Methods for processing paired end sequences
Inventors
Almogy, Gilad • Pratt, Mark • Oberstrass, Florian
Assignees
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Publication Number
US-12152277-B2
Publication Date
2024-11-26
Expiration Date
Abstract
Recognized herein is the need for methods and processes for increasing the efficiency and accuracy of paired end sequencing.
Core Innovation
The invention provides a method for processing a template nucleic acid by using a first set of beads and a second set of beads, where a first bead comprises a first primer having sequence complementarity with a first adaptor coupled to a first strand of the template nucleic acid, and a second bead comprises a second primer having sequence complementarity with a second adaptor coupled to a second strand of the template nucleic acid. The first bead and the second bead are coupled, and the template nucleic acid is coupled to the first bead and the second bead to provide a bead-template complex. Nucleic acid molecules attached to the first bead and the second bead are sequenced to generate sequencing reads corresponding to the second strand and the first strand, respectively.
The processing further addresses paired-end processing by configuring first- and second-strand copies such that the first- and second-strand copies are at least partially overlapping, or optionally have no overlap or limited overlap. This is supported by described embodiments including co-partitioning beads of different types with a biological sample in droplets in emulsion or in wells, followed by amplification of strand copies. Amplification is described to generate copies that enable generation of paired sequencing reads corresponding to opposite strands.
The invention also includes optional amplification workflows and adaptive adapter/primer structures, including adapter sub-parts with complementarity and embodiments using releasably or irreleasably coupled bead pairs, including coupling via covalent bonds or protein interactions. In alternative embodiments, synthetic double-stranded nucleic acid processing is described with cleavable element processing, ligation and circularization, cleavage, and gap filling as part of preparing material for sequencing. A clonal amplification embodiment with barcoded bead templates and a computer control system for sequencing and sequence analysis is also described, including modeling of paired-read efficiency versus redundancy.
Claims Coverage
The provided independent claim contains one inventive method aspect: bead-template complex construction using complementary primers/adaptors on coupled first and second beads, followed by sequencing to generate reads corresponding to opposite strands. Across dependent claims, additional inventive features concern strand overlap between first- and second-strand copy sequences, inter-bead amplification, sequencing by synthesis, and emulsion PCR.
Coupled first and second beads with complementary primers/adaptors
Providing a first set of beads and a second set of beads, wherein a first bead of the first set of beads comprises a first primer having sequence complementarity with a first adaptor coupled to a first strand of the template nucleic acid, wherein a second bead of the second set of beads comprises a second primer having sequence complementarity with a second adaptor coupled to a second strand of the template nucleic acid, wherein the first bead and the second bead are coupled
Bead-template complex coupling of template nucleic acid to both beads
Coupling the template nucleic acid to the first bead and the second bead, thereby providing a bead-template complex
Sequencing attached to first and second beads to generate opposite-strand reads
Sequencing nucleic acid molecules attached to the first bead and the second bead to generate sequencing reads corresponding to the second strand and the first strand, respectively
Inter-strand copy overlap between first- and second-strand copies
The method further comprises that nucleic acid molecules comprise one or more copies of the first strand and the second strand, where a first sequence from the first-strand copies overlaps a second sequence from the second-strand copies
Quantitative overlap length threshold for paired processing
The method further comprises that the overlap comprises at least 100 nucleotides
Inter-bead strand amplification that swaps strand ownership across beads
Prior to sequencing, apply amplification conditions sufficient to amplify the first strand on the second bead and the second strand on the first bead, generating one or more copies of each strand on the opposite bead
Sequencing by synthesis
The sequencing comprises sequencing by synthesis
Emulsion PCR as the amplification format
The PCR comprises emulsion PCR (emPCR)
Overall, the claim coverage centers on constructing a coupled bead-template complex using complementary primer/adaptor pairs on first and second beads and then sequencing molecules attached to each bead to produce paired sequencing reads corresponding to opposite strands. Dependent coverage further narrows the method by specifying overlap requirements between first- and second-strand copy sequences, inter-bead amplification that generates strand copies on opposite beads, and sequencing-by-synthesis and emulsion PCR as particular modalities.
Stated Advantages
Documented Applications
No documented applications found
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