This disclosure describes a technique for performing random access in a pool of polynucleotides by using one unique primer and one homopolymer primer to selectively amplify some but not all of the polynucleotides in the pool. The polynucleotides are synthesized by a template independent polymerase such as terminal deoxynucleotide transferase (TdT) rather than by phosphoramidite synthesis. Enzymatic synthesis efficiently creates homopolymer sequences through unregulated synthesis. Use of one homopolymer primer instead of two unique primers decreases the complexity, time, and cost of synthesizing the polynucleotides. Use of a unique primer provides a sequence that can be varied to uniquely identify multiple different groups of polynucleotides. This enables random access by polymerase chain reaction (PCR) amplification while still benefitting from the efficiency of homopolymer synthesis. The polynucleotides may include payload regions that use a sequence of nucleotides to encode digital data.

Methods for determining a location of a feature in a spatial array of features include: providing a spatial array including a plurality of features on a substrate, where a feature of the plurality of features has a capture probe, and where the capture probe has a spatial barcode and a constant sequence; hybridizing a first sequencing probe to the spatial barcode, where the first sequencing probe has a first label and a first nucleotide sequence; obtaining a first image of the first label with the first sequencing probe hybridized to the spatial barcode; determining, based on the first image, a first portion of a sequence of the spatial barcode; and associating the feature with a location in the spatial array based on a location of the first label in the first image.

Methods for determining a location of a feature in a spatial array of features include: providing a spatial array including a plurality of features on a substrate, where a feature of the plurality of features has a capture probe, and where the capture probe has a spatial barcode and a constant sequence; hybridizing a first sequencing probe to the spatial barcode, where the first sequencing probe has a first label and a first nucleotide sequence; obtaining a first image of the first label with the first sequencing probe hybridized to the spatial barcode; determining, based on the first image, a first portion of a sequence of the spatial barcode; and associating the feature with a location in the spatial array based on a location of the first label in the first image.

Processes and kits are provided for producing sequence specific fragments of nucleic acid molecules, whether from a genome or transcriptome, where one end of the molecule is highly diverse and/or the full-length molecule, whether a gene or a mRNA, is too long for it to be sequenced using currently available sequencing methods. Methods of preparing a sequencing library configured for 5′ or 3′ anchored sequencing, wherein the opposing termini of the library molecules are differentially truncated, and methods of parallel sequencing such libraries are described.

The present invention relates to a method of identifying a target genomic nucleic acid sequence including hybridizing a set of probes to the target genomic nucleic acid sequence, wherein the set of probes has a unique associated barcode sequence for identification of the target genomic nucleic acid sequence, wherein each probe of the set includes (1) a complementary sequence complementary to a first strand of the target genomic nucleic acid sequence and (2) the associated barcode sequence or a portion of the associated barcode sequence, sequencing the associated barcode sequence from probes hybridized to the target genomic nucleic acid sequence using a fluorescence-based sequencing method, and identifying the target genomic nucleic acid sequence by the sequenced barcode sequence.

The present invention relates to a method of identifying a target genomic nucleic acid sequence including hybridizing a set of probes to the target genomic nucleic acid sequence, wherein the set of probes has a unique associated barcode sequence for identification of the target genomic nucleic acid sequence, wherein each probe of the set includes (1) a complementary sequence complementary to a first strand of the target genomic nucleic acid sequence and (2) the associated barcode sequence or a portion of the associated barcode sequence, sequencing the associated barcode sequence from probes hybridized to the target genomic nucleic acid sequence using a fluorescence-based sequencing method, and identifying the target genomic nucleic acid sequence by the sequenced barcode sequence.

The present disclosure provides improved nucleic acid sequencing-by-synthesis (SBS) methods, related kits and reagents, and systems for performing such methods using such kits and reagents.

The present invention relates to a method of identifying a target genomic nucleic acid sequence including hybridizing a set of probes to the target genomic nucleic acid sequence, wherein the set of probes has a unique associated barcode sequence for identification of the target genomic nucleic acid sequence, wherein each probe of the set includes (1) a complementary sequence complementary to a first strand of the target genomic nucleic acid sequence and (2) the associated barcode sequence or a portion of the associated barcode sequence, sequencing the associated barcode sequence from probes hybridized to the target genomic nucleic acid sequence using a fluorescence-based sequencing method, and identifying the target genomic nucleic acid sequence by the sequenced barcode sequence.

The present invention relates to a method of identifying a target genomic nucleic acid sequence including hybridizing a set of probes to the target genomic nucleic acid sequence, wherein the set of probes has a unique associated barcode sequence for identification of the target genomic nucleic acid sequence, wherein each probe of the set includes (1) a complementary sequence complementary to a first strand of the target genomic nucleic acid sequence and (2) the associated barcode sequence or a portion of the associated barcode sequence, sequencing the associated barcode sequence from probes hybridized to the target genomic nucleic acid sequence using a fluorescence-based sequencing method, and identifying the target genomic nucleic acid sequence by the sequenced barcode sequence.

Compositions comprising covalently modified and mutated biotin-binding proteins, particularly biotin-binding proteins having a negative charge at physiological pH, are provided. Methods of producing such proteins are also provided, as are methods of immobilizing, sequencing, and making nucleic acids employing such proteins.