Journal of Visualized Experiments www.jove.com Copyright © 2014 Creative Commons Attribution-NonCommercial License December 2014 | 94 | e52117 | Page 1 of 15 Video Article Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples Yan Wei Lim 1 , Matthew Haynes 2 , Mike Furlan 1 , Charles E. Robertson 3 , J. Kirk Harris 4 , Forest Rohwer 1 1 Department of Biology, San Diego State University 2 DOE Joint Genome Institute 3 Department of Molecular, Cellular and Developmental Biology, University of Colorado 4 Department of Pediatrics, School of Medicine, University of Colorado Correspondence to: Yan Wei Lim at ywlim.s@gmail.com URL: http://www.jove.com/video/52117 DOI: doi:10.3791/52117 Keywords: Molecular Biology, Issue 94, virome, microbiome, metagenomics, metatranscriptomics, cystic fibrosis, mucosal-surface Date Published: 12/22/2014 Citation: Lim, Y.W., Haynes, M., Furlan, M., Robertson, C.E., Harris, J.K., Rohwer, F. Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples. J. Vis. Exp. (94), e52117, doi:10.3791/52117 (2014). 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. Video Link The video component of this article can be found at http://www.jove.com/video/52117/ Introduction Viral and microbial communities associated with the human body have been investigated extensively in the past decade through the application of sequencing technologies 1,2 . The outcomes have led to the recognition of the importance microbes in human health and disease. The major initiative came from the human microbiome project that describes the bacteria (and some archaea) residing on human skin, and within oral cavities, airways, urogenital tract, and gastrointestinal tract 3 . Further microbiome studies of healthy human airways through bronchoalveolar lavage (BAL) 4,5 and nasopharyngeal swabs 4 have shown that the lung can serve as an environmental sampling device, results in transient microbial colonization in the airways. However, the impact of microbial colonization in impaired airway surfaces can lead to severe and chronic lung infections, such as those seen in Cystic Fibrosis (CF) patients. CF is a lethal genetic disease caused by the mutation in Cystic Fibrosis Transmembrane Regulator (CFTR) gene 6 . These mutations give rise to defective CFTR proteins that in turn affect transepithelial ion transport across the apical surface of the epithelium. The disease affects multiple organ systems, but the majority of mortality and morbidity is attributable to CF lung disease 7 . The CF lung provides a unique ecosystem for microbial colonization. The defect in ion transport causes mucus to build up in the CF airways, creating microenvironments consisting of aerobic, microaerophilic, and anaerobic compartments anchored by a static nutrient-rich mucosal surface. This environment facilitates the colonization and proliferation of microbes, including viral, bacteria, and fungi. Acute and chronic pulmonary microbial infections lead to constant but ineffective immune responses, resulting in extensive airway remodeling, loss of pulmonary capacity, and ultimately pulmonary failure. Bacterial communities associated with the CF lung have been well described using both culture-dependent and culture-independent approaches, which include using 16S ribosomal RNA (rRNA) gene sequencing 8 and shotgun metagenomics 9,10 . The 16S rRNA-based approach is able to characterize a wide range of microbial species and capture broad shifts in community diversity. However, it is limited in its resolution in defining the communities (summarized in Claesson et al. 2010 11 ) and the predictions of metabolic potentials are limited to those general functions known