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 providing nucleic acid containing-cellular samples from blood or FFPE samples, lysing cells of the sample to liberate nucleic acids, and performing tagmentation without purifying the liberated nucleic acids.
1. A method of preparing a nucleic acid-containing cellular sample for library amplification comprising the following steps:
(a) providing a nucleic acid-containing cellular sample, wherein the sample is a blood sample or a formalin-fixed paraffin-embedded (FFPE) sample;
(b) lysing cells of the sample with a lysis reagent to liberate nucleic acids from within the cells of the cellular sample, thereby forming a lysate comprising liberated nucleic acids; and
(c) performing tagmentation on liberated nucleic acids in the lysate without purifying the liberated nucleic acids prior to tagmentation, thereby forming tagmented nucleic acids.
2. The method of claim 1 , wherein the nucleic acids are DNA.
3. The method of claim 1 , wherein the blood sample is a whole blood sample or a dried blood sample.
4. The method of claim 1 , wherein the sample is an FFPE sample.
5. The method of claim 1 , wherein the lysis reagent is water, purified water, or distilled water.
6. The method of claim 1 , wherein the lysis reagent is a detergent, a base, an acid, and/or an enzyme.
7. The method of claim 1 , wherein step (b) comprises treating the cells of the sample or the lysate with an enzyme to disrupt the structure of the nucleic acids.
8. The method of claim 7 , wherein the nucleic acids are DNA and the enzyme that disrupts the structure is proteinase K.
9. The method of claim 1 , further comprising neutralizing the lysis reagent prior to the tagmentation step (c) to inactivate the lysis reagent.
10. The method of claim 9 , wherein neutralizing the lysis reagent is carried out via a neutralizing agent or via heat.
11. The method of claim 1 , further comprising incubating the lysate before the tagmentation step.
12. The method of claim 1 , wherein the steps of lysing the sample and performing tagmentation on the liberated nucleic acids in the lysate are conducted in a single pot reaction.
13. The method of claim 1 , further comprising sequencing the tagmented nucleic acids to ascertain their nucleic acid sequences.
14. The method of claim 13 , wherein the sequencing is performed by high-throughput sequencing or a sequence-by-synthesis protocol.
15. The method of claim 1 , further comprising:
(d) exposing the tagmented nucleic acids to a solid surface with immobilized amplification primers, thereby immobilizing the tagmented nucleic acids on the solid surface; and
(e) clonally amplifying the immobilized, tagmented nucleic acids on the solid surface in a clustering reaction to generate clusters.
16. The method of claim 15 , wherein the clustering reaction is performed in the presence of one or more of proteinase K, formalin, paraffin, cellular components, protein, extracellular matrix components, collagen, and tissue debris.
17. The method of claim 16 , wherein the clustering reaction is performed in the presence of at least 0.01 pg, 0.1 pg, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, or at least 1 mg paraffin.
18. The method of claim 16 , wherein the sample is an FFPE sample and the clustering reaction is performed in the presence of at least 0.001 pg paraffin.
19. The method of claim 16 , wherein the clustering reaction is performed in the presence of at least 0.01 pg, 0.1 pg, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, or at least 1 mg proteinase K.
20. The method of claim 16 , wherein the clustering reaction is performed in the presence of at least 0.001 pg proteinase K.