US 11,773,511 B2
Immune profiling by primer extension target enrichment
Jan Berka, Pleasanton, CA (US); Carolina Dallett, Oakland, CA (US); Richard Dannebaum, Pleasant Hill, CA (US); Brian Godwin, Livermore, CA (US); Seoyoung Kim, Foster City, CA (US); Jainee Lewis, Oakland, CA (US); Khai Luong, Oakland, CA (US); Sedide Ozturk, Pleasanton, CA (US); Joseph Platzer, Oakland, CA (US); Florian Rubelt, Menlo Park, CA (US); and Dilduz Telman, Sunnyvale, CA (US)
Assigned to Roche Sequencing Solutions, Inc., Pleasanton, CA (US)
Filed by Roche Sequencing Solutions, Inc, Pleasanton, CA (US)
Filed on Aug. 18, 2020, as Appl. No. 16/996,733.
Application 16/996,733 is a continuation in part of application No. 15/611,507, filed on Jun. 1, 2017, granted, now 11,098,360.
Claims priority of provisional application 62/344,330, filed on Jun. 1, 2016.
Prior Publication US 2021/0172015 A1, Jun. 10, 2021
Int. Cl. C40B 50/06 (2006.01); C12Q 1/68 (2018.01); C12Q 1/6881 (2018.01); C12N 15/10 (2006.01); C12Q 1/6886 (2018.01)
CPC C40B 50/06 (2013.01) [C12N 15/1065 (2013.01); C12Q 1/68 (2013.01); C12Q 1/6881 (2013.01); C12Q 1/6886 (2013.01); C12Q 2600/16 (2013.01)] 19 Claims
OG exemplary drawing
 
1. A method for enriching a sample for a plurality of structurally different target polynucleotides comprising an immune gene sequence the method comprising:
a) contacting a sample with a plurality of immune cell receptor V gene specific primers, each primer including from 5′ to 3′: [SPLINT1], [BARCODE], and [V], wherein: [SPLINT] is a first adaptor sequence; [BARCODE] is a unique molecular identifier barcode; and [V] is a sequence capable of hybridizing to an immune cell receptor V gene;
b) hybridizing and extending the V gene specific primers to form a plurality of first double-stranded primer extension products;
c) contacting the sample with an exonuclease to remove unhybridized V gene specific primers from the first double stranded primer extension products;
d) contacting the sample with a plurality of immune cell receptor J gene specific primers, each primer including from 5′ to 3′: [SPLINT2], and [J], wherein: [SPLINT2] is a second adaptor sequence; and [J] is a sequence capable of hybridizing to an immune cell receptor J gene; and further contacting the sample with a first universal primer capable of hybridizing to the first adaptor sequence;
e) hybridizing and extending the J gene specific primers and the first universal primer to form a plurality of second double-stranded primer extension products;
f) contacting the sample with an exonuclease to remove unhybridized J gene specific primers and first universal primer from the second double-stranded primer extension products;
g) contacting the sample with first and second universal primers capable of hybridizing to the first and second adaptor sequences;
h) amplifying the plurality of second double-stranded primer extension products thereby enriching the plurality of structurally different target polynucleotides comprising an immune gene sequence.