METHODS FOR PREPARING A SAMPLE FOR NUCLEIC ACID AMPLIFICATION
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, exposing the amplified nucleic acids to a solid surface, and clonallyr amplifying the amplified nucleic acids to generate clusters.
1 . A flow cell comprising a solid surface comprising:
immobilized amplification primers;
proteinase K;
paraffin; and
one or more of formalin, cellular components, protein, extracellular matrix components, collagen, or tissue debris.
2 . The flow cell of claim 1 , wherein the proteinase K is present in an amount 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.
3 . The flow cell of claim 1 , wherein the proteinase K is present in an amount of at least 0.001 pg.
4 . The flow cell of claim 1 , wherein the paraffin is present in an amount of at least 0.001 pg.
5 . The flow cell of claim 1 , wherein the immobilized amplification primers are configured for clonal amplification and sequencing.
6 . The method of claim 5 , wherein sequencing comprises high throughput sequencing.
7 . The method of claim 5 , wherein sequencing comprises sequence-by-synthesis.
8 . The flow cell of claim 1 , wherein the solid surface is configured to form an array comprised of clusters of immobilized nucleic acid molecules.
9 . The flow cell of claim 1 , wherein the one or more of the formalin, cellular components, protein, extracellular matrix components, collagen, or tissue debris are components from an FFPE sample.
10 . The flow cell of claim 9 , wherein the FFPE sample is a tumor sample.
11 . The flow cell of claim 1 , further comprising an integrated system for preparing and detecting nucleic acids.
12 . The flow cell of claim 11 , wherein the integrated system further comprises fluidic components capable of delivering amplification reagents and sequencing reagents to one or more immobilized DNA fragments.
13 . The flow cell of claim 12 , wherein the fluidic system comprises components selected from pumps, valves, reservoirs, and fluidic lines.
14 . The flow cell of claim 12 , wherein one or more of the fluidic components of the integrated system are configured for an amplification method and for a detection method.
15 . The flow cell of claim 12 , wherein one or more of the fluidic components of the integrated system are configured for an amplification method and for the delivery of sequencing reagents in a sequencing method.
16 . The flow cell of claim 12 , wherein the integrated system comprises separate fluidic systems to carry out amplification methods and to carry out detection methods.
17 . The flow cell of claim 1 , wherein the flow cell comprises a MiSeq™ flow cell.
18 . The flow cell of claim 1 , wherein the cellular components comprise a nucleic acid.
19 . The flow cell of claim 18 , wherein the nucleic acid is DNA.