The invention provides a method for efficient DNA library construction and targeted genetic analyses of the libraries.

Methods of detecting target polynucleotide sequences may include introducing one or more nucleic acid analytes to a first reaction mixture comprising a single-stranded probe oligonucleotide A.sub.0, a pyrophosphorolysing enzyme, and a ligase. The analyte may anneal to the single-stranded probe oligonucleotide A.sub.0 to create a first intermediate product which is at least partially double-stranded, where the 3′ end of A.sub.0 forms a double-stranded complex with the analyte and where A.sub.0 is pyrophosphorylsed in the 3′-5′ direction from the 3′ end to create at least a partially digested strand A.sub.1. A.sub.1 may undergo ligation to form oligonucleotide A.sub.2. The methods may also include detecting a signal derived from the formed oligonucleotides, and inferring therefrom the presence or absence of the target polynucleotide sequence in the analyte.

Methods of detecting target polynucleotide sequences may include introducing one or more nucleic acid analytes to a first reaction mixture comprising a single-stranded probe oligonucleotide A.sub.0, a pyrophosphorolysing enzyme, and a ligase. The analyte may anneal to the single-stranded probe oligonucleotide A.sub.0 to create a first intermediate product which is at least partially double-stranded, where the 3′ end of A.sub.0 forms a double-stranded complex with the analyte and where A.sub.0 is pyrophosphorylsed in the 3′-5′ direction from the 3′ end to create at least a partially digested strand A.sub.1. A.sub.1 may undergo ligation to form oligonucleotide A.sub.2. The methods may also include detecting a signal derived from the formed oligonucleotides, and inferring therefrom the presence or absence of the target polynucleotide sequence in the analyte.

Provided herein are direct-to-library methods, systems, and compositions.

Provided herein are direct-to-library methods, systems, and compositions.

The present invention relates to systems, devices and methods for diagnosing cancer. In various embodiments, the present invention provides a method for performing sequencing of 5′-htRNA in an RNA sample including obtaining a DNA library of 5′-htRNA and sequencing the DNA library of 5′-htRNAs in the RNA sample. The invention also teaches a method for quantifying 5′-htRNA half molecules in an RNA sample including (a) treating an RNA sample containing 5′-htRNAs half molecules having a 3′ cyclic phosphate with a T4 polynucleotide kinase to form 3′-dephosphrylated 5′-tRNA halves, (b) 3′-AD ligating the 3′-dephosphrylated 5′-tRNA halves to form ligated RNA products, and (c) quantifying the ligated RNA products by RT-qPCR using a plurality of target primers and probes configured to simultaneously quantify at least two cP-containing 5′-tRNA half species. The treating of step (a) is simultaneously performed as one step in a single tube with the 3′-AD ligating of step (b).

The present invention relates to systems, devices and methods for diagnosing cancer. In various embodiments, the present invention provides a method for performing sequencing of 5′-htRNA in an RNA sample including obtaining a DNA library of 5′-htRNA and sequencing the DNA library of 5′-htRNAs in the RNA sample. The invention also teaches a method for quantifying 5′-htRNA half molecules in an RNA sample including (a) treating an RNA sample containing 5′-htRNAs half molecules having a 3′ cyclic phosphate with a T4 polynucleotide kinase to form 3′-dephosphrylated 5′-tRNA halves, (b) 3′-AD ligating the 3′-dephosphrylated 5′-tRNA halves to form ligated RNA products, and (c) quantifying the ligated RNA products by RT-qPCR using a plurality of target primers and probes configured to simultaneously quantify at least two cP-containing 5′-tRNA half species. The treating of step (a) is simultaneously performed as one step in a single tube with the 3′-AD ligating of step (b).

Methods for performing multiplex PCR-based enrichment of a target substrate are provided. Systems and methods for generating a sequencing library are also provided.

Methods for performing multiplex PCR-based enrichment of a target substrate are provided. Systems and methods for generating a sequencing library are also provided.

This disclosure provides systems and methods for sequencing nucleic acids using nucleotide analogues and translocation of tags from incorporated nucleotide analogues through a nanopore. In aspects, this disclosure is related to composition, method, and system for sequencing a nucleic acid using tag molecules and detection of translocation through a nanopore of tags released from incorporation of the molecule.