US 12,410,426 B2
Methods for generating, and sequencing from, asymmetric adaptors on the ends of polynucleotide templates comprising hairpin loops
Niall Anthony Gormley, Cambridge (GB)
Assigned to ILLUMINA CAMBRIDGE LIMITED, Cambridge (GB)
Appl. No. 17/600,046
Filed by Illumina Cambridge Limited, Cambridge (GB)
PCT Filed Oct. 23, 2020, PCT No. PCT/EP2020/079902
§ 371(c)(1), (2) Date Sep. 29, 2021,
PCT Pub. No. WO2021/078947, PCT Pub. Date Apr. 29, 2021.
Claims priority of provisional application 62/926,360, filed on Oct. 25, 2019.
Prior Publication US 2022/0259587 A1, Aug. 18, 2022
Int. Cl. C12Q 1/6806 (2018.01); C12N 15/10 (2006.01); C12Q 1/6869 (2018.01)
CPC C12N 15/1093 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6869 (2013.01)] 21 Claims
 
1. A method of generating an asymmetric double stranded nucleic acid template from tagmented DNA comprising complementary hairpin loops, comprising:
(I) generating tagmented DNA comprising complementary hairpin loops at the 5′ ends of each strand, wherein the hairpin loops comprise a base paired transposase recognition sequence, and wherein there is a gap of single stranded sequence between the 5′ ends and the 3′ ends of the tagmented DNA;
(II) filing in the gaps between the 5′ ends and the 3′ ends of the tagmented DNA using a gap-fill-ligation reaction to form closed ended tagmented DNA;
(III) generating a nick in the top strand at each hairpin region of the closed ended tagmented DNA;
(IV) extending from each nick a sequence complementary to the double stranded nucleic acid template using a processive polymerase to generate two long hairpin duplex templates, wherein one end of the duplex template comprises a closed hairpin (the “hairpin end”) and the other end of the duplex template comprises a 3′-strand end and a 5′-strand end (the “free end”); and
(V) closing the free end of each long hairpin duplex template to form an asymmetric closed-ended double stranded nucleic acid template by:
(a) ligating a nucleic acid-based hairpin or dumbbell adaptor to the free end of the duplex template; or
(b) using a TeIN Protelomerase to close the free end of the duplex template, wherein the free end of the duplex template is designed to include a TeIN recognition sequence.