Polymerase chain reaction normalization through primer titration
Disclosed herein include systems, methods, compositions, and kits for PCR normalization. In some embodiments, after barcoding copies of a higher abundance target (e.g., a cDNA species), the barcoded copies are amplified using a pair of forward primers comprising one or more mismatches and a reverse primer. The amplified copies can be further linearly amplified using a forward primer comprising the sequence of one of the pair of forward primers, and a reverse primer.
1 . A method for polymerase chain reaction (PCR), comprising:
amplifying copies of a target nucleic acid in a first amplification reaction to generate amplified nucleic acid molecules using:
a first forward primer and a first reverse primer to generate a first plurality of amplified nucleic acid molecules, wherein the first forward primer anneals to the target nucleic acid; and
a second forward primer and the first reverse primer to generate a second plurality of amplified nucleic acid molecules, wherein the second forward primer anneals to the target nucleic acid, wherein the first forward primer and the second forward primer anneal to the same sequence on the target nucleic acid,
wherein the sequence of the first forward primer incorporated into the amplified nucleic acid molecules and the sequence of the second forward primer incorporated into the amplified nucleic acid molecules are different,
wherein the first forward primer and the second forward primer each comprise a nucleic acid-hybridization region and an overhang region 5′ of the nucleic acid-hybridization region, and
wherein the sequence of the nucleic acid-hybridization region of the first forward primer is identical to the sequence of the nucleic acid-hybridization region of the second forward primer, and wherein the sequence of the overhang region of the first forward primer is different from the sequence of the overhang region of the second forward primer; and
subjecting the first and second pluralities of amplified nucleic acid molecules to a second amplification reaction using a third forward primer and a second reverse primer to generate a third plurality of amplified nucleic acid molecules,
wherein amplification of the second plurality of amplified nucleic acid molecules is reduced compared to amplification of the first plurality of amplified nucleic acid molecules in the second amplification reaction due to the difference in the sequence of the first forward primer incorporated into the first plurality of amplified nucleic acid molecules and the sequence of the second forward primer incorporated into the second plurality of amplified nucleic acid molecules.
2 . The method of claim 1 , comprising obtaining sequencing data of the third plurality of amplified nucleic acid molecules.
3 . The method of claim 1 , wherein the target nucleic acid comprises a cell label sequence and/or a molecular label sequence.
4 . The method of claim 3 , comprising determining the number of copies of the target nucleic acid based on the number of different molecular label sequences associated with the sequence of the target nucleic acid, a complementary sequence thereof, a portion thereof, or a combination thereof, in the sequencing data.
5 . The method of claim 1 , wherein the amplified nucleic acid molecules are amplified linearly in the second amplification reaction.
6 . The method of claim 1 , wherein the ratio of the first forward primer to the second forward primer ranges from 1:100 to 100:1.
7 . The method of claim 1 , wherein lengths of the first forward primer and the second forward primer are different.
8 . The method of claim 1 , wherein the sequence of the overhang region of the first forward primer differs from the sequence of the overhang region of the second forward primer by at least a plurality of nucleotides.
9 . The method of claim 8 , wherein the plurality of nucleotides are on the 3′ ends of the overhang regions of the first forward primer and the second forward primer.
10 . The method of claim 1 , wherein the sequence of at least one overhang region of the first forward primer and the second forward primer comprises a universal PCR primer binding site.
11 . The method of claim 1 , wherein the third forward primer comprises a nucleic acid-hybridization region and an overhang region 5′ of the nucleic acid-hybridization region.
12 . The method of claim 11 , wherein the nucleic acid-hybridization region of the third forward primer comprises the sequence of the overhang region of the first forward primer, a complementary sequence thereof, or a portion thereof.
13 . The method of claim 1 , wherein the third forward primer comprises the sequence of the overhang region of the first forward primer, a complementary sequence thereof, or a portion thereof.
14 . The method of claim 1 , wherein the third forward primer comprises the sequence of the first forward primer, a complementary sequence thereof, or a portion thereof.
