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Commercial DNA extraction kits are widely used for cultivation-free surveys of marine sediment. However, the consequences of popular extraction-kit choices for sequence-based biological inferences about marine sedimentary communities have not previously been exhaustively assessed. To address this issue, we extracted DNA from multiple paired subsamples of marine sediment using two popular commercial extraction kits (MO BIO Laboratories PowerSoil R DNA isolation kit and MP Biomedicals FastDNA Spin Kit for Soil). We report comparisons of (1) total DNA yield, (2) extract purity, (3) gene-targeted quantification, and (4) postsequencing ecological inferences in near-seafloor (< 1 meter below seafloor [mbsf]) and subsurface (> 1 mbsf) marine sediment. In near-seafloor sediment, the MP Biomedicals FastDNA Spin Kit for Soil exhibits higher extraction yields, higher 16S rRNA gene loads, higher taxonomic diversity, and lower contaminant loads. In subseafloor sediment, both kits yield similar values for all of these parameters. The MO BIO Laboratories PowerSoil R DNA isolation kit generally co-extracts less protein with the DNA in both near-seafloor and subseafloor sediment. For samples from all depths, both kits exhibit similar depth-dependent community richness patterns, taxonomic composition, and ordination-based similarity trends. We conclude that, despite kitspecific differences in extract yields, purity and reagent contaminant loads, ecological inferences based on next-generation sequencing of DNA extracted using these popular commercial kits are robustly comparable, particularly for subseafloor sediment samples. Microbes in marine sediment comprise a sizable fraction of Earth’s biosphere (D’Hondt et al. 2004; Kallmeyer et al. 2012). Community-wide adaptions to starvation (Jørgensen and D’Hondt 2006), extreme energy limitation (Hoehler and Jørgensen 2013), and slow biomass turnover rates (Lomstein et al. 2012) make cultivation-based ecological interrogation of this habitat prohibitively difficult (Cragg et al. 1990; Parkes et al. 2014; Jørgensen and Marshall 2016). In contrast, the cost of DNA sequencing, an alternative to cultivationbased surveys, is at an all-time low (Muir et al. 2016) and fuels the “next-generation sequencing (NGS)” revolution (Park and Kim 2016). NGS has contributed significantly to understanding of subseafloor sedimentary life (Biddle et al. 2008, 2018; Jørgensen et al. 2012; Orsi et al. 2013, 2018; Spang et al. 2015; Starnawski et al. 2017; Karst et al. 2018) and is now a standard tool for ecological studies of this and other marine habitats (Orcutt et al. 2011). The first step in NGS is nucleic-acid extraction from the sample matrix (Lombard et al. 2011). Given the remarkable complexity of marine sediment, a universally optimal DNA extraction method is unrealistic. Therefore, numerous protocols exist that aim to optimize specific aspects of the extraction process (Lipp et al. 2008; Kallmeyer and Smith 2009; Lloyd et al. 2010; Morono et al. 2013, 2014; Lever et al. 2015). Different methods of DNA extraction are known to differently affect DNA yield and quality (Mahmoudi et al. 2011; Knauth et al. 2013) and these consequences must be considered in cross-study comparisons (Felczykowska et al. 2015). Standardization of nucleic-acid extraction technique is a known challenge for surveying microbial biogeography across subseafloor habitats (Orcutt et al. 2011). Although formal efforts have been made to bring this issue to light (Orcutt et al. 2013), no consensus has been reached. Commercial DNA-extraction kits provide standardized, organic-solvent-free alternatives to the laborious tasks of inhouse reagent preparation and protocol optimization (Tan and *Correspondence: [email protected] Additional Supporting Information may be found in the online version of this article. Author Contribution Statement: GAR and SD conceived the experiment. GAR and DG collected and analyzed samples. GAR, DG, and SD interpreted results. GAR wrote the paper. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1 LIMNOLOGY and OCEANOGRAPHY: METHODS Limnol. Oceanogr.: Methods 00, 2018, 00–00 VC 2018 The Authors. Limnology and Oceanography: Methods published by Wiley Periodicals, Inc. on behalf of Association for the Sciences of Limnology and Oceanography doi: 10.1002/lom3.10264 Limnol. Oceanogr.: Methods 16, 2018, 525–536