FAQ: How can the percentage of dual-barcoded reads be increased

Dual-barcoded reads are reads with the sequencing adaptor at both ends. To increase the dual-barcoded reads percentage, the RT-PCR amplicon products can be diluted 3-fold, and 1 µl of the diluted amplicons used in NEBNext End Prep step 1.4.1. This will reduce the amount of DNA input and the final data throughput for each sample, but will provide improved End Prep efficiency and result in a higher percentage of dual-barcoded reads. We recommend performing this amplicon dilution when 48 or more samples are used.