FIGURE 1.

Amplicon library preparation methods for HTS sequencing. Traditional protocol is represented as the most common equimolar process. (1) Equimolar DNA inputs are prepared based on fluorimetric or spectrophotometric measures, all DNA samples are normalized to equivalent amounts (e.g., 5 ng/μL); (2) PCR amplifications are performed with single or two-step protocols with varying amplification cycles (most commonly 35 cycles); (3) Usually, PCR amplifications are then checked on agarose gel to confirm positive samples and discard negative ones; (4) PCR pooling for HTS sequencing is also performed in an equimolar manner through fluorometric quantification (e.g., pooling 20 ng from each sample). Equivolumetric protocol stands for equal volumes processed for each sample instead of equal concentration. In this protocol, samples retain their original differences in terms of concentrations of input DNA. (1) Equal volumes of each sample is used for PCR steps, regardless of its concentration (e.g., 2 μL); (2) Amplicon library preparation is carried out in a standardized, two-step PCR for 25 cycles using specific marker genes, then additional 10 cycles to add the sequencing adapter and indexes; (3) No agarose gel check is performed for these samples since we assume a wide variation in amplicon yield, related to the sample original DNA input; (4) PCR pooling for HTS sequencing is performed without specific sample normalizations. Equal volumes are used for each amplicon sample to assemble the HTS sequencing pool (e.g., pooling 20 μL from each sample).