Methods and compositions for processing samples containing nucleic acids

A method for processing nucleic acids in a biological sample, wherein the biological sample comprises nucleic acids from one or more microorganisms, comprising:

• (A) amplifying, for each respective subsample in one or more subsamples derived from the biological sample, a corresponding plurality of polynucleotides, or derivatives thereof, using:
• (i) a plurality of rounds of polymerase chain reaction (PCR), and (ii) a corresponding one or more primers for a target nucleic acid in the respective subsample, thereby obtaining amplified polynucleotides for the target nucleic acid, wherein for the respective subsample:
• the corresponding plurality of polynucleotides are derived from the nucleic acids of the biological sample,
• 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 plurality of rounds of PCR for the respective subsample, and
• a molar amount of the amplified polynucleotides for the target nucleic acid in the respective subsample is determined by the fixed molar amount of the one or more primers in the respective subsample;
• wherein the one or more primers and the plurality of rounds of PCR are in solution;
• and
• (B) using, after the amplifying, for each respective subsample in the one or more subsamples, the molar amount of the amplified polynucleotides for the target nucleic acid in the respective subsample to determine a relative portion of the respective subsample that has a target amount of the target nucleic acid.

A method for normalizing nucleic acids in a plurality of subsamples from a biological sample wherein the biological sample comprises nucleic acids, wherein the nucleic acids are from one or more microorganisms, comprising:

• (A) providing the plurality of subsamples derived from the biological sample, wherein each subsample comprises a corresponding plurality of polynucleotides that comprise a portion of the nucleic acids or are derived from the nucleic acids;
• (B) amplifying, for each respective subsample in the plurality of subsamples, the corresponding plurality of polynucleotides, or derivatives thereof, using a plurality of rounds of polymerase chain reaction (PCR), thereby obtaining amplified polynucleotides, wherein for the respective subsample:
• a corresponding one or more primers for the respective subsample is provided in a fixed molar amount,
• each primer of the corresponding one or more primers is completely or substantially completely consumed in the plurality of 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 at least one of the one or more primers is 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) a 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.

A method for obtaining a pooled sample comprising nucleic acids in a plurality of subsamples from a biological sample, wherein the nucleic acids are from one or more microorganisms, comprising:

• (A) providing the plurality of subsamples derived from the biological sample, 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;
• and
• (C) generating 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) a relative portion of each subsample added to the pooled sample.

A method for normalizing nucleic acids in a biological sample wherein the biological sample comprises nucleic acids and/or cells comprising nucleic acids, wherein the nucleic acids are from one or more microorganisms, comprising:

• (A) amplifying, for each respective subsample in one or more subsamples derived from the biological sample, a corresponding plurality of polynucleotides, or derivatives thereof, for the respective subsample using (i) a plurality of rounds of polymerase chain reaction (PCR) and (ii) a corresponding one or more primers for the respective subsample, wherein each primer of the corresponding one or more primers comprises a detectable label, thereby obtaining amplified polynucleotides;
• (B) using, for each respective subsample in the one or more subsamples, the detectable label to determine a molar amount of amplified polynucleotides in the respective subsample;
• and
• (C) generating a pooled sample from the one or more subsamples, wherein a relative molar amount of amplified polynucleotides from each subsample in the pooled sample is determined by (i) the molar amount of amplified polynucleotides determined using the detectable label, and (ii) a relative portion of each subsample added to the pooled sample.

A composition for preparing a biological sample comprising nucleic acids for sequencing of the nucleic acids, comprising:

• a buffer;
• a plurality of subsamples derived from the biological sample, wherein each subsample of the plurality of subsamples comprises a plurality of polynucleotides that comprise nucleic acids from one or more microorganisms, or are derived therefrom;
• dNTPs;
• a DNA polymerase;
• and
• a plurality of PCR primers comprising, for each respective subsample in the plurality of subsamples, a corresponding set of one or more PCR primers, wherein, for each respective subsample in the plurality of subsamples:
• each PCR primer in the corresponding set of one or more PCR primers is (i) present in an amount of from 5×10−14 to 2.5×10−11 mole, (ii) in solution, and (iii) not attached to a solid support, and
• the corresponding set of one or more PCR primers is for a different target nucleic acid for the respective subsample.