15 . The method of claim 1 , wherein the ratio of the first forward primer to the second forward primer in the first amplification reaction ranges from 1:1 to 100:1, wherein amplification of the second plurality of amplified nucleic acid molecules is reduced compared to amplification of the first plurality of amplified nucleic acid molecules in the second amplification reaction due to the difference in the sequence of the overhang region of the first forward primer and the sequence of the overhang region of the second forward primer.
16 . The method of claim 15 , wherein the sequence of the overhang region of the first forward primer differs from the sequence of the overhang region of the second forward primer by having a dU nucleotide where the sequence of the overhang region of the second forward primer has a dT nucleotide at a corresponding position.
17 . The method of claim 16 , comprising contacting the first and second pluralities of amplified nucleic acid molecules to uracil-DNA glycosylase (UDG) before subjecting the first and second pluralities of amplified nucleic acid molecules to the second amplification reaction.
18 . The method of claim 15 , wherein the sequence of the overhang region of the first forward primer differs from the sequence of the overhang region of the second forward primer by having an endonuclease restriction site, a Zinc-finger nuclease cleavage site, or a CRISPR-Cas9 cleavage site that is not present in the sequence of the overhang region of the second forward primer.
19 . The method of claim 15 , wherein the sequence of the overhang region of the first forward primer differs from the sequence of the overhang region of the second forward primer by 2-20 nucleotides.
20 . The method of claim 15 , wherein the overhang region of the first forward primer differs from the overhang region of the second forward primer by comprising a sequence homologous to a phage species.
21 . The method of claim 20 , wherein the phage species comprises a T7 phage or a PhiX phage.
22 . The method of claim 20 , wherein the ratio of the first forward primer to the second forward primer in the first amplification reaction ranges from 1:1 to 10:1.
23 . The method of claim 15 , wherein the ratio of the first forward primer to the second forward primer in the first amplification reaction ranges from 1:1 to 10:1.
24 . A method for polymerase chain reaction (PCR), comprising:
amplifying copies of a target nucleic acid in a first amplification reaction to generate amplified nucleic acid molecules using:
a first forward primer and a first reverse primer to generate a first plurality of amplified nucleic acid molecules, wherein the first forward primer anneals to the target nucleic acid; and
a second forward primer and the first reverse primer to generate a second plurality of amplified nucleic acid molecules, wherein the second forward primer anneals to the target nucleic acid, wherein the first forward primer and the second forward primer anneal to the same sequence on the target nucleic acid,
wherein the sequence of the first forward primer incorporated into the amplified nucleic acid molecules and the sequence of the second forward primer incorporated into the amplified nucleic acid molecules are different; and
subjecting the first and second pluralities of amplified nucleic acid molecules to a second amplification reaction using a third forward primer and a second reverse primer to generate a third plurality of amplified nucleic acid molecules, wherein the third forward primer comprises a nucleic acid-hybridization region and an overhang region 5′ of the nucleic acid-hybridization region, and
wherein amplification of the second plurality of amplified nucleic acid molecules is reduced compared to amplification of the first plurality of amplified nucleic acid molecules in the second amplification reaction due to the difference in the sequence of the first forward primer incorporated into the first plurality of amplified nucleic acid molecules and the sequence of the second forward primer incorporated into the second plurality of amplified nucleic acid molecules.
25 . The method of claim 24 , comprising obtaining sequencing data of the third plurality of amplified nucleic acid molecules.
26 . The method of claim 24 , wherein the target nucleic acid comprises a cell label sequence and/or a molecular label sequence.
27 . The method of claim 26 , comprising determining the number of copies of the target nucleic acid based on the number of different molecular label sequences associated with the sequence of the target nucleic acid, a complementary sequence thereof, a portion thereof, or a combination thereof, in the sequencing data.
28 . The method of claim 24 , wherein the amplified nucleic acid molecules are amplified linearly in the second amplification reaction.
29 . The method of claim 24 , wherein the ratio of the first forward primer to the second forward primer ranges from 1:100 to 100:1.
30 . The method of claim 24 , wherein lengths of the first forward primer and the second forward primer are different.