PRJNA435442
Adapterama II: Universal amplicon sequencing on Illumina platforms (TaggiMatrix)
Although next-generation sequencing (NGS) platforms allow many DNA sequences to be obtained at extremely low-cost per read, sequencing PCR amplicons via NGS remains overly expensive and inflexible. NGS amplicon library preparation strategies usually rely upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying (barcode/index/tag) sequence or ligating adapters onto PCR products. Herein we briefly review NGS amplicon library preparation methods and describe an approach that uses locus-specific PCR primers to produce up to millions of uniquely indexed Illumina libraries. We fuse partial adapter sequences (TruSeq or Nextera) onto the 5'' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers are used combinatorically to amplify samples within a 96-well plate (eight forward primers + 12 reverse primers yield 8x12=96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorically) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequence reads from the amplicon pools can be tuned, reducing sequencing cost per sample to an economically trivial amount. We demonstrate the utility and versatility of our approach with results from six projects, each with a different implementation of our protocol. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility.