Figure 1.

Next-generation sequencing (NGS)-ready library preparation workflow for the molecular barcode–containing 21-amplicon panel. The hairpin structure of the target-specific forward primers contains the partial P5 adaptor (5′ adaptor) sequence and a 12-bp random nucleotide sequence as a molecular barcode sequence. The target-specific reverse primers contain the partial P7 adaptor (3′ adaptor) sequence. The first stage of PCR was performed using target-specific forward and reverse primers, and a 130- to 211-bp region of interest was amplified. The second stage was performed with Illumina sample-indexing primers. The Illumina partial P5 and P7 adaptor sequences that were incorporated into the first-stage PCR product served as anchor sequences to the second-stage PCR primers; amplicons that contained full-length P5 and P7 adaptor sequences were generated by second-stage PCR. These amplicon libraries were subjected to sequencing on MiSeq with custom sequencing primers, and the data were analyzed with a custom bioinformatics pipeline and NextGENe NGS data–analysis software.