Granted Patent B2

US 12,522,869 · App. 18/534,929 · Granted Jan 13, 2026

Inventors: Richard P. Rava (Redwood City, CA); Manjula Chinnappa (Foster City, CA); David A. Comstock (Sunnyvale, CA); Gabrielle Heilek (Mountain View, CA); Brian Kent Rhees (Chandler, AZ)

Assignee: Verinata Health, Inc.

C12Q1/6869C12Q1/6806C12Q1/6809G16B20/10G16B30/10G16H10/40C12Q1/6883C12Q2600/106C12Q2600/112

Abstract

Claims (25)

1 . A method for nucleic acid sequencing comprising:

(a) providing a test sample comprising nucleic acid molecules, wherein said nucleic acid molecules are human cell-free DNA (cfDNA) molecules from a maternal plasma sample;

(b) performing end-repair of the nucleic acid molecules to generate blunt-ended nucleic acids;

(c) performing dA-tailing of the blunt-ended nucleic acids to generate dA-tailed nucleic acids;

(d) ligating adaptors to the dA-tailed nucleic acids to generate a library of adaptor-ligated polynucleotides; and

(e) subjecting the library to massively parallel sequencing;

wherein steps (b), (c), and (d) are consecutive steps that exclude purifying the end-repaired products prior to the dA-tailing step and exclude purifying the dA-tailing products prior to the adaptor-ligating step,

wherein said nucleic acid molecules of step (a), said blunt-ended nucleic acids of step (b), said dA-tailed nucleic acids of step (c), and said library of adaptor-ligated polynucleotides of step (d) are not subjected to fragmentation prior to consecutive steps (b), (c), and (d), and

wherein said consecutive steps (b), (c), and (d) are performed in less than 1 hour and in the same reaction tube.

2 . The method of claim 1 , wherein said consecutive steps (b), (c), and (d) are performed in the absence of polyethylene glycol.

3 . The method of claim 1 , wherein said massively parallel sequencing comprises solid phase amplification to create a high density sequencing flow cell with millions of clusters.

4 . The method of claim 1 , wherein the library is amplified on beads and wherein each bead comprises a forward and reverse amplification primer.

5 . The method of claim 1 , wherein:

said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators; or

said sequencing is massively parallel sequencing using sequencing-by-ligation.

6 . The method of claim 1 , wherein said sequencing comprises an amplification.

7 . The method of claim 1 , wherein said sequencing is single molecule sequencing.

8 . The method of claim 1 , further comprising amplifying the library using amplification primers prior to subjecting the library to massively parallel sequencing, said amplification primers comprising an adaptor-specific portion.

9 . The method of claim 1 , comprising obtaining sequencing reads of about 36 basepairs (bp) in length.

10 . The method of claim 1 , comprising amplifying a plurality of polymorphic target nucleic acids in a portion of the test sample.

11 . The method of claim 10 , wherein each of said plurality of polymorphic target nucleic acids comprises at least one single nucleotide polymorphism (SNP).

12 . The method of claim 11 , wherein said at least one SNP is a single SNP selected from rs560681, rs1109037, rs9866013, rs13182883, rs13218440, rs7041158, rs740598, rs10773760, rs4530059, rs7205345, rs8078417, rs576261, rs2567608, rs430046, rs99511 71, rs338882, rs10776839, rs9905977, rs1277284, rs258684, rs1347696, rs508485, rs9788670, rs8137254, rs3143, rs2182957, rs3739005, and rs530022.

13 . The method of claim 11 , wherein said at least one SNP is a tandem SNP selected from tandem SNP pairs rs7277033-rs2110153; rs2822654-rs1882882; rs368657-rs376635; rs2822731-rs2822732; rs1475881-rs7275487; rs1735976-rs2827016; rs447340-rs2824097; rs418989-rs13047336; rs987980-rs987981; rs4143392-rs4143391; rs1691324-rs13050434; rs11909758-rs9980111; rs2826842-rs232414; rs1980969-rs1980970; rs9978999-rs9979175; rs1034346-rs12481852; rs7509629-rs28283 58; rs481701 3-rs7277036; rs9981121-rs2829696; rs455921-rs2898102; rs2898102-rs458848; rs961301-rs2830208; rs2174536-rs458076; rs11088023-rs11088024; rs1011734-rs1011733; rs2831244-rs9789838; rs8132769-rs2831440; rs8134080-rs2831524; rs4817219-rs4817220; rs2250911-rs2250997; rs2831 899-rs2831900; rs2831902-rs2831903; rs11088086-rs2251447; rs2832040-rs11088088; rs2832141-rs2246777; rs2832959-rs9980934; rs2833734-rs2833735; rs933121-rs933122; rs2834140-rs12626953; rs2834485-rs3453; rs9974986-rs2834703; rs2776266-rs2835001; rs1984014-rs1984015; rs7281674-rs2835316; rs13047304-rs13047322; rs2835545-rs4816551; rs2835735-rs2835736; rs13047608-rs2835826; rs2836550-rs2212596; rs2836660-rs2836661; rs465612-rs8131220; rs9980072-rs8130031; rs418359-rs2836926; rs7278447-rs7278858; rs385787-rs367001; rs367001-rs386095; rs2837296-rs2837297; and rs2837381-rs4816672.

14 . The method of claim 10 , wherein each of said plurality of polymorphic target nucleic acids comprises at least one short tandem repeat (STR).

15 . The method of claim 14 , wherein said at least one STR is selected from CSF1PO, FGA, TH01, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D2S1338, Penta D, Penta E, D22S1045, D20S1082, D20S482, D18S853, D17S1301, D17S974, D14S1434, D12ATA63, D11S4463, D10S1435, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627, and D1GATA113.

Assignments (3)

Continuity (8)

References Cited (400)