@article{3827818e83b448658b1a4b98c47d8f9c,
title = "Detection and enumeration of Lak megaphages in microbiome samples by endpoint and quantitative PCR",
abstract = "Lak megaphages are prevalent across diverse gut microbiomes and may potentially impact animal and human health through lysis of Prevotella. Given their large genome size (up to 660 kbp), Lak megaphages are difficult to culture, and their identification relies on molecular techniques. Here, we present optimized protocols for identifying Lak phages in various microbiome samples, including procedures for DNA extraction, followed by detection and quantification of genes encoding Lak structural proteins using diagnostic endpoint and SYBR green-based quantitative PCR, respectively. For complete details on the use and execution of this protocol, please refer to Crisci et al., (2021).",
keywords = "Health Sciences, Immunology, Microbiology, Molecular Biology",
author = "Crisci, \{Marco A.\} and Corsini, \{Paula M.\} and Nicola Bordin and Chen, \{Lin Xing\} and Banfield, \{Jillain F.\} and Santini, \{Joanne M.\}",
note = "Funding Information: M.A.C. was funded by the BBSRC ( BB/M009513/1 ) through the London Interdisciplinary Doctoral Program. N.B. acknowledges funding by the BBSRC ( BB/R009597/1 ). Metagenomic sequencing that facilitated primer design for this protocol was funded by the Innovative Genomics Institute , UC Berkeley , the Chan Zuckerberg Biohub , and the National Institutes of Health ( RAI092531A ) to J.F.B. For assisting with pig microbiome sampling, the authors would like to thank Mick Bailey, Francesco Debenedetti, and Rachel Burt (University of Bristol). For assisting with racehorse sampling, the authors also thank Roger Ingram and Sharron Ingram (Wendover Stables, Epsom). Funding Information: M.A.C. was funded by the BBSRC (BB/M009513/1) through the London Interdisciplinary Doctoral Program. N.B. acknowledges funding by the BBSRC (BB/R009597/1). Metagenomic sequencing that facilitated primer design for this protocol was funded by the Innovative Genomics Institute, UC Berkeley, the Chan Zuckerberg Biohub, and the National Institutes of Health (RAI092531A) to J.F.B. For assisting with pig microbiome sampling, the authors would like to thank Mick Bailey, Francesco Debenedetti, and Rachel Burt (University of Bristol). For assisting with racehorse sampling, the authors also thank Roger Ingram and Sharron Ingram (Wendover Stables, Epsom). This protocol was designed and optimised by M.A.C. with input from J.M.S. and P.M.C. N.B. advised and assisted with Lak signature gene acquisition from metagenomic datasets. L.-X.C. and J.F.B. curated and provided sequences for the 660 kbp Lak variant from a horse gut metagenome. P.M.C. advised during qPCR optimization and analysis. M.A.C. wrote the protocol with input from J.M.S. P.M.C. N.B. L.-X.C. and J.F.B. The authors of this paper declare no competing interests. Publisher Copyright: {\textcopyright} 2021 The Author(s)",
year = "2022",
month = mar,
day = "18",
doi = "10.1016/j.xpro.2021.101029",
language = "English",
volume = "3",
journal = "STAR Protocols",
issn = "2666-1667",
publisher = "Cell Press",
number = "1",
}
