Methods and systems for decreasing amplification bias and primer-dimer formation in amplification reactions and for amplifying a plurality of target polynucleotides from a sample in a single reaction and for sequencing the target polynucleotides where samples can include forensic samples and where target polynucleotides can include identity- or ancestry-informative markers, short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). Methods of determining a nucleotide spacer sequence for disrupting primer dimer formation can include: receiving a set of primer sequences; determining a plurality of candidate spacers between an adapter sequence and a gene-specific portion of the primer sequence, the determined plurality of candidate spacers comprises sequences that disrupt stable interactions between sequences of the set of primer sequences; ranking candidate spacers that meet a predetermined threshold value of stable interactions in the extension sequences; and outputting a set of the ranked spacers that meet the predetermined threshold.

Methods and systems for decreasing amplification bias and primer-dimer formation in amplification reactions and for amplifying a plurality of target polynucleotides from a sample in a single reaction and for sequencing the target polynucleotides where samples can include forensic samples and where target polynucleotides can include identity- or ancestry-informative markers, short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). Methods of determining a nucleotide spacer sequence for disrupting primer dimer formation can include: receiving a set of primer sequences; determining a plurality of candidate spacers between an adapter sequence and a gene-specific portion of the primer sequence, the determined plurality of candidate spacers comprises sequences that disrupt stable interactions between sequences of the set of primer sequences; ranking candidate spacers that meet a predetermined threshold value of stable interactions in the extension sequences; and outputting a set of the ranked spacers that meet the predetermined threshold.

A method for specifically and efficiently quantifying the expression of targeted RNA variants with specific terminal sequences suitable to identify multiple isoforms bearing complex heterogeneity in terminal sequences by hybridizing a 5′-Dbs-adapter to the 5′-end of target RNAs, wherein the 5′-Dbs-adapter has a stem-loop structure whose protruding 5′-end base-pairs with the 5′-end of target RNAs, and wherein the loop region of 5′-Dbs-adapter contains a base-lacking spacer which will terminate reverse transcription in a subsequent step; hybridizing a 3′db-adapter to the 3′-end of target RNAs, wherein the 3′-db-adapter has a stem-loop structure whose protruding 3′-end base-pairs with the 3′-end of target RNAs; ligating both adapters with target RNAs by RN12 ligation to form a “dumbbell-like” structure; and, amplifying and quantifying the ligation product by RT-PCR.

The invention relates to a new method of characterising a target RNA polynucleotide by taking one or more measurements as the target RNA polynucleotide moves with respect to a transmembrane pore. The movement is controlled by a DNA helicase. The invention also relates to a modified RNA construct wherein the RNA polynucleotide has been modified to increase DNA helicase binding thereto.

The invention relates to a new method of characterising a target RNA polynucleotide by taking one or more measurements as the target RNA polynucleotide moves with respect to a transmembrane pore. The movement is controlled by a DNA helicase. The invention also relates to a modified RNA construct wherein the RNA polynucleotide has been modified to increase DNA helicase binding thereto.

The present invention concerns an oligonucleotide-functionalized hydrophobic polymer nanoparticle and method of its preparation. Said nanoparticle is a dye-loaded polymeric nanoparticle, and being functionalized by: (a) target-specific oligonucleotides, and/or (b) non-specific oligonucleotides.

The present invention concerns an oligonucleotide-functionalized hydrophobic polymer nanoparticle and method of its preparation. Said nanoparticle is a dye-loaded polymeric nanoparticle, and being functionalized by: (a) target-specific oligonucleotides, and/or (b) non-specific oligonucleotides.

A method for analyzing target nucleic acid from a cell, including: 1) providing a discrete partition: target nucleic acid derived from a single cell and added with an oligonucleotide adaptor sequence, and a solid support with at least one oligonucleotide tag attached, wherein each oligonucleotide tag includes a first and second strand, the first strand includes a barcode sequence and a hybridization sequence located at the 3′-end of the barcode sequence, the second strand includes a first portion, complementary to the hybridization sequence of the first strand, and a second portion, complementary to the oligonucleotide adaptor sequence attached to the target nucleic acid, and the first and second strand form a partial double-strand, or the second strand and target nucleic acid attached form a partial double-strand; and (2) in the discrete partition, the oligonucleotide tag is linked to the target nucleic acid attached, thereby producing barcoded target nucleic acid.

A method for analyzing target nucleic acid from a cell, including: 1) providing a discrete partition: target nucleic acid derived from a single cell and added with an oligonucleotide adaptor sequence, and a solid support with at least one oligonucleotide tag attached, wherein each oligonucleotide tag includes a first and second strand, the first strand includes a barcode sequence and a hybridization sequence located at the 3′-end of the barcode sequence, the second strand includes a first portion, complementary to the hybridization sequence of the first strand, and a second portion, complementary to the oligonucleotide adaptor sequence attached to the target nucleic acid, and the first and second strand form a partial double-strand, or the second strand and target nucleic acid attached form a partial double-strand; and (2) in the discrete partition, the oligonucleotide tag is linked to the target nucleic acid attached, thereby producing barcoded target nucleic acid.

The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.