Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

Abstract

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Analysing Microbial Community Composition through Amplicon Sequencing: From Sampling to Hypothesis Testing

“…It must also be noted that RNA can also be extracted and analyzed as complementary DNA (cDNA). DNA is a more stable molecule, so community signatures are less likely to experience radical change at the DNA level as a result of sample collection (Lim et al, 2014 ; Reck et al, 2015 ). On the other hand, different organisms, specially eukaryotes, can have an enormous range of copies of the rRNA gene in their genomes, which hinders a simple correlation between gene copies and cell numbers (Gong et al, 2013 ).…”

Pathogen Detection and Microbiome Analysis of Infected Wheat Using a Portable DNA Sequencer

“…For the purpose of microbial species identification, host sequences are unwanted and can overwhelm community DNA (Sharpton 2014). To enrich microbial sequences, one strategy is to apply intricate molecular methods during DNA extraction such as gradient centrifugation and sonication (Bulgarelli et al 2012; Lim et al 2014). As these techniques require sophisticated instruments, this can be a challenge to apply to environmental samples, especially in field conditions.…”

Peer Review #2 of "Benchmarking protocols for the metagenomic analysis of stream biofilm viromes (v0.2)"

“…To prevent heating, samples were put on ice during sonication. Finally, one subset of the samples were treated with 6.5 mM DTT and incubated for 1 hour at 37°C (Lim et al, 2014). At the end of the incubation period, samples were chilled on ice.…”