Normalization for sequencing libraries

A method for preparation of a normalized amplicon library for sequencing, the method comprising:

• generating a set of amplicon target molecules based on a first amplification process with at least one sample and a set of amplicon-generation primers associated with a set of targets corresponding to target molecules from the at least one sample;
• generating a set of normalized target molecules based on a second amplification process with the set of amplicon target molecules and a set of normalization-based primers;
• and
• generating a set of sequencing-ready target molecules based on a third amplification process with the set of normalized target molecules and a set of sequencing-based primers.

method of claim 7, where generating a set of amplicon target molecules further comprising as previous step at least one of:

• transforming microbial RNA from the sample into cDNA for facilitating preparation of a normalized metatranscriptomic-associated library for sequencing;
• transforming cDNA into double stranded cDNA for facilitating preparation of a normalized library for sequencing;
• and
• generating a set of amplicon target molecules from double stranded cDNA based on a first amplification process with at least one sample and a set of amplicon-generation primers associated with a set of targets corresponding to target molecules from at least one sample.

A method for preparation of a normalized microbial community-associated library for sequencing, the method comprising:

• generating a set of microbial community-associated fragments from total nucleic acids of a sample, wherein the set of microbial community-associated fragments comprises at least one of a set of rnetagenome-associated nucleic acid fragments and a set of metatranscriptomic-associated nucleic acid fragments;
• generating a set of ligated microbial community-associated fragments based on a ligation process with the set of microbial community-associated fragments and a set of ligation adapter molecules;
• generating a set of normalized microbial community-associated fragments based on a first amplification process with the set of ligated microbial community-associated fragments and a set of normalization-based primers;
• and
• generating a set of sequencing-ready microbial community-associated fragments based on a second amplification process with the set of normalized microbial community-associated fragments and a set of sequencing-based primers, wherein the set of sequencing-ready microbial community-associated fragments comprises at least one of a set of sequencing-ready metagenome-associated fragments and a set of sequencing-ready metatranscriptomic-associated fragments.