Adapterama II: Universal amplicon sequencing on Illumina platforms (TaggiMatrix)

Although next-generation sequencing (NGS) platforms allow massive quantities of DNA sequences to be obtained at low cost per read, sequencing PCR amplicons remains relatively expensive. NGS amplicon sequencing strategies have relied upon fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence (i.e., an index or tag) or ligating adapters onto pools of PCR products. Herein we describe an approach that uses locus-specific PCR primers to produce many indexed Illumina libraries ( 9216 samples). We fuse partial adapter sequences of variable length (Nextera or TruSeq) onto the 5' end of locus-specific PCR primers (fusion primers) with the option of adding tag sequences between the adapter and locus-specific sequences. We then use forward and reverse fusion primers combinatorically and then pooled for a subsequent PCR with dual-indexed iNext or iTru primers (also used combinatorically) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions so they can be pooled with any standard Illumina library for sequencing. Importantly, the number of sequence reads from the amplicon pools can be easily tuned, reducing the sequencing cost per sample to where it is economically trivial. We demonstrate the utility of our approach with results from five projects, each with a different implementation of our protocol. We show that by using fusion primers together with hundreds of available iTru or iNext primers, millions of potential combinations exist, thereby facilitating Illumina library construction of amplicons via simple PCR protocols and encouraging cooperation among researchers using NGS.