Methods for rapid DNA extraction from tissue and library preparation for nanopore-based sequencing
Inventors
Williams, Samuel Zev • Wei, Shan
Assignees
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Publication Number
US-12630875-B2
Publication Date
2026-05-19
Expiration Date
Abstract
The present disclosure relates to improved methods for preparing, sequencing and analyzing short DNA fragments using handheld, nanopore-based sequencing technology as well as improved methods for extracting DNA, in particular genomic DNA, for any downstream application.
Core Innovation
The invention relates to a method of preparing or constructing a sequencing library for high-throughput nanopore sequencing using DNA fragments of 1000 bp or less. DNA is obtained from a sample and used at an amount of less than 90 ng. The library is prepared by incubating the DNA fragments with a Tn5 transposase and barcoded duplexes for a time and at a temperature to allow a reaction to occur.
Tn5-barcoded DNA fragments are then pooled and purified. After pooling and purifying the Tn5-barcoded DNA fragments, the method ligates the Tn5-barcoded DNA fragments to a sequencing adaptor, with no purification step following the ligation step. This approach is presented as part of expedited library preparation for nanopore sequencing of short DNA fragments.
The documented workflows combine handheld nanopore-based short-read sequencing with rapid DNA extraction and expedited library preparation that includes direct-ligation and tagmentation-based rapid nanopore sequencing (rapid tag). The background problem addressed is the need for rapid and high-throughput nanopore sequencing workflows, including reducing overall workflow time while improving multiplexing and throughput for short-read applications.
Claims Coverage
The independent claim is directed to a sequencing library preparation method for high-throughput nanopore sequencing, with five core inventive features.
Short DNA fragments under 1000 bp with less than 90 ng
Providing fragments of DNA having a size of 1000 bp or less, wherein the DNA was obtained from a sample, and wherein an amount of the DNA is less than 90 ng.
Tn5 transposase incubation with barcoded duplexes
Incubating the DNA fragments with a Tn5 transposase and barcoded duplexes for a time and at a temperature to allow a reaction to occur.
Pooling and purifying Tn5-barcoded DNA fragments
Pooling and purifying Tn5-barcoded DNA fragments.
Ligation to a sequencing adaptor without post-ligation purification
Ligating the Tn5-barcoded DNA fragments to a sequencing adaptor, wherein no purification step follows the ligation step.
Tn5-barcoded duplexes with defined barcode region or defined sequences (dependent)
Barcoded duplexes including a barcode region of about 8 bp and/or having nucleotide sequences selected from SEQ ID NOs.
Overall, the claim coverage centers on library preparation for high-throughput nanopore sequencing using short DNA fragments and low DNA input, generating Tn5-barcoded fragments via Tn5 transposase and barcoded duplexes, followed by pooling and purifying and then ligating to a sequencing adaptor with no purification step after ligation.
Stated Advantages
Reducing overall workflow time for nanopore sequencing by expediting DNA extraction and library preparation.
Improving multiplexing and throughput for short-read nanopore sequencing workflows.
Enabling rapid aneuploidy screening from chorionic villus samples, embryo biopsies, and products of conception.
Providing performance results for aneuploidy screening with concordance/sensitivity/specificity versus G-banding as reported in the document.
Supporting preliminary cancer panel screening using nanopore-compatible barcoding and variant calling in FFPE tissues as reported in the document.
Documented Applications
Rapid aneuploidy screening from chorionic villus samples (CVS), embryo samples / PGT-A, and products of conception (POC), with reported concordance/sensitivity/specificity versus G-banding.
Preliminary cancer panel screening in FFPE tissues using long nanopore-compatible barcoding adapters and variant calling, including lung/colorectal cancer panels.
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