High coverage stlfr
AssigneesMGI Tech Co., Ltd., Bgi Shenzhen
InventorsBrock A. Peters, Ou Wang, Radoje T. Drmanac
Life of this patent
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Abstract
Described herein are high coverage single tube Long Fragment Read (stLFR) technology which uses performs stLFR on target DNA fragments that have already been amplified before they are co-barcoded, which provides higher amount of DNA for sequencing and increases sequencing coverage. In some embodiments, the high coverage stLFR described in this application uses two rounds of stLFR. In some embodiments, the target DNA fragments are transposed with transposons having particular positional barcodes that can be used to order sequence reads.
Claims30
A method for preparing a library of barcoded polynucleotides for determining a sequence of a target nucleic acid(s) comprising
- (a) providing fragments derived from the target nucleic acid, wherein the fragments are double-stranded or partially double-stranded;
- (b) introducing staggered single-stranded breaks into at least some double-stranded fragments, thereby producing a plurality of first complexes, wherein each first complex comprises a plurality of first subfragments, and
- (c) associating first capture oligonucleotide sequences with at least some of the first subfragments,
- wherein each first capture oligonucleotide sequence comprises a first barcode, and optionally comprises a promoter sequence or a primer binding sequence, and
- wherein the associating comprises combining the double-stranded fragments in (a) or the complexes in (b) with a plurality of individual first beads, wherein each individual first bead comprises a plurality of first capture oligonucleotides immobilized thereon, wherein each capture oligonucleotides comprises a first capture oligonucleotide sequence, wherein the first capture oligonucleotides immobilized on each individual first bead comprises the same first capture oligonucleotide sequence, wherein a majority of different first beads have different first capture oligonucleotides immobilized thereon, and wherein each different first capture oligonucleotide sequence comprises a different first barcode,
- thereby providing barcoded first subfragments;
- (d) amplifying at least a portion of the barcoded first subfragments to produce amplified barcoded first subfragments, wherein the amplified barcoded first subfragments are double-stranded or partially double-stranded;
- (e) introducing staggered single-strand breaks into some of the amplified barcoded first subfragments to generate second complexes, each comprising a plurality of second subfragments;
- and
- (f) associating second capture oligonucleotide sequences with at least some of the second subfragments;
- wherein the associating comprises combining the amplified barcoded first subfragments in (d) or the second complexes in (e) with a plurality of individual second beads, wherein each individual second bead comprises a plurality of second capture oligonucleotides immobilized thereon, wherein each second capture oligonucleotide comprises a second capture oligonucleotide sequence, wherein the second capture oligonucleotides immobilized on each individual second bead comprises the same second capture oligonucleotide sequence, wherein a majority of different second beads have different second capture oligonucleotides immobilized thereon, and wherein each different second capture oligonucleotide sequence comprises a different second barcode,
- thereby providing a library of barcoded second subfragments.
7-10. (canceled)
12-25. (canceled)
(canceled)
A method of analyzing the full-length sequence of one or more target region comprising
- (a) amplifying each target region,
- (b) ligating an adaptor oligonucleotide to both ends of amplified nucleic acid target fragments comprising the target region,
- (c) introducing staggering single-stranded breaks in at least some of the amplified nuceic acid fragments from (b), to produce a plurality of first complexes each comprising a plurality of first subfragments,
- (d) introducing first capture oligonucleotides to at least some of the first subfragments, wherein each first capture oligonucleotides comprises
- (1) optionally a promoter sequence or a primer binding sequence, and
- (2) a first barcode, wherein first capture oligonucleotides immobilized on the same individual bead comprise the same first barcode, and a majority of beads have different first barcodes, thereby providing barcoded first subfragments.
29-38. (canceled)
41-43. (canceled)
A method of inserting oligonucleotides into fragments of a target nucleic acid comprising:
- (a) introducing staggered single-stranded breaks into the fragments,
- (b) contacting the fragments from (a) with an insertion scaffold, wherein the adaptors are anchored to the scaffold and separated by predetermined spacing, wherein the insertion scaffold comprises a plurality of double-stranded or partially double-stranded adaptors and a scaffold,
- wherein each adaptor comprises an insertion oligonucleotide comprising a unique positional barcode, and
- wherein the contacting results in the plurality of insertion oligonucleotides being introduced into the fragments at the single-stranded breaks, thereby producing first insertion complexes, each comprising a plurality of first subfragments.
The method of claim 41, wherein the second insertion complexes are mixed with a population of second beads, wherein each bead comprises second capture oligonucleotides immobilized hereon, said oligo nucleotides comprising a second barcode, wherein the oligonucleotides immobilized on the same individual bead comprise the same second barcode and a majority of beads have different second barcodes,
- (g) for each of at least some of the second insertion complexes, introducing multiple copies of the second capture oligonucleotide comprising a second barcode, wherein the multiple copies are from a single bead, and thereby producing a plurality of barcoded second subfragments, each is linked to at least one copy of the second barcode.
53-56 (canceled)
A plurality of insertion scaffolds,
- wherein each of a plurality of insertion scaffolds comprises
- (1) a plurality of adaptors, wherein the adaptors are double-stranded or partially double-stranded, and
- (2) a scaffold, and the adaptors are anchored to the scaffold and separated by predetermined spacing,
- wherein for each insertion scaffold, each adaptor in the insertion scaffold carries a unique positional barcode and a common scaffold barcode, and
- wherein adaptors in different insertion scaffolds have different scaffold barcodes.
58-59. (Canceled)
Dependencies are inferred from the claim text.