SPOT Microbial Observatory Myovirus g23 3' TRFLP relative peak abundance data from 2014 (Bacterial, Archaeal, and Protistan Biodiversity project, Marine Viral Dynamics project, Mar. Microbial Communities project)

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T4-like myovirus communities were analyzed by terminal restriction fragment length polymorphism (TRFLP) of g23, which encodes the major capsid protein (Chow and Fuhrman, 2012). Viral fingerprints were obtained from both terminal fragments (5’ and 3’). g23-TRFLP products were run in duplicate on non-adjacent lanes on an ABI377 by slab gel electrophoresis with internal size standards (Bioventures, Murfreesboro, TN, USA) every 25 bp (50–900 bp) or 50 bp (900–1400 bp). Peaks were identified in DAx (van Mierlo, Inc, Eindhoven, The Netherlands). Fragments (50–500 bp) were rounded to the nearest 0.1 bp and dynamically binned (Ruan, et al., 2006b; Chow and Fuhrman, 2012). The resulting bins were manually curated to merge bins <0.1 bp wide with the nearest neighbor. Terminal fragments from in silico analysis of publicly available T4-like viral genomes were used to assign identities to environmental g23-TRFLP OTUs. Unique fragment lengths were considered as individual OTUs. Relative abundance of each OTU was calculated by dividing a peak’s area by the total area within the monthly fingerprint. Viral OTUs <0.1% of the community were removed from further analysis, and the remaining peaks were normalized by sample to determine relative abundance per month; each community thus totaled to 100%. Relevant References: * Chow CET, Kim DY, Sachdeva R, Caron DA, and Fuhrman JA. (2014). Top-down controls on bacterial community structure: microbial network analysis of bacteria, T4-like viruses and protists. ISME Journal, 8: 816-829 and  Brown MV, Schwalbach MS, Hewson I, Fuhrman JA.(2005). Coupling 16S-ITS rDNA clone libraries and automated ribosomal intergenic spacer analysis to show marine microbial diversity: development and application to a time series. Environ Microbiol 7: 1466–1479.  Chow C-ET, Fuhrman JA. (2012). Seasonality and monthly dynamics of marine myovirus communities. Environ Microbiol 14: 2171–2183.  Chow C-ET, Sachdeva R, Cram JA, Steele JA, Needham DM, Patel A et al. (2013). Temporal variability and coherence of euphotic zone bacterial communities over a decade in the Southern California Bight. ISME J; epub ahead of print 18 July 2013; doi:10.1038/ismej.2013.122.  Countway PD, Gast RJ, Savai P, Caron DA. (2005). Protistan diversity estimates based on 18S rDNA from seawater incubations in the western north Atlantic1. J Eukaryot Microbiol 52: 95–106.  Countway PD, Vigil PD, Schnetzer A, Moorthi SD, Caron DA. (2010). Seasonal analysis of protistan community structure and diversity at the USC Microbial Observatory (San Pedro Channel, North Pacific Ocean). Limnol Oceanogr 55: 2381–2396.  Fisher MM, Triplett EW. (1999). Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities. Appl Environ Microbiol 65: 4630–4636.

本研究针对T4样肌尾噬菌体（T4-like myovirus）群落，通过靶向编码主要衣壳蛋白的g23基因的末端限制性片段长度多态性（terminal restriction fragment length polymorphism, TRFLP）技术进行分析（Chow与Fuhrman, 2012）。分别从5'和3'末端片段获取病毒指纹图谱。将g23-TRFLP产物以重复样形式在ABI377测序仪的非相邻泳道中进行平板凝胶电泳检测，电泳所用内参分子量标准（Bioventures, 美国田纳西州默弗里斯伯勒）在50–900 bp区间每25 bp设置一个刻度，900–1400 bp区间每50 bp设置一个刻度。峰型识别在DAx软件（van Mierlo公司，荷兰埃因霍温）中完成。将50–500 bp区间的片段数值修约至最近的0.1 bp，并进行动态分箱（Ruan等, 2006b；Chow与Fuhrman, 2012）。对生成的分箱结果进行人工校正，将宽度小于0.1 bp的分箱与相邻最近的分箱进行合并。通过对公开可用的T4样噬菌体基因组进行计算机模拟分析得到的末端片段，用于为环境样本的g23-TRFLP操作分类单元（Operational Taxonomic Unit, OTU）赋予分类身份。将具有独特片段长度的序列视为独立的OTU。每个OTU的相对丰度通过将该峰的面积除以月度指纹图谱内的总峰面积计算得到。将占群落丰度小于0.1%的病毒OTU剔除，不纳入后续分析；剩余峰通过样本标准化处理，以计算月度相对丰度，最终每个群落的总丰度归一为100%。相关参考文献：* Chow CET、Kim DY、Sachdeva R、Caron DA与Fuhrman JA. (2014). 细菌群落结构的下行调控：细菌、T4样病毒与原生生物的微生物网络分析. ISME期刊, 8: 816-829及 Brown MV、Schwalbach MS、Hewson I与Fuhrman JA. (2005). 结合16S-ITS rDNA克隆文库与自动化核糖体基因间区分析表征海洋微生物多样性：方法开发及其在时间序列研究中的应用. 环境微生物学, 7: 1466–1479. Chow C-ET与Fuhrman JA. (2012). 海洋肌尾噬菌体群落的季节节律与月度动态. 环境微生物学, 14: 2171–2183. Chow C-ET、Sachdeva R、Cram JA、Steele JA、Needham DM、Patel A等. (2013). 南加州近岸海域真光层细菌群落十年间的时间变异与一致性. ISME期刊；2013年7月18日预印版在线发表；DOI:10.1038/ismej.2013.122. Countway PD、Gast RJ、Savai P与Caron DA. (2005). 基于西北大西洋西部海水培养物18S rDNA的原生生物多样性评估. 真核微生物学报, 52: 95–106. Countway PD、Vigil PD、Schnetzer A、Moorthi SD与Caron DA. (2010). 美国南加州大学微生物观测站（北太平洋圣佩德罗海峡）原生生物群落结构与多样性的季节分析. 湖沼学与海洋学, 55: 2381–2396. Fisher MM与Triplett EW. (1999). 用于微生物多样性核糖体基因间区分析的自动化方法及其在淡水细菌群落中的应用. 应用与环境微生物学, 65: 4630–4636.

创建时间：

2021-12-05