Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples.
Next-generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI-based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post-adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250-bp, paired-end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian-specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing.
Museum of Vertebrate Zoology and Department of Integrative Biology, University of California, Berkeley, CA, 94720-3140, USA.
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D000818:Animals; D002988:Cloaca / Q000382:microbiology*; D004247:DNA / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D056186:Metagenomics / Q000379:methods*; D009061:Mouth Mucosa / Q000382:microbiology*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D020308:Songbirds / Q000382:microbiology
D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D004247:DNA
cloacal; faeces/feces; microbial ecology; oral; solid phase reversible immobilization beads; trophic ecology