Journal
FRONTIERS IN MICROBIOLOGY
Volume 6, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2015.00731
Keywords
MiSeq; functional gene; 16S rRNA; amoA; nifH; dsrA; dsrB; nxrB
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Funding
- Austrian Science Fund (FWF) [P25111-B22, P26127-B20]
- Vienna Science and Technology Fund (WWTF) [LS12-001]
- Austrian Science Fund (FWF) [P25111, P26127] Funding Source: Austrian Science Fund (FWF)
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High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. 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 Illumine MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology.
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