Targeted amplicon sequencing on Illumina sequencing systems is a cost-effective method in many fields of genomics including metagenomics and genetic barcoding. The technique combined with multiplexing through the use of standard Illumina sample indices enables the sequencing of amplicons from up to 384 samples in one lane. However, this approach is inefficient for sequencing of single locus PCR amplicons due to the need for parallel processing of samples prior to multiplexing and low sequence diversity. This necessitates addition of a high diversity library such as PhiX and clustering in low densities and results in the inability to multiplex sufficiently high numbers of samples to take advantage of the high yield of current sequencing systems. 

To overcome low diversity and increase multiplexing level and library prep efficiency we have developed MegaPlex library preparation and a demegaplexing bioinformatics pipeline. We have designed colour balanced sample-specific PCR primers by addition of variable length inline barcodes which increases single locus sequence diversity during sequencing at the same time as uniquely marking sample amplicons at the first step in library preparation. We use a pool of these amplicons as input into PCR-free library prep allowing multiplexing of thousands of samples in one sequencing lane. This technique using 236 barcoded primers and 44 commercially available Illumina adapters theoretically allows multiplexing amplicons from millions of samples in one lane. Here we demonstrate the utility of the MegaPlex method by amplifying the 18S region with a subset of designed barcoded primers from samples containing a mix of different marine species.