Delaying real-time sequencing

Patent metadata

Field	Value
Publication number	US-12473596-B2
Application number	US-202318120014-A
Country	US
Kind code	B2
Filing date	Mar 10, 2023
Priority date	Oct 24, 2013
Publication date	Nov 18, 2025
Grant date	Nov 18, 2025

Abstract

Methods, compositions, and systems are provided that allow for reliable sequencing of the initial sequence region of a sequence of interest. The methods of the invention allow for more reliable barcoding of subpopulations of nucleic acids to be sequenced.

First claim

What is claimed is:

1 . A composition comprising a plurality of circular nucleic acid templates, wherein each of the plurality of circular nucleic acid templates comprise:
an adaptor comprising a priming region and at least one modified base, a primer complementary to at least a portion of the priming region, an insert region comprising a sequence of interest, wherein the insert region is double stranded, and wherein the adaptor is a hairpin connected to both strands of the double stranded insert region at a first end of the double-stranded insert region, a hairpin adaptor at a second end of the double-stranded insert region; and a strand displacing polymerase bound to the template at the priming region to form a polymerase-template complex, wherein the modified base is between the priming region and the insert region, whereby, when the polymerase extends the primer, it extends through the modified base and then extends through the insert region, wherein the modified base slows the rate of incorporation.

2 . The composition of claim 1 , wherein the modified base is an unnatural base.

3 . The composition of claim 1 , wherein the modified base has a modified sugar backbone.

4 . The composition of claim 1 , wherein the modified base comprises hydroxymethylcytosine.

5 . The composition of claim 1 , wherein the modified base comprise glucosylated 5-hydroxymethylcytosine.

6 . The composition of claim 1 , wherein the stand displacing polymerase is at least 5x slower at extending past the modified base.

7 . The composition of claim 1 , wherein the adaptor comprises a plurality of modified bases.

8 . The composition of claim 1 , wherein the adaptor further comprises a runway region.

9 . The composition of claim 8 , wherein the runway region has a length that delays sequencing the insert region greater than about 60 seconds.

10 . The composition of claim 8 , wherein the length of the runway region is 200 nucleotides or greater.

11 . The composition of claim 8 , wherein each of the plurality of circular nucleic acid templates further comprises a barcode region between the runway region and the double-stranded insert region.

12 . The composition of claim 11 , wherein the plurality of circular nucleic acid templates comprises multiple sub-populations of circular nucleic acid templates, wherein the barcode region in each of the sub-populations of circular nucleic acid templates is different.

13 . The composition of claim 1 , wherein the adaptor comprises at least one modified base that is absent from the insert region.

14 . The composition of claim 1 , wherein the modified base comprises 6-mA.

15 . The composition of claim 1 , wherein the modified base comprises 8-oxoguanosine.

Assignees

• Pacific Biosciences California Inc

Inventors

• Clark Tyson A
• Korlach Jonas
• Heiner Cheryl
• Travers Kevin
• Miller Erik

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2527/113

  Time · CPC title

• C12Q2525/301

  Hairpin oligonucleotides · CPC title

• C12Q2525/204

  specific length of the oligonucleotides · CPC title