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 containing 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, wherein there is no purification of the nucleic acids from the lysate prior to the amplification, wherein amplifying comprises tagmentation, and sequencing the nucleic acids, wherein the sample is a blood sample or a formalin-fixed paraffin-embedded (FFPE) sample.
1. A method of sequencing nucleic acids from a cellular sample, comprising:
lysing cells from a nucleic acid-containing cellular sample with a lysis reagent to form a lysate of nucleic acids;
amplifying the nucleic acids in the lysate wherein there is no purification of the nucleic acids from the lysate prior to the amplification, wherein amplifying comprises tagmentation; and
sequencing the nucleic acids,
wherein the sample is a blood sample or a formalin-fixed paraffin-embedded (FFPE) sample.
2. The method of claim 1 , wherein the FFPE sample is a tissue sample, a biopsy, or an aspirate.
3. The method of claim 1 , wherein the sample is a tumor sample.
4. The method of claim 1 , wherein the blood sample is a whole blood sample or a dried blood sample.
5. The method of claim 1 , wherein the nucleic acid is DNA.
6. The method of claim 1 , wherein the lysis reagent is water, purified water, or distilled water.
7. 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.
8. The method of claim 7 , wherein the lysis reagent comprises a reducing agent, stabilizing agent, 2-mercaptoethanol, or dithiothreitol.
9. The method of claim 1 , wherein the amplifying comprises treating the cells of the sample or the lysate with an enzyme that disrupts the structure of the nucleic acid.
10. The method of claim 9 , wherein the enzyme that disrupts the structure of the nucleic acid is proteinase K.
11. The method of claim 1 , further comprising neutralizing the lysis reagent prior to the amplification step to inactivate the lysis reagent.
12. 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.
13. The method of claim 1 , wherein sequencing comprises high throughput sequencing or sequence-by-synthesis.
14. The method of claim 1 , wherein the tagmentation is performed using bead-based tagmentation.
15. The method of claim 14 , wherein the tagmentation comprises fragmenting the nucleic acid in the lysate.
16. The method of claim 1 , wherein the lysate of nucleic acids further comprises components from the FFPE sample.
17. The method of claim 16 , wherein the FFPE components are selected from the group consisting of formalin, paraffin, cellular components, protein, extracellular matrix components, collagen, and tissue debris.
18. The method of claim 17 , wherein the amplifying step is performed in the presence of at least 0.01 pg, at least 0.1 pg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 1 μg, at least 10 μg, at least 100 μg, or at least 1 mg paraffin.
19. The method of claim 17 , wherein the amplifying step is performed in the presence of at least 0.001 pg paraffin.