Methods for the rapid amplification of extremely low quantity nucleic acids in a sample are provided. The disclosed methods are capable of amplifying less than 1 pg of DNA and/or RNA from a biological sample using a single tube and one-step or two-step preparation.

Methods for the rapid amplification of extremely low quantity nucleic acids in a sample are provided. The disclosed methods are capable of amplifying less than 1 pg of DNA and/or RNA from a biological sample using a single tube and one-step or two-step preparation.

Disclosed here is a composition comprising a primer that is (a) a loopable primer comprising a target-specific section, an adaptor section, and a stem-forming section, wherein the stem-forming section is hybridizable to a portion of the target-specific section to form a stem structure, or (b) a split primer comprising a first target-specific section, a second target-specific section, and an adaptor section positioned between the first target-specific section and the second target-specific section, or (c) a split-loopable primer comprising a first target-specific section, a second target-specific section, a stem-forming section positioned between the first target-specific section and the second target-specific section, and an adaptor section, or comprising a first adaptor section, a second adaptor section, a stem-forming section positioned between the first adaptor section and the second adaptor section, and a target-specific section. Also disclosed is a method for amplifying a target locus of interest from a template DNA, comprising at least two pre-amplification cycles using the loopable primer, the split primer and/or the split-loopable primer, wherein each amplification cycle comprises annealing the primer to the template DNA or pre-amplification product thereof and elongating the annealed primer. Further disclosed is a kit for amplifying a target locus of interest, comprising the loopable primer, the split primer, and/or the split-loopable primer.

Disclosed here is a composition comprising a primer that is (a) a loopable primer comprising a target-specific section, an adaptor section, and a stem-forming section, wherein the stem-forming section is hybridizable to a portion of the target-specific section to form a stem structure, or (b) a split primer comprising a first target-specific section, a second target-specific section, and an adaptor section positioned between the first target-specific section and the second target-specific section, or (c) a split-loopable primer comprising a first target-specific section, a second target-specific section, a stem-forming section positioned between the first target-specific section and the second target-specific section, and an adaptor section, or comprising a first adaptor section, a second adaptor section, a stem-forming section positioned between the first adaptor section and the second adaptor section, and a target-specific section. Also disclosed is a method for amplifying a target locus of interest from a template DNA, comprising at least two pre-amplification cycles using the loopable primer, the split primer and/or the split-loopable primer, wherein each amplification cycle comprises annealing the primer to the template DNA or pre-amplification product thereof and elongating the annealed primer. Further disclosed is a kit for amplifying a target locus of interest, comprising the loopable primer, the split primer, and/or the split-loopable primer.

Provided are methods of nucleic acid sequencing. The methods include producing a circularized DNA including a full-length cDNA and a known heterologous sequence, and performing rolling circle amplification using the circularized DNA as template to produce a concatemer including repeating segments including the full-length cDNA and the known heterologous sequence. The methods further include obtaining a raw sequencing read of the concatemer using a nanopore, identifying the repeating segments in the raw sequencing read, and producing a consensus sequence of the full-length cDNA based on the sequences of the repeating segments. Computer-readable media, computing devices, and systems that find use, e.g., in practicing the methods of the present disclosure are also provided.

Provided are methods of nucleic acid sequencing. The methods include producing a circularized DNA including a full-length cDNA and a known heterologous sequence, and performing rolling circle amplification using the circularized DNA as template to produce a concatemer including repeating segments including the full-length cDNA and the known heterologous sequence. The methods further include obtaining a raw sequencing read of the concatemer using a nanopore, identifying the repeating segments in the raw sequencing read, and producing a consensus sequence of the full-length cDNA based on the sequences of the repeating segments. Computer-readable media, computing devices, and systems that find use, e.g., in practicing the methods of the present disclosure are also provided.

The present invention provides compositions, kits, and method for determining the levels of expression of human polyoma or papillomavirus species and RNA transcripts. These levels can be used for the prognosis of risk of developing virally-induced cancers. The ratio (R) between early and late transcripts is indicative of HPV infections associated with higher risk of developing genital neoplasia and cancer.

The present invention provides compositions, kits, and method for determining the levels of expression of human polyoma or papillomavirus species and RNA transcripts. These levels can be used for the prognosis of risk of developing virally-induced cancers. The ratio (R) between early and late transcripts is indicative of HPV infections associated with higher risk of developing genital neoplasia and cancer.

The present invention provides a method for genotyping alleles in at least one homologous genetic loci set, comprising: (i) providing a DNA-containing sample that includes said at least one homologous genetic loci set; (ii) performing PCR amplification of regions of said homologous genetic loci set using consensus sequence-specific primers, wherein said consensus sequence-specific primers bind to consensus sequences that are common to a plurality of genes within the genetic loci set, thereby generating a pool of amplification products; (iii) sequencing a plurality of said amplification products in order to determine the relative proportion of each nucleotide at each position in a sequencing read; (iv) performing a sequence alignment between the sequencing read results of (iii) and at least one reference sequence, which reference sequence corresponds to one of the genes in said homologous genetic loci set; and (v) performing genotype calling of the allele or alleles in said sample based on the relative proportion of each nucleotide at each of a plurality of discriminant positions in said alignment. Also disclose are related products, kits and systems for performing the method.

The present invention provides a method for genotyping alleles in at least one homologous genetic loci set, comprising: (i) providing a DNA-containing sample that includes said at least one homologous genetic loci set; (ii) performing PCR amplification of regions of said homologous genetic loci set using consensus sequence-specific primers, wherein said consensus sequence-specific primers bind to consensus sequences that are common to a plurality of genes within the genetic loci set, thereby generating a pool of amplification products; (iii) sequencing a plurality of said amplification products in order to determine the relative proportion of each nucleotide at each position in a sequencing read; (iv) performing a sequence alignment between the sequencing read results of (iii) and at least one reference sequence, which reference sequence corresponds to one of the genes in said homologous genetic loci set; and (v) performing genotype calling of the allele or alleles in said sample based on the relative proportion of each nucleotide at each of a plurality of discriminant positions in said alignment. Also disclose are related products, kits and systems for performing the method.