Provided herein are automated apparatus for the identification of microorganisms in various samples. The disclosure solves existing challenges encountered in identifying and distinguishing various types of microorganisms, including viruses and bacteria in a timely, efficient, and automated manner by sequencing.

Provided herein are automated apparatus for the identification of microorganisms in various samples. The disclosure solves existing challenges encountered in identifying and distinguishing various types of microorganisms, including viruses and bacteria in a timely, efficient, and automated manner by sequencing.

Described herein are methods for determining a sequence of a region of interest from an mRNA molecule. Sequenced polynucleotides can include a barcode region, a homopolymer region (e.g., a poly-A region), and a target region associated with the mRNA molecule. According to some methods, the barcode region omits the same base present in the homopolymer region. According to some methods, extension of the primer used for sequencing is stalled within the homopolymer region. According to some methods, sequencing flow cycles and the different barcode regions of the polynucleotides configured are such that the primer is extended to the end of the barcode region across the plurality of polynucleotides before being extended into the homopolymer region. According to some methods, two primers or a cleavable primer is used to separately sequence the barcode region and the target region.

Described herein are methods for determining a sequence of a region of interest from an mRNA molecule. Sequenced polynucleotides can include a barcode region, a homopolymer region (e.g., a poly-A region), and a target region associated with the mRNA molecule. According to some methods, the barcode region omits the same base present in the homopolymer region. According to some methods, extension of the primer used for sequencing is stalled within the homopolymer region. According to some methods, sequencing flow cycles and the different barcode regions of the polynucleotides configured are such that the primer is extended to the end of the barcode region across the plurality of polynucleotides before being extended into the homopolymer region. According to some methods, two primers or a cleavable primer is used to separately sequence the barcode region and the target region.

Described herein are methods for determining a sequence of a region of interest from an mRNA molecule. Sequenced polynucleotides can include a barcode region, a homopolymer region (e.g., a poly-A region), and a target region associated with the mRNA molecule. According to some methods, the barcode region omits the same base present in the homopolymer region. According to some methods, extension of the primer used for sequencing is stalled within the homopolymer region. According to some methods, sequencing flow cycles and the different barcode regions of the polynucleotides configured are such that the primer is extended to the end of the barcode region across the plurality of polynucleotides before being extended into the homopolymer region. According to some methods, two primers or a cleavable primer is used to separately sequence the barcode region and the target region.

Provided is a method including detecting an incorporation of a labelled nucleotide into a nascent polynucleotide strand complementary to a template polynucleotide strand by a polymerase, wherein the polymerase is tethered to a solid support conductive channel by a tether and the labelled nucleotides is a compound of Formula I: ##STR00001##

Provided is a method including detecting an incorporation of a labelled nucleotide into a nascent polynucleotide strand complementary to a template polynucleotide strand by a polymerase, wherein the polymerase is tethered to a solid support conductive channel by a tether and the labelled nucleotides is a compound of Formula I: ##STR00001##

Provided is a method including detecting an incorporation of a labelled nucleotide into a nascent polynucleotide strand complementary to a template polynucleotide strand by a polymerase, wherein the polymerase is tethered to a solid support conductive channel by a tether and the labelled nucleotides is a compound of Formula I: ##STR00001##

Provided are methods of producing a nucleic acid complex. In certain aspects, the methods include combining a sample including ribosomal RNA (rRNA) and a probe complement oligonucleotide with an oligonucleotide probe. The oligonucleotide probe includes a 3′ region complementary to a 3′ region of a rRNA, and a 5′ region complementary to the probe complement oligonucleotide. The combining is under conditions in which the 3′ region of the oligonucleotide probe hybridizes to the 3′ region of the rRNA and the 5′ region of the oligonucleotide probe hybridizes to the probe complement oligonucleotide, thereby producing a nucleic acid complex. In certain aspects, the methods find use in producing rRNA libraries that find use, e.g., in rRNA sequencing applications. Oligonucleotide probes, libraries thereof, compositions, and kits that find use, e.g., in practicing the methods of the present disclosure, are also provided.

Provided are methods of producing a nucleic acid complex. In certain aspects, the methods include combining a sample including ribosomal RNA (rRNA) and a probe complement oligonucleotide with an oligonucleotide probe. The oligonucleotide probe includes a 3′ region complementary to a 3′ region of a rRNA, and a 5′ region complementary to the probe complement oligonucleotide. The combining is under conditions in which the 3′ region of the oligonucleotide probe hybridizes to the 3′ region of the rRNA and the 5′ region of the oligonucleotide probe hybridizes to the probe complement oligonucleotide, thereby producing a nucleic acid complex. In certain aspects, the methods find use in producing rRNA libraries that find use, e.g., in rRNA sequencing applications. Oligonucleotide probes, libraries thereof, compositions, and kits that find use, e.g., in practicing the methods of the present disclosure, are also provided.