PACIFIC BIOSCIENCES OF CALIFORNIA, INC.

Integrated target waveguide devices and optical analytical systems comprising such devices are provided. The target devices include an optical coupler that is optically coupled to an integrated waveguide and that is configured to receive optical input from an optical source through free space, particularly through a low numerical aperture interface. The devices and systems are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices provide for the efficient and reliable coupling of optical excitation energy from an optical source to the optical reactions. Optical signals emitted from the reactions can thus be measured with high sensitivity and discrimination. The devices and systems are well suited for miniaturization and high throughput.

Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.

The invention provides a novel class of cyanine dyes that are functionalized with sulfonic acid groups and a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.

A method for determining the presence of an allele, including (a) binding a polymerase to a double stranded nucleic acid that includes a primer hybridized to a template, the template including a first allele of a locus; (b) adding a nucleotide to the primer via catalytic activity of the polymerase, thereby producing an extended nucleic acid; (c) dissociating the polymerase from the extended nucleic acid; (d) detecting dissociation of the polymerase from the extended nucleic acid; and (e) comparing the dissociation of the polymerase from the extended nucleic acid to dissociation of the polymerase from a second double stranded nucleic acid, the second double stranded nucleic acid including a primer hybridized to the same position of the locus as the primer of the extended nucleic acid.

Methods of processing pooled samples of single template molecules for sequencing are provided. Template nucleic acid segments are prepared and pooled. The prepared template nucleic acids include double stranded segments and a linking oligonucleotide with a unique, identifiable barcode sequence. The template nucleic acid segments are prepared by ligating a linking oligonucleotide segment to join the 3′ end of the first strand segment to the 5′ end of the second strand segment.

Disclosed herein is a high throughput optical scanning system to generate super resolution images and methods of use. The optical scanning device and methods of use provided herein can allow high throughput scanning of a continuously moving object with a high resolution despite fluctuations in stage velocity. This can aid in high throughput scanning of a substrate, such as a biological chip comprising fluorophores. Also provided herein are improved optical relay systems and scanning optics.

Systems and methods for mapping a plurality of sequence reads to a genomic region are provided. A plurality of sequence reads mappable to the genomic region are obtained. An initial Markov model for the genomic region is obtained. The initial Markov model comprises at least (i) a first repeat for a first repeat region, (ii) a second repeat for a second repeat region, and (iii) an intermediate region linking the first repeat to the second repeat. The initial Markov model is refined using the plurality of sequence reads, thereby obtaining a refined Markov model. For each respective sequence read in the plurality of sequences, the respective sequence read is used to find a highest probability path through the Markov model. This highest probability path is then used to map the respective sequence read to the genomic region.

Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.

Provided herein are highly multiplexed optical analytical systems for improved nucleic acid sequencing. The systems include a plurality of highly multiplexed optical chips, at least one optical source, and a plurality of optical delivery devices for illuminating an array of nanoscale rection regions on each of the optical chips. In use, the reaction regions contain fluorescent nucleic acid sequencing reagents and are arranged to report nucleic acid sequence information to optical detectors associated with the multiplexed optical chips in real time. The systems enable a massive increase in the scale of nucleic acid sequencing reactions capable of being performed within a single instrument without a corresponding increase in size, complexity, or cost of the instrument.

Arrays of integrated analytical devices are provided. The arrays are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. In particular, the arrays provide increased efficiency of optical collection and decreased background signal as the lateral dimensions of the unit cell of devices within the array are decreased, for example as they are decreased to 2 μm, or even less.