Methods and kits for nucleic acid sample preparation for sequencing
The present disclosure relates to methods and kits for DNA library construction, particularly for consistent and reproducible DNA sequencing.
1. A method for DNA library construction, comprising:
modifying nucleic acid fragments; and
ligating nucleic acid adaptors to the modified nucleic acid fragments;
wherein one or more of the reactions are rehydrated from a lyophilized form in a reaction volume comprising the nucleic acid fragments.
2. A method for DNA library construction, comprising:
modifying DNA fragments with a polymerase and a polynucleotide kinase in a first reaction mixture sufficient for end repair of the DNA fragments;
contacting the end repaired DNA fragments with a (3′→5′ exo-) polymerase in a second reaction mixture sufficient for A-tailing the end-repaired DNA fragments;
ligating DNA adaptors to the A-tailed DNA fragments in a third reaction mixture comprising a nucleic acid ligase;
wherein one or more of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from a lyophilized form in a reaction volume comprising the DNA fragments.
3. The method of claim 2 , wherein at least the first reaction mixture is rehydrated from a lyophilized form.
4. The method of claim 3 , wherein the lyophilized form is one or more lyophilized pellets, and wherein each lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm.
5. The method of claim 4 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.
6. The method of claim 3 , wherein the lyophilized form comprises T4 DNA polymerase, T4 DNA polynucleotide kinase, deoxynucleotide triphosphates, a source of magnesium, and a buffer.
7. The method of claim 3 , wherein the DNA fragments in the reaction volume are added to a single use reaction tube containing the lyophilized first reaction mixture.
8. The method of claim 2 , wherein at least the second reaction mixture is rehydrated from a lyophilized form.
9. The method of claim 8 , wherein the lyophilized form is one or more lyophilized pellets, and wherein each lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm.
10. The method of claim 9 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.
11. The method of claim 8 , wherein the lyophilized form comprises Klenow (3′→5′ exo-), a source of magnesium, a source of sodium, dATP, and a buffer.
12. The method of claim 8 , wherein the end repaired DNA fragments in the reaction volume are added to a single use reaction tube containing the lyophilized second reaction mixture.
13. The method of claim 2 , wherein at least the third reaction mixture is rehydrated from a lyophilized form.
14. The method of claim 13 , wherein the lyophilized form is one or more lyophilized pellets, and wherein each lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm.
15. The method of claim 14 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.
16. The method of claim 13 , wherein the lyophilized form comprises T4 DNA ligase, a source of magnesium, ATP, and a buffer.
17. The method of claim 13 , wherein the A-tailed DNA in the reaction volume are added to a single use reaction tube containing the lyophilized third reaction mixture.
18. The method of claim 2 , wherein at least two of the first reaction mixture, second reaction mixture, and third reaction mixture are rehydrated from lyophilized form.
19. A method of for DNA library construction, comprising:
modifying DNA fragments with T4 DNA polymerase and T4 polynucleotide kinase in a first reaction mixture sufficient for end repair of the DNA fragments;
contacting the end repaired DNA fragments with Klenow (3′→5′ exo-) in a second reaction mixture sufficient for A-tailing the end-repaired DNA fragments;
ligating DNA adaptors to the A-tailed DNA in a third reaction mixture comprising T4 DNA ligase,
wherein one or more of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from a lyophilized form in a reaction volume comprising the DNA fragments.