Disclosed is a method for sequencing a nucleotide sequence of a target nucleic acid. The method comprises: providing a pool of amplicons, wherein the pool of amplicons is prepared by attaching a unique identifier to the target nucleic acid, and amplifying by PCR the target nucleic acid to which the unique identifier is attached; and sequencing the amplicons comprising the unique identifier and the target nucleic acid. In the method, a nucleotide sequence of the unique identifier comprises both a random nucleotide (N) and a predetermined nucleotide.

Disclosed is a method for sequencing a nucleotide sequence of a target nucleic acid. The method comprises: providing a pool of amplicons, wherein the pool of amplicons is prepared by attaching a unique identifier to the target nucleic acid, and amplifying by PCR the target nucleic acid to which the unique identifier is attached; and sequencing the amplicons comprising the unique identifier and the target nucleic acid. In the method, a nucleotide sequence of the unique identifier comprises both a random nucleotide (N) and a predetermined nucleotide.

Provided herein are methods of amplifying nucleic acids. In particular, methods are provided for amplifying circular RNA molecules. In certain embodiments, circular DNA molecules for amplification are generated from circular RNA molecules. Provided herein are methods for amplifying a nucleic acid. In certain embodiments, a method comprises priming a circular RNA template molecule with one or more DNA primers and extending the primers with a reverse transcriptase to generate a cDNA strand that is a copy of the circular RNA molecule. In certain embodiments, the cDNA strand generated is linear.

Provided herein are methods of amplifying nucleic acids. In particular, methods are provided for amplifying circular RNA molecules. In certain embodiments, circular DNA molecules for amplification are generated from circular RNA molecules. Provided herein are methods for amplifying a nucleic acid. In certain embodiments, a method comprises priming a circular RNA template molecule with one or more DNA primers and extending the primers with a reverse transcriptase to generate a cDNA strand that is a copy of the circular RNA molecule. In certain embodiments, the cDNA strand generated is linear.

A method for preparing a homopolymer recalibration panel includes: extracting, from a set of amplicons used in sequencing-by-synthesis, a set of candidate amplicons satisfying a first set of criteria, wherein the first set of criteria includes amplicons known to belong to high-confidence regions of a reference genome with no variants; and selecting, from the set of candidate amplicons, a reduced set of amplicons satisfying a second set of criteria, wherein the second set of criteria includes amplicons that together comprise at least a minimal threshold number of homopolymers of each homopolymer length between a predetermined minimal homopolymer length and a predetermined maximal homopolymer length for one or more of homopolymer types A, T, C, and G.

A method for preparing a homopolymer recalibration panel includes: extracting, from a set of amplicons used in sequencing-by-synthesis, a set of candidate amplicons satisfying a first set of criteria, wherein the first set of criteria includes amplicons known to belong to high-confidence regions of a reference genome with no variants; and selecting, from the set of candidate amplicons, a reduced set of amplicons satisfying a second set of criteria, wherein the second set of criteria includes amplicons that together comprise at least a minimal threshold number of homopolymers of each homopolymer length between a predetermined minimal homopolymer length and a predetermined maximal homopolymer length for one or more of homopolymer types A, T, C, and G.

Described herein are methods for enriching test samples for target nucleic acid molecules for further genetic screening. Methods may comprise isolating nucleic acid from test subjects, preparing nucleic acid libraries wherein the nucleic acid molecules are tagged or barcoded to identify sample of origin, determining fragment size distribution, determining abundance of a target nucleic acid population, calculating numerical offset values to determine amount of libraries to add for fragment size selection, performing fragment size selection, and performing a diagnostic assay on a sample enriched for a target nucleic acid.

Described herein are methods for enriching test samples for target nucleic acid molecules for further genetic screening. Methods may comprise isolating nucleic acid from test subjects, preparing nucleic acid libraries wherein the nucleic acid molecules are tagged or barcoded to identify sample of origin, determining fragment size distribution, determining abundance of a target nucleic acid population, calculating numerical offset values to determine amount of libraries to add for fragment size selection, performing fragment size selection, and performing a diagnostic assay on a sample enriched for a target nucleic acid.

Methods for non-invasive assessment of genetic variations that make use of nucleic acid fragment length information, in particular length of fragments in circulating cell-free nucleic acids and compares the number of counts from fragments with different length.

Methods for non-invasive assessment of genetic variations that make use of nucleic acid fragment length information, in particular length of fragments in circulating cell-free nucleic acids and compares the number of counts from fragments with different length.