synthetic metagenome Raw sequence reads

High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities that thrive in the environment or are associated with humans, animals, or plants. Here, we describe a highly adaptable and economical PCR approach to barcoding amplicons of diverse target genes that alleviates the need for gene- and sequencing platform-specific fusion primers and enables nearly limitless customized barcode-primer combinations. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon targets ranged in size from approximately 290 to 720 bp, we generally recovered a sufficient number of high-quality sequences per sample-specific and gene target-specific dataset and found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach can be applied to produce large and diverse sets of amplicon sequence data needed for modern studies in microbial ecology.