Normalization of NGS library concentration
A bottleneck in the Next Generation Sequencing (NGS) workflow is the quantification of libraries for accurate pooling and loading of the sequencing instrument flow cell or chip. Disclosed herein are methods that improve performance and reduce time compared to existing methods.
1. A kit comprising:
a first primer comprising a first portion, a second portion and a third portion, wherein the first portion is located at a 3′ end of the first primer, wherein the second portion comprises 3 or more consecutive ribonucleotide bases and is located 5′ to the first portion, and wherein the third portion is located 5′ adjacent to the second portion and comprises a low complexity sequence selected from the group consisting of poly(A), poly(T), poly(G), poly(C), poly(AG), poly(AC), poly(GT), poly(CT), poly(AT), poly(GC), a trinucleotide, a tetranucleotide, and a pentanucleotide;
a second primer, wherein the second primer comprises a second primer first portion, a second primer second portion comprising 3 or more consecutive ribonucleotide bases located 5′ adjacent to the second primer first portion and a second primer third portion located 5′ adjacent to the second primer second portion and comprising a low complexity sequence selected from the group consisting of poly(A), poly(T), poly(G), poly(C), poly(AG), poly(AC), poly(GT), poly(CT), poly(AT), poly(GC), a trinucleotide, a tetranucleotide, and a pentanucleotide;
a ligase selected from the group consisting of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and E. coli DNA ligase;
an enzyme with 3′-5′ exonuclease activity; and
a probe, wherein the probe comprises a modification to provide resistance to digestion by the enzyme with 3′-5′ exonuclease activity, and wherein at least a portion of the probe is complementary to at least a portion of the second and third portion of the first primer including the three or more consecutive ribonucleotide bases and at least a portion of the low complexity sequence.
2. The kit of claim 1 , wherein the enzyme with 3′-5′ exonuclease activity is exonuclease III.
3. The kit of claim 1 , wherein the enzyme with 3′-5′ exonuclease activity is T4 DNA Polymerase.
4. The kit of claim 1 , wherein the ligase is T4 DNA ligase.
5. The kit of claim 1 , wherein the low complexity sequence of the first primer is poly(T).
6. The kit of claim 5 , wherein the low complexity sequence of the first primer is 10-20 bases.
7. The kit of claim 1 , wherein the low complexity sequence of the first primer is 10-20 bases.
8. The kit of claim 1 , wherein the enzyme with 3′-5′ exonuclease activity is exonuclease III, wherein the ligase is T4 DNA ligase, and wherein the low complexity sequence of the first primer is 10-20 bases.
9. The kit of claim 8 , wherein the low complexity sequence of the first primer is poly(T).
10. The kit of claim 8 , wherein the modification to provide resistance to digestion by the enzyme with 3′-5′ exonuclease activity comprises phosphorothioate linkages at the 5′ end of the probe.
11. The kit of claim 1 , wherein the low complexity sequence of the second primer is poly(T).
12. The kit of claim 11 , wherein the low complexity sequence of the second primer is 10-20 bases.
13. The kit of claim 1 , wherein the low complexity sequence of the second primer is 10-20 bases.
14. The kit of claim 1 , wherein the enzyme with 3′-5′ exonuclease activity is exonuclease III, wherein the ligase is T4 DNA ligase, and wherein the low complexity sequence of the second primer is 10-20 bases.
15. The kit of claim 14 , wherein the low complexity sequence of the second primer is poly(T).
16. The kit of claim 14 , wherein the modification to provide resistance to digestion by the enzyme with 3′-5′ exonuclease activity comprises at least four consecutive phosphorothioate linkages at the 5′ end of the probe.
17. The kit of claim 1 , wherein the enzyme with 3′-5′ exonuclease activity is exonuclease III, wherein the ligase is T4 DNA ligase, wherein the low complexity sequence of the first primer is 10-20 bases, wherein the low complexity sequence of the second primer is 10-20 bases, wherein the low complexity sequence of the first primer is poly(T), and wherein the low complexity sequence of the second primer is poly(T).
18. The kit of claim 17 , wherein the modification to provide resistance to digestion by the enzyme with 3′-5′ exonuclease activity comprises at least four consecutive phosphorothioate linkages at the 5′ end of the probe.
19. The kit of claim 18 , wherein the probe is single-stranded DNA.
20. The kit of claim 1 , wherein the probe is single-stranded DNA and wherein the modification to provide resistance to digestion by the enzyme with 3′-5′ exonuclease activity comprises phosphorothioate linkages at the 5′ end of the probe, and wherein the enzyme with 3′-5′ exonuclease activity is selected from the group consisting of T4 DNA Polymerase and exonuclease III.