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 two or more deoxynucleotides;
a second primer;
a ligase;
a probe, wherein the probe comprises a modification to provide resistance to digestion by an enzyme with 3′-5′ exonuclease activity, and wherein at least a portion of the probe is complementary to at least a portion of the third portion of the first primer and to the three or more consecutive ribonucleotide bases; and
an exonuclease.
2. The kit of claim 1 , wherein the ligase is T4 DNA ligase.
3. The kit of claim 1 , wherein the exonuclease is Exonuclease III.
4. The kit of claim 1 , wherein the second primer further comprises a fourth portion, a fifth portion comprising 3 or more consecutive ribonucleotide bases located 5′ adjacent to the fourth portion and a sixth portion located 5′ adjacent to the fifth portion and comprising two or more deoxynucleotides.
5. The kit of claim 1 , further comprising a thermostable polymerase with 3′-5′ exonuclease proofreading activity.
6. The kit of claim 1 , wherein the third portion of the first primer 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.
7. The kit of claim 1 , wherein the probe comprises single-stranded DNA, and wherein the probe comprises a 5′ phosphate.
8. 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;
a ligase;
a probe, wherein the probe comprises a modification to provide resistance to digestion by an enzyme with exonuclease activity; and
an exonuclease.
9. The kit of claim 8 , wherein the probe comprises a sequence complimentary to the low complexity sequence.