Methods for preparing a sample for nucleic acid amplification using tagmentation
Presented are methods and compositions for preparing samples for amplification and sequencing. Particular embodiments relate to methods of preparing nucleic acid-continuing cellular samples for library amplification, wherein the methods include lysing cells of the sample to form a lysate, amplifying the nucleic acids from the lysed samples, exposing the amplified nucleic acids to a solid surface, and clonally amplifying the amplified nucleic acids to generate clusters.
1. A method of preparing a sample for library amplification and subsequent amplification comprising the following steps:
on a provided nucleic acid-containing cellular sample,
(a) lysing cells of the sample with a lysis reagent to liberate nucleic acid from within the cells of the cellular sample, thereby forming a lysate;
(b) amplifying the nucleic acids from the lysate to form amplified nucleic acids;
(c) exposing the amplified nucleic acids to a solid surface with immobilized amplification primers, thereby immobilizing the amplified nucleic acids on the solid surface; and
(d) clonally amplifying the immobilized, amplified nucleic acids on the solid surface to generate clusters;
wherein there is no purification of the nucleic acid from the lysate prior to beginning the amplification step (b); and
wherein amplifying the nucleic acid in step (b) comprises tagmentation,
wherein the sample is a blood sample, a whole blood sample, or a dried blood sample.
2. The method of claim 1 , wherein the nucleic acid is DNA.
3. The method of claim 1 , wherein the lysis reagent is water, purified water, or distilled water.
4. The method of claim 1 , wherein the lysis reagent is a detergent, a non-denaturing lytic detergent, a base, an acid, and/or an enzyme.
5. The method of claim 4 , wherein the lysis reagent comprises a reducing agent, stabilizing agent, 2-mercaptoethanol, or dithiothreitol.
6. The method of claim 1 , further comprising, prior to step (a), treating the nucleic acid-containing cellular sample with an enzyme that disrupts the structure of the nucleic acid.
7. The method of claim 6 , wherein the enzyme that disrupts the structure of the nucleic acid is proteinase K.
8. The method of claim 1 , further comprising neutralizing the lysis reagent prior to the amplification step (b) to inactivate the lysis reagent.
9. The method of claim 1 , wherein the steps of lysing the sample and amplifying the nucleic acid contained in the lysate are conducted in a single pot reaction.
10. The method of claim 1 , wherein the immobilized, amplified nucleic acids are sequenced by high throughput sequencing or by a sequence-by-synthesis protocol to ascertain their sequences.
11. The method of claim 1 , wherein clonally amplifying the immobilized, amplified nucleic acids on the solid surface to generate clusters is performed in the presence of (a) proteinase K and/or (b) one or more of formalin, paraffin, cellular components, protein, extracellular matrix components, collagen, and tissue debris.
12. The method of claim 1 , wherein the tagmentation is performed using bead-based tagmentation.
13. A method of preparing a sample for library amplification and subsequent amplification comprising the following steps:
on a provided nucleic acid-containing cellular sample,
(a) lysing cells of the sample with a lysis reagent to liberate nucleic acid from within the cells of the cellular sample, thereby forming a lysate;
(b) amplifying the nucleic acids from the lysate to form amplified nucleic acids;
(c) exposing the amplified nucleic acids to a solid surface with immobilized amplification primers, thereby immobilizing the amplified nucleic acids on the solid surface; and
(d) clonally amplifying the immobilized, amplified nucleic acids on the solid surface to generate clusters;
wherein there is no purification of the nucleic acid from the lysate prior to beginning the amplification step (b); and
wherein amplifying the nucleic acid in step (b) comprises tagmentation,
wherein the sample is a formalin fixed paraffin embedded (FFPE) sample.
14. The method of claim 13 , wherein the nucleic acid is DNA.
15. The method of claim 13 , wherein the lysis reagent is water, purified water, or distilled water.
16. The method of claim 13 , wherein the lysis reagent is a detergent, a non-denaturing lytic detergent, a base, an acid, and/or an enzyme.
17. The method of claim 16 , wherein the lysis reagent comprises a reducing agent, stabilizing agent, 2-mercaptoethanol, or dithiothreitol.
18. The method of claim 13 , further comprising, prior to step (a), treating the nucleic acid-containing cellular sample with an enzyme that disrupts the structure of the nucleic acid.
19. The method of claim 18 , wherein the enzyme that disrupts the structure of the nucleic acid is proteinase K.
20. The method of claim 13 , further comprising neutralizing the lysis reagent prior to the amplification step (b) to inactivate the lysis reagent.
21. The method of claim 13 , wherein the steps of lysing the sample and amplifying the nucleic acid contained in the lysate are conducted in a single pot reaction.
22. The method of claim 13 , wherein the immobilized, amplified nucleic acids are sequenced by high throughput sequencing or by a sequence-by-synthesis protocol to ascertain their sequences.
23. The method of claim 13 , wherein clonally amplifying the immobilized, amplified nucleic acids on the solid surface to generate clusters is performed in the presence of at least 0.001 pg paraffin.
24. The method of claim 13 , wherein clonally amplifying the immobilized, amplified nucleic acids on the solid surface to generate clusters is performed in the presence of (a) proteinase K and/or (b) one or more of formalin, paraffin, cellular components, protein, extracellular matrix components, collagen, and tissue debris.
25. The method of claim 13 , wherein the tagmentation is performed using bead-based tagmentation.