US 12,410,469 B2
Methods and compositions for sequencing library normalization
Martin Ranik, Boulder, CO (US); Eric van der Walt, Cape Town (ZA); Clara Ross, Lafayette, CO (US); Craig Marshall, Boulder, CO (US); Lindsay Peterkin, Longmont, CO (US); Brian Kudlow, Boulder, CO (US); and Travis J. Sanders, Boulder, CO (US)
Assigned to Watchmaker Genomics, Inc., Boulder, CO (US)
Filed by Watchmaker Genomics, Inc., Boulder, CO (US)
Filed on Oct. 23, 2023, as Appl. No. 18/492,763.
Application 18/492,763 is a continuation of application No. PCT/US2023/077435, filed on Oct. 20, 2023.
Claims priority of provisional application 63/516,033, filed on Jul. 27, 2023.
Claims priority of provisional application 63/380,488, filed on Oct. 21, 2022.
Prior Publication US 2024/0132942 A1, Apr. 25, 2024
Prior Publication US 2024/0229115 A9, Jul. 11, 2024
Int. Cl. C12Q 1/6834 (2018.01); C12N 9/22 (2006.01)
CPC C12Q 1/6834 (2013.01) [C12N 9/22 (2013.01); C12N 2310/20 (2017.05); C12Q 2600/166 (2013.01)] 25 Claims
 
1. A method for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the method comprising, for each sample of the at least two samples:
(i) obtaining the sample, wherein the target polynucleotides of the sample comprise:
a first adapter sequence comprising a proximal portion, and a distal portion comprising a sequence that binds to an adapter binding site of a next-generation sequencing platform during next-generation sequencing; and
a second adapter sequence;
(ii) contacting the target polynucleotides with primers that are complementary to the proximal portion of the first adapter sequence;
(iii) extending the primers to produce reverse complements of the target polynucleotides that do not comprise a reverse complement of the distal portion of the first adapter sequence, thereby producing partially double stranded polynucleotides comprising a double stranded second adapter sequence;
(iv) producing a solution, the producing comprising combining: (a) the partially double stranded polynucleotides, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to a strand of the double stranded second adapter sequence; and (c) a predetermined concentration of a dCas or a dArgonaute comprising an affinity tag, wherein the dCas or the dArgonaute is cognate to the guide polynucleotide;
(v) contacting the solution with a solid phase comprising an affinity tag binding molecule that is capable of binding to the affinity tag;
(vi) separating the solution from the solid phase; and
(vii) extracting the target polynucleotides from the solid phase to normalize the concentration of target polynucleotides between the two or more samples.