US 12,071,659 B2
Multiple tagging of long DNA fragments
Radoje Drmanac, Los Altos Hills, CA (US); Brock A. Peters, San Francisco, CA (US); and Andrei Alexeev, Woodland, CA (US)
Assigned to Complete Genomics, Inc., San Jose, CA (US)
Filed by Complete Genomics, Inc., San Jose, CA (US)
Filed on Mar. 26, 2018, as Appl. No. 15/936,123.
Application 15/936,123 is a continuation of application No. 14/782,307, granted, now 10,557,166, previously published as PCT/US2014/030649, filed on Mar. 17, 2014.
Application 14/782,307 is a continuation in part of application No. 14/205,145, filed on Mar. 11, 2014, granted, now 9,328,382, issued on May 3, 2016.
Claims priority of provisional application 61/801,052, filed on Mar. 15, 2013.
Prior Publication US 2019/0002969 A1, Jan. 3, 2019
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6869 (2018.01); C12N 15/10 (2006.01); C12Q 1/6806 (2018.01)
CPC C12Q 1/6869 (2013.01) [C12Q 1/6806 (2013.01); C12N 15/1065 (2013.01); C12Q 2525/204 (2013.01)] 24 Claims
 
1. A method of producing tagged long fragments comprising target DNA sequences in a homogeneous reaction without the use of physical compartmentalization, the method comprising:
(a) obtaining a plurality of double-stranded long fragments comprising target DNA sequences;
(b) inserting multiple copies of a common adaptor sequence into at least some long fragments of the plurality of double-stranded long fragments to produce a plurality of adaptored long fragments that comprise the target DNA sequences and the common adaptor sequence, wherein said inserting comprises nicking on both strands of said at least some long fragments to produce nick sites with free 3′ termini, and
i) introducing gaps at the nick sites and ligating the common adaptor sequence at the free 3′ termini, or
ii) using controlled nick translation (CNT) to translate the nick sites and ligating the common adaptor sequence at 3′ termini produced by the CNT;
(c) combining at least some adaptored long fragments of the plurality of adaptored long fragments in a single mixture with a population of beads under conditions that promote interaction of individual adaptored long fragments with single beads, thereby producing a plurality of individual adaptored long fragments that are each associated with a single bead, wherein
(i) each bead in the population of beads comprises at least 1000 copies of the same oligonucleotide immobilized thereon, said oligonucleotide comprising a tag-containing sequence and a sequence complementary to the common adaptor sequence,
(ii) each tag-containing sequence comprises a tag sequence, and
(iii) the population of beads comprises, in aggregate, at least 100,000 different tag sequences;
(d) producing tagged long fragments in the single mixture, wherein said producing comprises incorporating into each of a plurality of the individual adaptored long fragments multiple copies of the tag sequence from the single bead with which the individual adaptored long fragment is associated.