and methods for determining the quality of nucleic acids in a sample that includes a first set of primers and a second set of primers. Additionally, provided herein are compositions and methods for determining nucleic the acid quality in a sample including sets of primers for amplification of repetitive nucleic acid sequences.

The present invention provides a method for amplifying a sequence adjacent to a specific sequence, comprising the steps of: annealing a first forward primer to the specific sequence to synthesize a complementary strand; sequentially polymerically adding a first deoxynucleotide and a second deoxynucleotide to a 3′-end of the complementary strand; annealing a first reverse primer to a binding site between the 3′-end of the complementary strand and a polydeoxynucleotide strand composed of the first deoxynucleotide to synthesize a double-stranded DNA; performing a PCR with the double-stranded DNA as a template by using a second forward primer complementary to the specific sequence and a first reverse primer; and further performing a PCR by using a third forward primer complementary to the specific sequence and a second reverse primer.

The present invention provides a method for amplifying a sequence adjacent to a specific sequence, comprising the steps of: annealing a first forward primer to the specific sequence to synthesize a complementary strand; sequentially polymerically adding a first deoxynucleotide and a second deoxynucleotide to a 3′-end of the complementary strand; annealing a first reverse primer to a binding site between the 3′-end of the complementary strand and a polydeoxynucleotide strand composed of the first deoxynucleotide to synthesize a double-stranded DNA; performing a PCR with the double-stranded DNA as a template by using a second forward primer complementary to the specific sequence and a first reverse primer; and further performing a PCR by using a third forward primer complementary to the specific sequence and a second reverse primer.

A method for generating sequence ready fragments of nucleotide sequences is described, the method making use of “bubble primers” which include first and third portions which hybridise to a target, and a second partly self-complementary portion which forms an unhybridised loop. The loop contains generic sequences allowing use of sequencing primers. The first portion may be degradable so as to generate an amplicon of sequence of interest flanked by the third portion and the generic sequences of the second portion. In preferred embodiments, the second portion, or the region between the second portion and the third portion, also comprises a tetrad of nucleotides A, C, G, T, allowing calibration of the sequencing reaction.

A method for generating sequence ready fragments of nucleotide sequences is described, the method making use of “bubble primers” which include first and third portions which hybridise to a target, and a second partly self-complementary portion which forms an unhybridised loop. The loop contains generic sequences allowing use of sequencing primers. The first portion may be degradable so as to generate an amplicon of sequence of interest flanked by the third portion and the generic sequences of the second portion. In preferred embodiments, the second portion, or the region between the second portion and the third portion, also comprises a tetrad of nucleotides A, C, G, T, allowing calibration of the sequencing reaction.

Provided herein is a method of detecting short nucleic acids, such as microRNAs, in a sample, such as urine, and a kit for use in detection of the short nucleic acids.