Hybrid targeted and whole transcriptome amplification
Inventors
Shum, Eleen • Fan, Christina • Walczak, Elisabeth Marie
Assignees
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Publication Number
US-12071617-B2
Publication Date
2024-08-27
Expiration Date
Abstract
Disclosed herein include systems, methods, compositions, and kits for the labeling of nucleic acids targets. In some embodiments, the methods comprise the use of target-specific primers and target-non-specific primers during library preparation (e.g., hybrid library preparation). In some embodiments, the methods increase the abundance of select low abundance targets during cDNA library preparation and/or sequencing library preparation.
Core Innovation
The disclosure describes a method of labeling nucleic acid targets by hybridizing a plurality of nucleic acid targets with oligonucleotides that each include a first universal sequence and a barcode sequence. The hybridized oligonucleotides are extended to generate first strand barcoded polynucleotides, and second strand barcoded polynucleotides are synthesized to generate double-stranded barcoded polynucleotides.
An adaptor comprising a second universal sequence is added to the double-stranded barcoded polynucleotides. The double-stranded barcoded polynucleotides are amplified using primers capable of hybridizing to the first universal sequence and the second universal sequence to generate a first plurality of barcoded amplicons, and using primers capable of hybridizing to two or more of the plurality of nucleic acid targets and a primer capable of amplifying the first universal sequence to generate a second plurality of barcoded amplicons.
The method further includes random priming and extension using random primers and the first plurality of barcoded amplicons and the second plurality of barcoded amplicons as templates. The disclosure frames this hybrid labeling workflow as applicable to nucleic acid targets including mRNAs, including low-expressing gene mRNAs, and as increasing relative abundance and sequencing read allocation while preserving overall expression and protein profiles and correlation (R2).
Claims Coverage
The provided independent claim coverage defines one hybrid labeling method with first universal sequence and barcode hybridization, second-universal adaptor addition, generation of first and second pluralities of barcoded amplicons, and random priming and extension using both pluralities as templates. The inventive features are those recited in the method steps.
Universal-sequence barcoded hybridization of multiple targets
Hybridizing a plurality of nucleic acid targets with oligonucleotides each comprising a first universal sequence and a barcode sequence, where the plurality of nucleic acid targets comprises two or more nucleic acid targets.
First-strand barcoded polynucleotide extension
Extending the hybridized oligonucleotides to generate first strand barcoded polynucleotides.
Second-strand synthesis to form double-stranded barcoded polynucleotides
Synthesizing second strand barcoded polynucleotides using the first strand barcoded polynucleotides as templates to generate double-stranded barcoded polynucleotides.
Adaptor with second universal sequence addition
Adding an adaptor to the double-stranded barcoded polynucleotides, where the adaptor comprises a second universal sequence.
Dual-plurality barcoded amplification using universal-sequence primers
Amplifying the double-stranded barcoded polynucleotides using primers capable of hybridizing to the first universal sequence and the second universal sequence to generate a first plurality of barcoded amplicons, and using primers capable of hybridizing to two or more of the plurality of nucleic acid targets and a primer capable of amplifying the first universal sequence to generate a second plurality of barcoded amplicons.
Random priming and extension from barcoded amplicons
Performing random priming and extension using random primers and the first plurality of barcoded amplicons and the second plurality of barcoded amplicons as templates.
The inventive concept is a hybrid workflow that attaches a first universal sequence and barcode to nucleic acid targets, converts this to double-stranded barcoded polynucleotides with a second universal adaptor, amplifies into first and second pluralities of barcoded amplicons using different primer logics, and performs random priming and extension using both amplified pluralities as templates.
Stated Advantages
Increases relative abundance and sequencing read allocation for low and intermediate abundance targets.
Preserves overall expression and protein profiles.
Maintains high correlation (R2) for expression and protein profiles.
Documented Applications
Single-cell RNA sequencing (scRNA-seq) barcoded sequencing for nucleic acid targets including mRNAs, including low-expressing gene mRNAs.
Barcoded sequencing workflows that use hybrid targeted and whole-transcriptome amplification approaches, including whole-transcriptome amplification (WTA) products and a second target-enriched plurality.
Hybrid versus non-hybrid nucleic acid cDNA/sequencing library workflows intended for low abundance and intermediate abundance targets to increase sequencing read allocation while preserving overall expression and protein profiles, including correlation (R2).
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