Haplotagging—haplotype phasing and single-tube combinatorial barcoding of nucleic acid molecules using bead-immobilized TN5 transposase

Inventors

CHAN, Yingguang Frank • KUCKA, Marek • DREAU, Andreea

Assignees

Max-Planck-Gesellschaft zur Förderung der Wissenschaften eV

Abstract

The present invention relates to methods for producing solid supports. The present invention further provides a mixture of said solid supports for tagmentation of target DNA for DNA sequencing approaches, a corresponding kit comprising the same and methods employing said mixture of solid supports and/or kit. Specifically, methods for producing sequencing libraries and corresponding DNA sequencing methods for analyzing the generated sequencing libraries and tools used therein are provided. In particular, DNA sequencing approaches allowing preservation of contiguity information of long DNA fragments even when using short read sequencing approaches are disclosed. A key concept of the present invention is to employ segmented barcodes, with every barcode segmented allowing for barcode error detection and correction on a segment level. Preferred barcode sequences employed are characterized in that they comprise no linker sequences or only linker sequences of one or two nucleotides in length between the barcode segments.

Core Innovation

The invention relates to haplotagging, using short-read sequencing to preserve long-range contiguity information by associating DNA fragments that originate from an individual contiguous target DNA molecule with a common DNA barcode tag. The approach is positioned as a way to assign haplotypes while maintaining contiguity using barcoded indexing compatible with index reads and demultiplexing.

A mixture of at least one million solid supports is provided, where each solid support carries multiple identical copies of a solid-support-specific set of two transposons having a transfer strand and a non-transfer strand. Each transposon is configured so that a transposase can bind to the 3′ end of the transfer strand, and the non-transfer strands are 5′ phosphorylated.

The distinguishing barcode tag on each solid support consists of first and second barcode sequences B1 and B2 included in adapters A1 and A2. The barcode architecture uses a segmented barcode structure in which B1 comprises z barcode segments and B2 comprises g barcode segments, with predefined barcode nucleic acid sequences that pairwise differ in at least two nucleotide positions, enabling detecting and error-correcting barcode segments and assigning fragments that share identical barcode tags to a contiguous target DNA molecule.

The patent also describes producing solid supports with directly linked segmented DNA barcode sequences by providing solid supports bearing single stranded DNA oligonucleotides with barcode segment A, ligating predefined polynucleotides B, and removing strands originating from the single stranded section by exonuclease digestion. In another described workflow, barcode-tagged DNA sequencing libraries are generated via on-bead tagmentation and transposon-mediated strand end ligations, followed by sequencing and computational error detection and correction on each barcode segment.

Claims Coverage

The independent claims cover four inventive features: a solid-support mixture with segmented barcode architectures, on-bead tagmentation and transposon-mediated end ligation for barcode-tagged sequencing libraries, sequencing-based detection with error detection and correction to assign barcode tags and contiguity, and production of solid supports bearing directly linked segmented DNA barcode sequences.

Across the independent claims, the coverage centers on segmented barcode architectures embedded in adapter-associated transposons on solid supports, coupled to sequencing workflows that detect and error-correct barcode segments to assign barcode tags and contiguity, and on producing solid supports with directly linked segmented DNA barcode sequences.

Stated Advantages

Documented Applications