The present disclosure provides methods for optimizing barcode design for multiplex DNA sequencing. Also disclosed are DNA barcodes optimized for use with particular sequencing technologies.

The present invention provides compositions, apparatuses and methods for detecting one or more nucleic acid targets present in a sample. Methods of the invention include utilizing two or more ligation probes that reversibly bind a target nucleic acid in close proximity to each other and possess complementary reactive ligation moieties. When such probes have bound to the target in the proper orientation, they are able to undergo a spontaneous chemical ligation reaction that yields a ligation product that is directly detected or that is amplified to produce amplicons that are then detected. The present invention also provides methods to stabilize sample RNA so that degradation does not significantly affect the results of the analysis.

The present invention provides compositions, apparatuses and methods for detecting one or more nucleic acid targets present in a sample. Methods of the invention include utilizing two or more ligation probes that reversibly bind a target nucleic acid in close proximity to each other and possess complementary reactive ligation moieties. When such probes have bound to the target in the proper orientation, they are able to undergo a spontaneous chemical ligation reaction that yields a ligation product that is directly detected or that is amplified to produce amplicons that are then detected. The present invention also provides methods to stabilize sample RNA so that degradation does not significantly affect the results of the analysis.

Provided herein are methods and compositions for performing PCR with primers with blocked 3′-ends that are unblocked when these primers anneal to the template. The multiplexed PCR can be used as real-time qPCR, for end-point detection or as enrichment method for next generation sequencing (NGS). Also described herein are methods and compositions to improve sensitivity of mutation-specific PCR when targeting closely-spaced mutations.

Provided herein are methods and compositions for performing PCR with primers with blocked 3′-ends that are unblocked when these primers anneal to the template. The multiplexed PCR can be used as real-time qPCR, for end-point detection or as enrichment method for next generation sequencing (NGS). Also described herein are methods and compositions to improve sensitivity of mutation-specific PCR when targeting closely-spaced mutations.

The present invention relates to amplification primers with a universal tag and sequencing primers comprising at least one non-natural nucleobase capable of hybridizing to a complementary non-natural nucleobase. The present invention further relates to amplification methods of nucleic acid amplification and sequencing using an amplification primer with a universal tag and sequencing primers, as well as kits and solid supports comprising such primers and tags.

The present invention relates to amplification primers with a universal tag and sequencing primers comprising at least one non-natural nucleobase capable of hybridizing to a complementary non-natural nucleobase. The present invention further relates to amplification methods of nucleic acid amplification and sequencing using an amplification primer with a universal tag and sequencing primers, as well as kits and solid supports comprising such primers and tags.

Methods and containers are provided for identifying a species, illustratively a bacterial species. Illustrative methods comprise amplifying various genes in the nucleic acid from the bacterial species in a single reaction mixture using pairs of outer first-stage primers designed to hybridize to generally conserved regions of the respective genes to generate a plurality of first-stage amplicons, dividing the reaction mixture into a plurality of second-stage reactions, each using a unique pair of second-stage primers, each pair of second-stage primers specific for a target bacterial species or subset of bacterial species, detecting which of the second-stage reactions amplified, and identifying the bacterial species based on second-stage amplification. Methods for determining antibiotic resistance are also provided, such methods also using first-stage primers for amplifying genes known to affect antibiotic resistance a plurality of the second-stage reactions wherein each pair of second-stage primers specific for a specific gene for conferring antibiotic resistance.

The present disclosure relates to methods, kits and products for barcoding of nucleic acids. In certain embodiments, the present disclosure provides a method of producing nucleic acid for sequencing utilising clonal amplification on a solid substrate, the method comprising: (a) providing a nucleic acid sample for sequencing; (b) amplifying the nucleic acid sample using an amplifying primer comprising a degenerate nucleotide sequence and a 5′ fixed nucleotide sequence, to produce amplified nucleic acids; and (c) further amplifying the amplified nucleic acids with (i) a first primer comprising the 5′ fixed nucleotide sequence and a first adapter nucleotide sequence, and (ii) a second primer comprising the 5′ fixed nucleotide sequence and a second adapter nucleotide sequence, wherein the first adapter nucleotide sequence or the second adapter nucleotide sequence provides a sequence for subsequent priming of DNA synthesis from a nucleotide sequence attached to the solid substrate and the other adapter nucleotide sequence provides a sequence for subsequent priming of DNA synthesis from a template produced from the subsequent priming, and wherein one or more of the first primer, the second primer and the amplifying primer comprise a specific identifier sequence to identify nucleic acids amplified with the first primer, the second primer and/or the amplifying primer; thereby producing nucleic acid for sequencing utilising clonal amplification on the solid substrate.

The present disclosure relates to methods, kits and products for barcoding of nucleic acids. In certain embodiments, the present disclosure provides a method of producing nucleic acid for sequencing utilising clonal amplification on a solid substrate, the method comprising: (a) providing a nucleic acid sample for sequencing; (b) amplifying the nucleic acid sample using an amplifying primer comprising a degenerate nucleotide sequence and a 5′ fixed nucleotide sequence, to produce amplified nucleic acids; and (c) further amplifying the amplified nucleic acids with (i) a first primer comprising the 5′ fixed nucleotide sequence and a first adapter nucleotide sequence, and (ii) a second primer comprising the 5′ fixed nucleotide sequence and a second adapter nucleotide sequence, wherein the first adapter nucleotide sequence or the second adapter nucleotide sequence provides a sequence for subsequent priming of DNA synthesis from a nucleotide sequence attached to the solid substrate and the other adapter nucleotide sequence provides a sequence for subsequent priming of DNA synthesis from a template produced from the subsequent priming, and wherein one or more of the first primer, the second primer and the amplifying primer comprise a specific identifier sequence to identify nucleic acids amplified with the first primer, the second primer and/or the amplifying primer; thereby producing nucleic acid for sequencing utilising clonal amplification on the solid substrate.