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Publication Number
US-12319910-B2
Publication Date
2025-06-03
Expiration Date
Abstract
Methods and compositions are provided herein for preparing high-throughput cDNA sequencing libraries.
Core Innovation
The invention relates to a method for tagging a cDNA polynucleotide using a double-stranded mRNA:cDNA hybrid in which the 5′ end of a first strand cDNA polynucleotide comprises a first adapter sequence or complement thereof. One or more second strand cDNA polynucleotides are synthesized to be complementary to and hybridized to the first strand cDNA polynucleotide by hybridizing random primers to the first strand cDNA and extending the hybridized random primers. The resulting double-stranded cDNA comprises a first end and a second end.
The method then contacts the double-stranded cDNA polynucleotide with an adapter-loaded tagmentase to form a reaction mixture comprising a tagged double-stranded cDNA polynucleotide. In the tagged double-stranded cDNA, the first end comprises the first adapter sequence and the second end comprises a second adapter sequence. The adapter-loaded tagmentase may be homo- or heteroadapter-loaded.
The invention further includes forming partitions that comprise amplification primers including at least one primer comprising a partition-specific barcode and the first strand cDNA and the second strand cDNAs. The first and second strand cDNAs are then amplified with the amplification primers. In described embodiments reflected in dependent claims, the amplification can be selectively performed to generate a sequencing-platform-specific cDNA amplicon, with amplification primers that hybridize to adapter sequences and add sequencing-platform-specific adapter sequences.
Claims Coverage
The provided independent claim set out a multi-stage tagging workflow with four inventive features: adapter positioning at the 5′ end, random-primed second-strand synthesis, adapter-loaded tagmentase tagging at both ends, and partitioning with barcode-containing amplification primers followed by amplification.
Adapter at the 5′ end of first-strand cDNA in an mRNA:cDNA hybrid
A double-stranded mRNA:cDNA hybrid comprising a first strand cDNA polynucleotide hybridized to a complementary mRNA, where the 5′ end of the first strand cDNA polynucleotide comprises a first adapter sequence or complement thereof.
Random-primed synthesis of complementary second-strand cDNA
Synthesizing one or more second strand cDNA polynucleotides complementary to and hybridized to the first strand cDNA polynucleotide by hybridizing random primers to the first strand cDNA of the mRNA:cDNA hybrid and extending the hybridized random primers.
Adapter-loaded tagmentase tagging to add adapter sequences at both ends
Contacting the double-stranded cDNA polynucleotide with an adapter-loaded tagmentase to form a reaction mixture comprising a tagged double-stranded cDNA polynucleotide with a first end comprising the first adapter sequence and a second end comprising a second adapter sequence.
Partitioning with partition-specific barcoded amplification primers and amplifying first and second strands
Forming partitions comprising amplification primers including at least one primer with a partition-specific barcode and the first strand cDNA and the second strand cDNAs, and amplifying the first and second strand cDNAs with the amplification primers.
Selective primer scheme generating sequencing-platform-specific cDNA amplicons
Using amplification primers comprising first and second primers that selectively hybridize to first and second adapter sequences, and adding sequencing-platform-specific adapter sequences to produce a sequencing-platform-specific cDNA amplicon.
Homoadapter-loaded tagmentase configuration
A homoadapter-loaded tagmentase carrying two identical polynucleotide adapters to add one adapter to the second end of the double-stranded cDNA in the reaction mixture.
Heteroadapter- or structurally distinct-adapter tagmentase configuration
A heteroadapter-loaded tagmentase having two structurally distinct polynucleotide adapters or two homoadapter-loaded tagmentases with structurally distinct adapters, to attach one polynucleotide adapter to the second end of the double-stranded cDNA.
Partition format as droplets in an emulsion
The partitions are droplets in an emulsion.
Cell-input constraint for mRNA quantity in the DNA polymerase reaction
Performing an mRNA template-directed DNA polymerase reaction in a reaction mixture containing mRNA from at least 10 cells.
The core coverage centers on adapter positioning at the 5′ end of first-strand cDNA within an mRNA:cDNA hybrid, complementary second-strand synthesis via random-primed extension, end tagging using adapter-loaded tagmentase, and partitioning with barcode-containing amplification primers followed by amplification. Additional dependent features specify sequencing-platform-specific cDNA amplicons, tagmentase adapter arrangements, partition format, and mRNA quantity constraints.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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