Our cost-efficient, labor-reducing method begins with amplification of each gene library by two-step PCR using barcoded primers, followed by pooling the libraries together ( Figure 1). Our protocol was validated by sequencing archaeal 16S rRNA genes from environmental samples using the Ion Torrent (Life Technologies Corporation, Carlsbad, CA) chemistry (with sequencing adapters IonA and P1), but the same template preparation principles are also valid for the other NGS chemistries. Here we show how library preparation can be simplified with a two-step PCR protocol with M13-tagged primers and how the sample pool can be cut to a certain length all at once instead of performing shearing, adapter ligation, and size selection for each sample separately. Many previously established PCR primer pairs produce much longer amplicons, which must be cut for optimal NGS sequencing. Two commonly used NGS platforms, Illumina and Ion Torrent, recommend maximum fragment lengths of about 300 and 400 nucleotides, respectively ( 2). Another challenge when amplifying target genes with well-established universal primer pairs is related to fragment size optimization. When analyzing the sequence diversity of 10 genes in 100 samples, barcoding with fusion primers would require 1000 barcoded primers (10 genes × 100 samples), making the task both laborious and expensive without dual indexing, which is only available on the Illumina MiSeq platform. Barcodes can be added to the PCR amplicons either by ligation or by performing the PCR amplification with fusion primers, which include both the barcodes and sequencing adapters.

The efficiency of next-generation sequencing (NGS) of PCR amplicons has increased via sample barcoding ( 1), which facilitates multiplex sequencing of numerous samples and genes (such as ribosomal RNA or protein-coding genes) in the same run.

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