Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples

Abstract

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.

Effects of Specimen Collection Methodologies and Storage Conditions on the Short-Term Stability of Oral Microbiome Taxonomy

“…The number of reads mapped to a sample ranged from 240 to 9,533,688, with 147/152 samples mapped to more than 10,000 reads. The wide distribution of reads to a multiplexed sample is not uncommon and is seen in other studies using Illumina platforms (24,25). Samples where more than 50% of the reads could not be assigned a bacterial phylum were deemed to be artifacts of sequencing and subsequently excluded from analysis.…”

Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

“…The Mu-DNA method can be easily converted to SPRI purification by replacing the silica binding step with an SPRI protocol. However Vo and Jedlicka (2014) found SPRI to only have improved performance with less contaminated samples, such as avian oral and cloacal swab extractions. SPRI DNA purification is therefore best reserved for relatively clean environmental sample types, in particular clear lake and stream waters or tissue samples (see Mayjonade et al 2016).…”

“…However we found that neither DNeasy Blood and Tissue nor the adapted Mu-DNA: Tissue/Water protocol could achieve inhibition-free DNA from turbid, algal rich waters (Water B) as effectively as DNeasy PowerWater or Mu-DNA: Water (data not shown). Solid phase reversible immobilisation (SPRI) DNA purification, based on Rohland and Reich (2012), can achieve higher DNA yield and purity than spin column based protocols (Vo and Jedlicka 2014). The Mu-DNA method can be easily converted to SPRI purification by replacing the silica binding step with an SPRI protocol.…”

“…Although designed for specific sample types, many studies have adapted their use across sample types. DNeasy PowerSoil, or aspects thereof, has been used for stomach, gut or faecal analysis of invertebrates (Knapp et al 2010, O'Rorke et al 2015, fish (Koinari et al 2013, Bolnick et al 2014, reptiles (Lau et al 2013, Colston et al 2015, birds (Vo andJedlicka 2014, Lewis et al 2016), mammals (Parfrey et al 2014, Ishaq andWright 2014) and, in particular, the Human Microbiome Project (Aagaard et al 2013). DNeasy Blood and Tissue has been used for studies of environmental DNA (eDNA) from water samples (Rees et al 2014, Spens et al 2016, Niemiller et al 2017.…”

Full Issue

“…While publications analyzing bat diets have dominated the field (Symondson and Harwood 2014), significant advances in establishing effective protocols for processing minimally invasive avian samples now show it is possible to recover high quantities and qualities of DNA from avian fecal samples for downstream analysis (Vo and Jedlicka 2014). Trevelline et al (2016) successfully applied molecular techniques to reveal that Louisiana Waterthrush (Parkesia motacilla) nestlings consumed larger percentages of lepidopterans and dipterans than was expected.…”