Methods and compositions for processing samples containing nucleic acids
Provided herein are methods and composition for processing samples that contain nucleic acids, or cells containing nucleic acids, of a microbiome, using amounts of primers within a range of mole values and rounds of polymerase chain reaction (PCR) within a range of numbers of rounds.
1. A primer-limited method for processing and normalizing nucleic acids in a plurality of subsamples from a sample of a microbiome of a host wherein the sample comprises nucleic acids and/or cells comprising nucleic acids, wherein the nucleic acids are from one or more microorganisms in the microbiome, comprising:
(A) providing the plurality of subsamples derived from the sample of the microbiome of the host, wherein each subsample comprises a corresponding plurality of polynucleotides that comprise the nucleic acids or derived from the nucleic acids;
(B) performing, for each respective subsample in the plurality of subsamples, a plurality of rounds of polymerase chain reaction (PCR) on the corresponding plurality of polynucleotides, or derivatives thereof, using a corresponding one or more primers for a different target nucleic acid in the respective subsample, thereby obtaining amplified polynucleotides, wherein for the respective subsample:
2-50 rounds of PCR are performed,
each primer of the corresponding one or more primers is provided in a fixed molar amount,
each primer of the corresponding one or more primers is completely or substantially completely consumed in the 2-50 rounds of PCR for the respective subsample, and
a molar amount of the amplified polynucleotides for the respective subsample is determined by the fixed molar amount of each primer of the one or more primers in that subsample;
wherein the one or more primers and PCR are in solution; and
(C) generating, after the amplifying, a pooled sample from the plurality of subsamples, wherein a relative molar amount of amplified polynucleotides from each subsample in the pooled sample is determined by (i) the fixed molar amount of primers used to amplify each subsample, and (ii) the relative portion of each subsample added to the pooled sample, and wherein the amplified polynucleotides are not washed or isolated from a solid support prior to pooling.
2. The method of claim 1 wherein the host is a human.
3. The method of claim 1 wherein the sample comprises a stool sample.
4. The method of claim 1 wherein the polynucleotides comprise RNA and the method further comprises producing cDNA from the RNA to provide cDNA derivatives of the RNA.
5. The method of claim 4 further comprising removing noninformative RNA before producing the cDNA.
6. The method of claim 1 wherein the plurality of subsamples comprise at least 10 subsamples.
7. The method of claim 1 wherein the method further comprises sequencing the amplified polynucleotides.
8. The method of claim 1 wherein 20-40 rounds of PCR are performed.
9. The method of claim 1 wherein 24-28 rounds of PCR are performed.
10. The method of claim 1 wherein one or more primers is provided in an amount between 7.5×10 −13 to 2×10 −12 mole.
11. The method of claim 1 wherein one or more primers is provided in an amount between 1×10 −12 to 1.75×10 −12 mole.