Barcoded molecular standards
Inventors
KINDE, Isaac A. • Kaufman, Howard B. • HAGMANN, Leonardo
Assignees
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Publication Number
US-12091705-B2
Publication Date
2024-09-17
Expiration Date
Abstract
High throughput personal genomic testing has created a need for robust quality control mechanisms to track sample identity, reagent integrity, and other factors with significant influence on assay performance. A method of massively parallel sequencing using an accompanying barcoded molecular standard enables one to track nucleic acid analytes to identify them by project, lot, batch, or patient. The molecular standard contains sequences present in the analyte, allowing it to be processed simultaneously without any other additional reagents. Within the molecular standard, a calibrator sequence permits assessment of fidelity of sequence determination. Additional sequences in the molecular standard may be used to manipulate the molecular standard separate from the analyte. The molecular standard can be used to benchmark sequencing platforms and assess error rates.
Core Innovation
The invention relates to barcoded molecular standards for massively parallel sequencing (MPS), including a calibrator DNA molecule that is co-amplified with a DNA analyte in the same reaction vessel. The calibrator is amplified using a pair of primers that are complementary to a first and a second strand of the DNA analyte at respective ends of a region to be amplified of the DNA analyte. The primers also interact with two non-contiguous, primer-complementary regions on opposite strands of the calibrator double-stranded DNA nucleic acid molecule.
The calibrator double-stranded DNA molecule includes an input calibrator region that is distinct from the region of the DNA analyte to be amplified, and the input calibrator region is located between the two non-contiguous regions. The calibrator further includes a pair of priming sites distal to and surrounding the two non-contiguous regions, where the pair of priming sites is not present in the analyte DNA and is capable of priming amplification of the calibrator nucleic acid molecule without priming amplification of the DNA analyte.
The disclosed barcoded molecular standards use barcodes to enable identity tracking by patient/project/batch/lot, including a unique molecular barcode associated with the calibrator. The calibrator can include configurable sequence features associated with amplification and sequencing errors, including error-prone motifs and regions such as homopolymer repeats, tandem repeats, and GC-rich regions.
Claims Coverage
The provided material centers on one independent kit claim with inventive calibrator structure and primer interactions that prevent calibrator priming from priming the analyte. Dependent claims refine the calibrator with modified nucleotide bases, distinct unique molecular barcodes, damaged DNA, GC-rich content, and homopolymer repeat constraints.
Kit with calibrator DNA and primers complementary to analyte ends
A kit comprising a calibrator double-stranded DNA nucleic acid molecule and a pair of primers, where the pair of primers is complementary to a first and a second strand of a DNA analyte at respective ends of a region to be amplified of the DNA analyte.
Calibrator structure with two non-contiguous primer-complementary regions
The calibrator double-stranded DNA nucleic acid molecule comprises two non-contiguous regions respectively complementary to the primers on opposite strands.
Distinct input calibrator region between non-contiguous regions
The calibrator includes an input calibrator region that is distinct from the region of the DNA analyte to be amplified, with the input calibrator region located between the two non-contiguous regions.
Primer-surrounding priming sites distal from analyte and capable of calibrator-only priming
The calibrator includes a pair of priming sites distal to and surrounding the two non-contiguous regions, where the pair of priming sites is not present in the analyte DNA and is capable of priming amplification of the calibrator nucleic acid molecule without priming amplification of the DNA analyte.
Modified nucleotide bases in the calibrator
The kit further includes one or more modified nucleotide bases in the calibrator molecule that are not naturally occurring in the DNA analyte.
Multiple calibrator molecules with distinct unique molecular barcodes
The kit further includes a plurality of calibrator nucleic acid molecules having distinct unique molecular barcodes.
Damaged DNA as the input calibrator region
The input calibrator region comprises damaged DNA.
GC-rich input calibrator region with overall GC content constraint
The input calibrator region has an overall GC content over 60%.
Homopolymer repeat constraint in the input calibrator region
The input calibrator region contains a homopolymer repeat consisting of at least 7 identical nucleotides in a sequence.
Overall, the claim coverage is centered on a kit architecture where primers that amplify a DNA analyte region also engage a calibrator structured with two non-contiguous primer-complementary regions on opposite strands, while a distinct input calibrator region and distal priming sites prevent priming amplification of the analyte DNA. Dependent claims further refine the calibrator by specifying damaged DNA, error-prone sequence characteristics, modified nucleotide bases, and multiple calibrator molecules with distinct unique molecular barcodes.
Stated Advantages
Identity tracking by patient/project/batch/lot using barcodes, including a unique molecular barcode.
Assay fidelity and error-rate determination by comparing input versus output calibrator region sequencing.
Monitoring amplification, purification, lysis, and sequencing errors using configurably error-prone calibrator sequence features.
Benchmarking platforms and tuning base-calling or bias models.
Documented Applications
Calibration for massively parallel sequencing by co-amplifying a calibrator DNA molecule with a DNA analyte and sequencing the input versus output calibrator region to determine assay fidelity and error-rate.
Monitoring amplification, purification, lysis, and sequencing errors in massively parallel sequencing by using calibrator sequence features associated with error-prone motifs and regions.
Benchmarking platforms and tuning base-calling or bias models for massively parallel sequencing.
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