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Modern Methods for Delineating Metagenomic Complexity

2015; Elsevier BV; Volume: 1; Issue: 1 Linguagem: Inglês

10.1016/j.cels.2015.07.007

2639-5460

Ebrahim Afshinnekoo, Cem Meydan, Shanin Chowdhury, Dyala Jaroudi, Collin Boyer, Nick Bernstein, Julia M. Maritz, Darryl Reeves, Jorge Gandara, Sagar Chhangawala, Sofia Ahsanuddin, Amber Simmons, Timothy Nessel, Bharathi Sundaresh, Elizabeth Pereira, Ellen Jorgensen, Sergios‐Orestis Kolokotronis, Nell Kirchberger, Isaac Garcia, David R. Gandara, Sean Dhanraj, Tanzina Nawrin, Yogesh Saletore, Noah Alexander, Priyanka Vijay, Elizabeth Hénaff, Paul Zumbo, Michael Walsh, G. D. O’Mullan, Scott Tighe, Joel T. Dudley, Anya Dunaif, Sean Ennis, Eoghan O’Halloran, Tiago R. Magalhães, Braden Boone, Angela Jones, Theodore R. Muth, Katie Schneider Paolantonio, Elizabeth Alter, Eric Schadt, Jeanne Garbarino, Robert J. Prill, Jane M. Carlton, Shawn Levy, Christopher E. Mason,

Zoonotic diseases and public health

We appreciate the comments of Ackelsberg et al. (Ackelsberg et al., 2015Ackelsberg J. Rakeman J. Hughes S. Peterson J. Mead P. Schriefer M. Kingry L. Hoffmaster A. Gee J. Cell Syst. 2015; 1 (this issue): 4-5Abstract Full Text Full Text PDF Scopus (19) Google Scholar) and have decided to revise the paper (Afshinnekoo et al., 2015Afshinnekoo E. Meydan C. Chowdhury S. Jaroudi D. Boyer C. Bernstein N. Maritz J.M. Reeves D. Gandara J. Chhangawala S. et al.Cell Syst. 2015; 1 (this issue): 72-87Abstract Full Text Full Text PDF Scopus (183) Google Scholar) as follows:Figure 3B has been corrected to show the general coverage of the Yersinia pestis pMT1 plasmid, but not the murine toxin gene (yMT). The initial claim of "…consistent 20× coverage across the murine toxin gene…" was erroneously based on looking at annotations from related plasmids and comparing different reference sequences. In actuality no reads mapped to the yMT gene.The result of low coverage to the Bacillus anthracis plasmids (pXO1 and pXO2) and no evidence of plcR SNP—an often defining feature of anthrax—is now reported in the Results section.The language in the Summary, Results, and Discussion has been revised, and speculative text about pathogenic organisms has been deleted. We now state that although all our metagenomic analysis tools identified reads with similarity to B. anthracis and Y. pestis sequences, there is minimal coverage to the backbone genome of these organisms, and there is no strong evidence to suggest these organisms are in fact present and no evidence of pathogenicity. Furthermore, in regards to the concerns of the culture methods we have posted subsequent details on the study website (http://www.pathomap.org/2015/04/13/culture-methods/) and below. A second culture experiment was performed to address the question of antibiotic resistance (Afshinnekoo et al., 2015Afshinnekoo E. Meydan C. Chowdhury S. Jaroudi D. Boyer C. Bernstein N. Maritz J.M. Reeves D. Gandara J. Chhangawala S. et al.Cell Syst. 2015; 1 (this issue): 72-87Abstract Full Text Full Text PDF Scopus (183) Google Scholar, Figure 4A). Bacteria were cultured in LB agar and then spread onto LB plates, after lawn growth, single colonies were picked and then plated onto antibiotic plates (kanamycin – 50 ug/ml, chloramphenicol – 35 ug/ml, and ampicillin – 100 ug/ml) and growth was assessed. Plates were incubated at 37°C. As a control, air samples were taken and cultured at every location. In all cases, these did not yield growth. The non-selective plate done last when replica plating also serves as a control. There was no quantitative confirmation of bacterial versus non-bacterial organisms, although there was no observable fungal growth in the samples. Further experiments are being done to dive deeper into the question of viability of microorganisms on the subway system as well as the presence of antibiotic-resistant bacteria. The field of metagenomics is relatively new but has great potential to serve an incredibly important role both in our understanding of the world around us—with key applications in the built environment—as well as the clinical realm. Nevertheless, there are still major hurdles and challenges that the field faces in order to realize this potential. We welcome and appreciate the discussion (http://microbe.net/2015/02/17/the-long-road-from-data-to-wisdom-and-from-dna-to-pathogen/) prompted by our study, and we anticipate that this large dataset will enable further experimentation, additional testing of taxonomic tools, and hopefully help in developing methodologies for metagenomic analysis. Geospatial Resolution of Human and Bacterial Diversity with City-Scale MetagenomicsAfshinnekoo et al.Cell SystemsMarch 3, 2015In BriefAfshinnekoo et al. describe a city-scale molecular profile of DNA collected from a city's subway system, public surfaces, and one waterway. These data enable a baseline analysis of bacterial, eukaryotic, and aracheal organisms in the built environment of mass transit and urban life. Full-Text PDF Open AccessLack of Evidence for Plague or Anthrax on the New York City SubwayAckelsberg et al.Cell SystemsJuly 29, 2015In BriefAckelsberg et al. point out a lack of evidence in the dataset of Afshinekoo et al. for the presence of plague and anthrax on the New York City subway. Full-Text PDF Open Archive