Linked dual barcode insertion constructs

Patent metadata

Field	Value
Publication number	US-12553077-B2
Application number	US-202018027085-A
Country	US
Kind code	B2
Filing date	Oct 1, 2020
Priority date	Oct 1, 2020
Publication date	Feb 17, 2026
Grant date	Feb 17, 2026

Abstract

Contemporary gene sequencing techniques, including “Next Generation Sequencing” techniques, can include sequencing a plurality of fragments of a target polynucleotide. However, tire limitations of existing sequencing techniques, and the often repetitive or otherwise difficult-to-sequence structure of natural polynucleotides, means that it can be difficult and/or expensive to generate accurate sequences. Methods provided herein include inserting dual polynucleotide ‘barcodes,’ along with neighboring primer sequences, into a target polynucleotide prior to other sequencing processes. These inserted barcodes can improve the accuracy of sequences generated for the target by adding ‘noise’ into the target, allowing subsequent sequencing techniques (e.g., alignment, stitching, etc.) to more accurately estimate the target-plus-barcodes sequence. The primers can cause the fragments to begin at points within the target that correspond to the beginning of other sequences, facilitating the stitching of sequence ends together. The barcodes can then be removed to provide the sequence of the target polynucleotide.

First claim

What is claimed is:

1 . A method, comprising:
adding a probe to a sample that contains a target polynucleotide, wherein the probe comprises a payload polynucleotide and an insertion vector, wherein the payload polynucleotide comprises a first polynucleotide barcode, a reverse primer, a forward primer, and a second polynucleotide barcode, and wherein the insertion vector inserts the payload polynucleotide into the target polynucleotide; applying an amplification agent to generate: (i) a first copy of the target polynucleotide, with the payload polynucleotide inserted, beginning from the reverse primer such that the first copy includes a complement of the first polynucleotide barcode proximate to an end of the first copy, and (ii) a second copy of the target polynucleotide, with the payload polynucleotide inserted, beginning from the forward primer such that the second copy includes a complement of the second polynucleotide barcode proximate to an end of the second copy; subsequent to adding the probe to the sample, sequencing at least a portion of the sample a plurality of times to obtain a read of the first copy of the target polynucleotide and a read of the second copy of the target polynucleotide; subsequent to applying the amplification agent, inserting, into the target polynucleotide, an additional plurality of polynucleotide barcodes to obtain additional copies of the target polynucleotide with the additional plurality of polynucleotide barcodes inserted therein; and subsequent to inserting the additional plurality of polynucleotide barcodes into the target polynucleotide, sequencing at least a portion of the sample a plurality of times to obtain reads of the additional copies of the target polynucleotide, wherein determining the sequence for the target polynucleotide comprises determining the sequence based on the read of the first copy of the target polynucleotide, the read of the second copy of the target polynucleotide, and the reads of the additional copies of the target polynucleotide.

2 . The method of claim 1 , wherein the insertion vector comprises Tn5 transposase.

3 . The method of claim 1 , wherein the insertion vector comprises CRISPR-Cas9.

4 . The method of claim 1 ,
wherein determining the sequence for the target polynucleotide comprises:
determining a preliminary sequence for the target polynucleotide based on the read of the first copy and the read of the second copy; and
removing the sequence of the first and second polynucleotide barcodes from the preliminary sequence.

5 . The method of claim 4 , wherein determining the preliminary sequence comprises stitching together the end of the first copy to the end of the second copy.

6 . The method of claim 5 , further comprising:
sequencing the probe to generate a probe sequence; and based on the probe sequence, determining respective sequences for the first and second polynucleotide barcodes and determining that the first and second polynucleotide barcodes correspond to each other, wherein stitching together the end of the first copy to the end of the second copy is performed responsive to determining that the first copy contains the determined sequence for the first polynucleotide barcode proximate to the end of the first copy and that the second copy contains the determined sequence for the second polynucleotide barcode proximate to the end of the second copy.

7 . The method of claim 4 , further comprising:
prior to adding the probe to the sample, sequencing at least a portion of the sample a plurality of times to obtain a plurality of unmodified reads of the target polynucleotide, wherein removing the first and second polynucleotide barcodes from the preliminary sequence comprises comparing the preliminary sequence to at least one read of the plurality of unmodified reads of the target polynucleotide.

8 . The method of claim 1 , wherein the target polynucleotide comprises DNA.

9 . The method of claim 1 , wherein the target polynucleotide comprises RNA, wherein the target polynucleotide is a first isoform of an RNA sequence, and wherein the sample contains a second isoform of the RNA sequence, and wherein the first isoform differs from the second isoform.

10 . The method of claim 1 , wherein the first and second polynucleotide barcodes have the same sequence.

11 . The method of claim 1 , wherein the second polynucleotide barcode is a reversed, complementary version of the first polynucleotide barcode.

12 . A method for generating a payload polynucleotide, the method comprising:
obtaining a template polynucleotide that includes a first polynucleotide barcode and a first amplification primer; forming, on the template polynucleotide, a complementary polynucleotide such that the complementary polynucleotide includes a second amplification primer and a second polynucleotide barcode; forming a hairpin between the template polynucleotide and the complementary polynucleotide; and dissociating the complementary polynucleotide from the template polynucleotide via a denaturing process.

13 . A non-transitory computer readable medium having stored therein instructions executable by a computing device to cause the computing device to perform operations to determine a sequence for a target polynucleotide based on a read of a first copy of the target polynucleotide, a read of a second copy of the target polynucleotide, and reads of additional copies of the target polynucleotide, wherein the read of the first copy of the target polynucleotide, the read of the second copy of the target polynucleotide, and the reads of the additional copies of the target polynucleotide are obtained by:
adding a probe to a sample that contains the target polynucleotide, wherein the probe comprises a payload polynucleotide and an insertion vector, wherein the payload polynucleotide comprises a first polynucleotide barcode, a reverse primer, a forward primer, and a second polynucleotide barcode, and wherein the insertion vector inserts the payload polynucleotide into the target polynucleotide; applying an amplification agent to generate: (i) the first copy of the target polynucleotide, with the payload polynucleotide inserted, beginning from the reverse primer such that the first copy includes a complement of the first polynucleotide barcode proximate to an end of the first copy, and (ii) the second copy of the target polynucleotide, with the payload polynucleotide inserted, beginning from the forward primer such that the second copy includes a complement of the second polynucleotide barcode proximate to an end of the second copy; subsequent to adding the probe to the sample, sequencing at least a portion of the sample a plurality of times to obtain the read of the first copy of the target polynucleotide and the read of the second copy of the target polynucleotide; subsequent to applying the amplification agent, inserting, into the target polynucleotide, an additional plurality of polynucleotide barcodes to obtain additional copies of the target polynucleotide with the additional plurality of polynucleotide barcodes inserted therein; and subsequent to inserting the additional plurality of polynucleotide barcodes into the target polynucleotide, sequencing at least a portion of the sample a plurality of times to obtain the reads of the additional copies of the target polynucleotide; wherein determining the sequence for the target polynucleotide comprises: determining a preliminary sequence for the target polynucleotide based on the read of the first copy and the read of the second copy; and removing the sequence of the first and second polynucleotide barcodes from the preliminary sequ

Assignees

• Google Llc

Inventors

• Bashir Ali
• Berndl Marc
• Pawlosky Annalisa
• Kim Jun

Classifications

• G16B30/10

  Sequence alignment; Homology search · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title