HIGH MULTIPLEX PCR WITH MOLECULAR BARCODING

• US 20180002738A1
• Filed: 01/21/2016
• Published: 01/04/2018
• Est. Priority Date: 01/23/2015
• Status: Active Grant

• Current Assignee: QIAGEN Sciences, LLC (Qiagen NV)
• Original Assignee: QIAGEN Sciences, LLC (Qiagen NV)
• Inventors: Wang, Yexun, Peng, Quan
• Granted Patent: US 10,760,120 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The present disclosure provides methods and kits for performing high multiplex PCR using molecular barcodes. The methods disclosed herein separately extend a set of primers (BC primers) that each comprise a target-specific sequence, a molecular barcode and a universal sequence, and amplify the resulting extension products using another set of primers (LA primers) that each comprise another target-specific sequence and a universal sequence. The methods may further comprise amplification using universal primers (preferably comprising an adapter).

Claims

1. A method for amplifying target nucleic acids in a nucleic acid sample, comprising:

(a) extending each of a plurality of barcode primers (BC primers) to obtain extension products using the target nucleic acids as templates, wherein

(i) each barcode primer comprises, from 5′

to 3′

, a 1^st universal primer sequence (US1), a molecular tag sequence (MT), and a 1^st target-specific sequence (TS1),

(ii) a plurality of barcode primers comprise at least 20 different barcode primers, and

(iii) among the plurality of barcode primers (BC primers), the 1^st universal primer sequences (US1) are the same, but the 1^st target-specific sequences (TS1) are different;

(b) separating the plurality of barcode primers that have not been extended in step (a) from the extension products; and

(c) amplifying the extension products of step (b) in the presence of a plurality of limited amplification primers (LA primers) to obtain a plurality of 1^st amplification products, wherein

(i) each limited amplification primer comprises, from 5′

to 3′

, a 2^nd universal primer sequence (US2) and a 2^nd target-specific sequence (TS2), and

(ii) among the plurality of limited amplification primers, the 2^nd universal primer sequences (US2) are the same, but the 2^nd target-specific sequences (TS2) are different.

2. The method of claim 1, further comprising:

(d) separating the plurality of unused limited amplification primers (LA primers) that have not been extended in step (c) from the 1^st amplification products.

3. The method of claim 2, further comprising:

(e) amplifying the 1^st amplification products in the presence of a pair of universal primers to obtain 2^nd amplification products, wherein one of the universal primers comprises at its 3′

-terminus the 1^st universal primer sequence (US1), and the other of the universal primers comprises at its 3′

-terminus the 2^nd universal primer sequence (US2).

4. The method of claim 3, wherein each of the universal primers comprises an adapter sequence.

5. The method of claim 4, further comprising:

(f) sequencing the 2^nd amplification products.

6. The method of claim 1, further comprising:

(g) determining the copy number of one or more target nucleic acids.

7. The method of claim 1, further comprising:

(h) identifying one or more genetic variations of interest in one or more target nucleic acids.

8. The method of claim 1, wherein each of the 1^st target-specific sequences of the plurality of barcode primers (BC primers) and the 2^nd target-specific sequences of the plurality of limited amplification primers (LA primers) does not contain more than 10 nucleotides that are complementary to any other barcode primers (BC primers) or to any other limited amplification primers (LA primers).

9. The method of claim 1, wherein when two of the target nucleic acids overlap with each other in genomic DNA, the TS1 sequences in the BC primers for the two target nucleic acids anneal to different strands of the genomic DNA.

10. The method of claim 1, wherein the molecular tag sequences (MT) in the plurality of barcode primers are completely or semi-defined.

11. The method of claim 1, wherein the molecular tag sequences (MT) in the plurality of barcode primers are completely random.

12. The method of claim 1, wherein the molecular tag sequences (MT) in the plurality of barcode primers are 5 to 15 nucleotides in length.

13. The method of claim 1, wherein the plurality of barcode primers (BC primers) comprises at least 500 different barcode primers, and the plurality of limited amplification primers (LA primers) comprises at least 500 different limited amplification primers.

14. The method of claim 1, wherein the number of different barcode primers (BC primers) of step (a) is the same as the number of the different limited amplification primers (LA primers) of step (c).

15. The method of claim 1, wherein the number of different barcode primers (BC primers) of step (a) is different from the number of the different limited amplification primer (LA primers) of step (c).

16. The method of claim 1, wherein step (a) is performed in the presence of a DNA polymerase that does not have strand displacement activity, flap endonuclease or 5′

→

3′

exonuclease activity.

17. The method of claim 16, wherein the DNA polymerase is KOD DNA polymerase.

18. The method of claim 1, wherein step (b) is carried out by size selection purification.

19. The method of claim 1, wherein step (c) is performed in the presence of another primer comprising the 1^st universal primer sequence.

20. The method of claim 19, wherein step (c) is performed by 2 to 16 cycles of polymerase chain reaction (PCR).

21. The method of claim 3, wherein either one or both of the universal primers comprise an index sequence (IDX) located 5′

to the 1^st universal primer sequence (US1) or the 2^nd universal primer sequence (US2).

22. The method of claim 1, wherein the target nucleic acids are cDNA molecules.

23. The method of claim 1, wherein the target nucleic acids are microbial DNA molecules or mitochondrial DNA molecules.

24. The method of claim 1, wherein the target nucleic acids are genomic DNA molecules.

25. The method of claim 1 wherein the target nucleic acids are purified from a formalin-fixed, paraffin-embedded (FFPE) sample.

26. The method of claim 5, wherein the target nucleic acids are genomic DNA, and wherein step (f) determines the sequence of an at least 40 kb region of the genomic DNA.

27. The method of claim 26, wherein the genomic DNA is human genomic DNA, and the total amount of human genomic DNA used in step (a) is about about 0.1 ng to about 1 μ

g.

28. The method of claim 6, wherein at least one target nucleic acid is of low abundance in the nucleic acid sample.

29. The method of claim 6, wherein its coefficient of variation (CV) of the copy number determination of substantially all of the one or more target nucleic acids is less than 20%.

30. The method of claim 7, wherein at least one genetic variation of interest has an allelic frequency of 5% or less in the nucleic acid sample.

31. The method of claim 7, having one or both of the following features:

the sensitivity of step (h) is at least about 60%, and

the specificity of step (h) is at least about 60%.

32. A kit comprising:

(1) a plurality of barcode primers (BC primers), wherein

(i) each barcode primer comprises, from 5′

to 3′

, a 1^st universal primer sequence (US1), a molecular tag sequence (MT), and a 1^st target-specific sequence (TS1),

(ii) a plurality of barcode primers comprise at least 20 different barcode primers, and

(iii) among the plurality of barcode primers, the 1^st universal primer sequence (US1) are the same, the molecular tag sequences (MT) are different, and the 1^st target-specific sequence (TS1) are different; and

(2) a plurality of limited amplification primers (LA primers), wherein

(i) each limited amplification primer comprises, from 5′

to 3′

, a 2^nd universal primer sequence (US2) and a 2^nd target-specific sequence (TS2), and

(ii) among the plurality of limited amplification primers, the 2^nd universal primer sequences (US2) are the same, but the 2^nd target-specific sequence (TS2) are different.

33-47. -47. (canceled)