Direct-to-library methods, systems, and compositions
Inventors
BERCOVICI, Sivan • BLAIR, Lily • Blauwkamp, Timothy A. • EUGSTER, Peter J. • HONG, David K. • KAWLI, Trupti • Rosen, Michael J. • Spacek, Damek • Vilfan, Igor D.
Assignees
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Publication Number
US-12320036-B2
Publication Date
2025-06-03
Expiration Date
Abstract
Provided herein are direct-to-library methods, systems, and compositions.
Core Innovation
The invention provides a method of preparing a nucleic acid library from an initial sample in which nucleic acids are not extracted from the initial sample before preparing the nucleic acid library. The method generates a nucleic acid library from initial samples comprising plasma, synovial fluid, bronchoalveolar lavage, cerebrospinal fluid, or urine, and includes dephosphorylating the nucleic acids and denaturing the dephosphorylated nucleic acids.
The method attaches a 3′-end adapter to the denatured nucleic acids to produce adapted nucleic acids, anneals a primer to the adapted nucleic acids and extends the primer with a polymerase to generate complementary strands, attaches a 5′-end adapter, and amplifies the complementary strands. Dependent refinements specify splint oligonucleotide attachment, ligation using T4 DNA ligase, and non-templated activity polymerase with template switching for adapter attachment.
The disclosure further describes process control molecules for monitoring library-processing steps, including dephosphorylation, denaturation, ligation, and non-templated extension and template switching controls. The document also provides sequencing and bioinformatics scope, including assembling sequence data and identifying genetic variants and microbial/pathogen signals.
Claims Coverage
The independent claim coverage comprises one independent method claim directed to preparing a nucleic acid library from a biological initial sample without extracting nucleic acids prior to library preparation. Across the claims, the inventive features center on the no-extraction constraint, specified clinical sample types, and a dephosphorylation/denaturation/adaptor/primer/amplification workflow.
No nucleic-acid extraction prior to library preparation
Nucleic acids used to generate the nucleic acid library are not extracted from the initial sample before preparing the nucleic acid library.
Library generation from specified clinical initial samples
The initial sample comprises a plasma sample, a synovial fluid sample, a bronchoalveolar lavage sample, a cerebrospinal fluid sample, or a urine sample.
Dephosphorylation and denaturation to create processable nucleic acids
The method dephosphorylates the nucleic acids in the initial sample to produce dephosphorylated nucleic acids and denatures the dephosphorylated nucleic acids to produce denatured nucleic acids.
Adapter attachment, primer extension, and complementary-strand amplification
The method attaches a 3′-end adapter to the denatured nucleic acids to produce adapted nucleic acids, anneals a primer to the adapted nucleic acids and extends the primer with a polymerase to generate complementary strands, attaches a 5′-end adapter, and amplifies the complementary strands.
Overall, the claim set centers on preparing a nucleic acid library from specified bodily-fluid samples without extracting nucleic acids, using dephosphorylation, denaturation, 3′-adapter attachment, primer extension to complementary strands, 5′-adapter attachment, and amplification. Dependent claims further narrow adapter attachment and add process control molecule spiking while preserving the no-extraction requirement.
Stated Advantages
Improved sensitivity and specificity for low-abundance and low-quality nucleic acids.
Reduced length, GC, and secondary-structure bias.
Reduced turnaround time and hazardous chemical use.
Reduced contamination effects and potentially elimination of negative controls.
Improved discrimination of endogenous versus environmental signal.
Ability to process lower sample volumes.
Ability to process both DNA/RNA and single- or double-stranded forms.
Improved normalization of recovery profiles using process controls.
Environmental contamination correction using fragment-length distribution modeling/deconvolution.
Improves library yield and short-fragment recovery in direct-to-library preparation.
Reduces environmental contamination.
Improves detection in clinical samples.
Documented Applications
Microbial/pathogen detection and quantification using process control molecules and recovery-profile normalization.
Environmental contamination correction based on fragment-length distribution modeling/deconvolution.
Personalized medicine.
Cancer detection, diagnosis, and prognosis.
Fetal health, including IVF embryo/fetus.
Organ transplant acceptance/rejection.
Microbe/pathogen detection and quantification, including DNA/RNA discrimination, latent versus active infection, and drug-resistance/risk assessment.
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