Diagram of fusion primers and dual-indexing approach for library construction. Diagram of fusion primers and dual-indexing approach for library construction.

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 16S rRNA gene marker  intra-gene variability  primer selection  size & information content Primer selection, information content, alignment and length.

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Diagram of fusion primers and dual-indexing approach for library construction. Diagram of fusion primers and dual-indexing approach for library construction. Illumina Nextera left (P5) and right (P7) adaptors contain the i5 (S5xx series) and i7 (N7xx series) indices in their respective middle sections, respectively. All nucleotide sizes within fusion primers are the same for different target amplicons, with the exception of the target-specific primer regions (F+R) which here demonstrate the sizes of the 16S rRNA gene V6-V8 primers (B969F+BA1406R [27]) employed in the presented protocol. André M. Comeau et al. mSystems 2017; doi:10.1128/mSystems.00127-16