Roche Sequencing Solutions, Inc.

Techniques described herein relate to systems and methods for parallel DNA molecules sequencing. A preprocessor can receive raw data frames from a sensor chip including 100,000 or more cells, where each raw data frame can include detection signals from the 100,000 or more cells at a given time during the formation of the 100,000 or more cells or during the DNA molecules sequencing using the 100,000 or more cells. The preprocessor can then extract relevant information for determining states of the cells from the raw data frames, generate one or more digested frames that includes the extracted information, and send the digested frames to a processor for processing, such as base determination. Because the number of digested frames sent to the processor is less than a number of the raw data frames and the digested frames include preprocessed data, the amount of data being transferred to the processor and the amount of data processing by the processor can be reduced.

The present disclosure is directed to compositions, kits, and methods of target enrichment by unidirectional primer extension, whereby the compositions, kits, and methods utilize both poison primers and target capture primers.

Techniques described herein relate to systems and methods for parallel DNA molecules sequencing. A preprocessor can receive raw data frames from a sensor chip including 100,000 or more cells, where each raw data frame can include detection signals from the 100,000 or more cells at a given time during the formation of the 100,000 or more cells or during the DNA molecules sequencing using the 100,000 or more cells. The preprocessor can then extract relevant information for determining states of the cells from the raw data frames, generate one or more digested frames that includes the extracted information, and send the digested frames to a processor for processing, such as base determination. Because the number of digested frames sent to the processor is less than a number of the raw data frames and the digested frames include preprocessed data, the amount of data being transferred to the processor and the amount of data processing by the processor can be reduced.

The present disclosure provides a method for enrichment of at least one target nucleic acid in a library of nucleic acids. This present disclosure is also directed to a faster and easier method of target capture using primer extension reactions that can improve ease of use, turnaround time, and variant allele specificity by designing target enrichment primers to specifically enrich library fragments based on the relative location of the variant base(s) in the primer, the utilization of polymerases with better priming specificity, designing the variant bases in the capture primer, designing the variant bases in the release primer, and/or designing variant specific primers to the both the plus and minus strands of the target library fragment.

The invention includes improved methods and compositions for reduction of a C5-C6 double bond of a cytosine. In particular, the improved methods and compositions for reduction of a C5-C6 double bond of a cytosine is via enzymatic means, not via chemical means. In particular, the disclosure is directed to methods of converting 5,6-dihydro-fC (fC) and/or 5,6-dihydro-caC to 5,6-dihydro-U (DHU). In particular, the disclosure is directed to methods of converting 5fC and/or 5caC to DHU. In addition, the disclosure is directed to methods for detection of epigenetic cytosine modification, particularly cytosine methylation, using ene reductases to reduce the C5-C6 double bond of cytosine.

The disclosure relates to tagged nucleoside compounds comprising a nucleotide polyphosphate covalentiy attached to a tag, wherein the compound is a polymerase substrate and the polymer moiety is capable of entering a nanopore linked to the polymerase and thereby altering the flow of ions through the nanopore. The disclosure also provides methods for preparing the tagged nucleoside compounds and for their use as tags in nanopore-based nucleic acid detection and sequencing.

Methods and materials are provided for detecting nucleic acid sequence differences including single nucleotide mutations or polymorphisms, one or more nucleotide insertions, and one or more nucleotide deletions in single molecule target members present in a test population of nucleic acid fragments. Heteroduplexes are formed between members of the test nucleic acid population and their corresponding complements provided in a pool of mismatch cleavage probes. Mismatched base pairs in the heteroduplexes are specifically cleaved and cleaved probe fragments are electronically detected to signal the present of the target members in the test population.

A method for sequencing a nucleic acid molecule includes providing a sequencing cell having a nanopore in a membrane that resides over a well, a first electrode at a bottom of the well, a second electrode in a chamber above the membrane, and an electrolyte in the well and the chamber. The first electrode is configured to facilitate non-Faradaic conduction of ionic current and forms a capacitance with ions in the electrolyte. A first voltage signal is applied across the first electrode and second electrodes, thereby creating a force that moves a nucleic acid molecule in the sequence cell through the nanopore. The first voltage signal increases to compensate for changes in the capacitance at the first electrode during application of the first voltage signal. The method further includes determining signal values measured during the first voltage signal, which correspond to one or more nucleotides in the nucleic acid molecule.

Described herein are variants of alpha-hemolysin having at least one mutation, such as a mutation to a positive charge. In certain examples, the mutation is selected from V149K, E287R, H35G, T109K, P151K, K147N, E111N, M113A, or combinations thereof in the mature, wild-type alpha-hemolysin amino acid sequence. The α-hemolysin variants may also include a substitution at H144A and/or a series of glycine residues spanning residues 127 to 131 of the mature, wild-type alpha hemolysin. Also provided are nanopore assemblies including the alpha-hemolysin variants, the assembly having a decreased time-to-thread. The decreased time-to-thread, for example, increases DNA sequencing efficiency and accuracy.

The present disclosure provides a method for enrichment of at least one target nucleic acid in a library of nucleic acids. A first oligonucleotide is hybridized to a target nucleic acid in library of nucleic acids having first and second adapters. The hybridized first oligonucleotide is extended with a first polymerase, thereby producing a first primer extension complex including the target nucleic acid and the extended first oligonucleotide. The first primer extension complex is captured, enriched relative to the library of nucleic acids, and a second oligonucleotide is hybridized to the target nucleic acid. The hybridized second oligonucleotide is extended with a second polymerase, thereby producing a second primer extension complex including the target nucleic acid and the extended second oligonucleotide and further liberating the extended first oligonucleotide from the first primer extension complex.