Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same

Inventors

Hardenbol, Paul • Patel, Pranav • Hindson, Benjamin • Wyatt, Paul William • Bjornson, Keith • Wu, Indira • Belhocine, Zahra Kamila

Assignees

10X Genomics Inc

Abstract

This disclosure provides methods for preparing a sequencing library including the steps of providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads including oligonucleotide adapter sequence segments; amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence including occasional dUTPs; and excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.

Core Innovation

The invention relates to nucleic acid processing in which a double-stranded nucleic acid molecule comprising at least one uracil is fragmented at one or more sites corresponding to the at least one uracil. In the presence of barcode molecules each comprising a barcode sequence, the fragmentation generates a plurality of nucleic acid fragments that comprise different sequences, while the barcode molecules and the double-stranded nucleic acid molecule remain separate molecules.

The processing then generates nucleic acid products using the plurality of nucleic acid fragments and the barcode molecules. Each nucleic acid product comprises a sequence corresponding to one nucleic acid fragment of the plurality of nucleic acid fragments and comprises the barcode sequence or reverse complement thereof, thereby associating fragment sequence information with a barcode sequence derived from the separate barcode molecule.

The document further describes controlling fragment generation at uracil sites and linking barcode molecules to produced fragments, including approaches that include nicking at one or more sites corresponding to uracil followed by one or more nucleic acid extension reactions. The document also describes downstream library-related processing using barcode sequences and sequencing-adapter structures, including approaches involving adaptors (P5 and P7) and read structures (read1/read2), together with sample multiplexing based on non-overlapping barcode sets and sample index sequences.

Claims Coverage

The independent claim coverage is concentrated in a single independent method claim that defines fragmentation at uracil-associated sites using separate barcode molecules and then producing nucleic acid products that contain both fragment-derived sequence information and a barcode sequence or its reverse complement. No additional independent claims are provided in the partial content.

Across the provided independent-claim content, the inventive concept centers on forming uracil-associated nucleic acid fragments in the presence of separate barcode molecules and then producing nucleic acid products that each include a fragment-derived sequence together with a barcode sequence or its reverse complement.

Stated Advantages

Documented Applications