Mullaloo aquifer 16S and 18S rRNA data

Groundwater samples for eDNA analysis were collected in June 2015 approximately 18 months after the formal commissioning of the Pawsey Centre GWC system located in suburban Perth, Western Australia, in November 2013. DNA was extracted from filtered bore water samples from Production bores and monitoring bores from the Pawsey bores and Water Corporation bores. \nLineage: Groundwater was filtered on 0.1 µm Durapore® membrane filters using a peristaltic pump. Cells were harvested on the on 0.1 µm Durapore® membrane filters and 0.2 g biomass from the filters was used for extracting DNA the Powersoil DNA isolation kit with extended incubation steps and an extra ethanol wash before the final elution in 100 µL of C6 (elution buffer). DNA samples were amplified using EMP 16S rRNA primers to analyse bacteria communities and EMP 18S v4 rRNA primers for eukaryote community analysis. Bacterial and archaeal 16S rRNA genes were amplified using the standard Earth Microbiome project (EMP) 16S Illumina amplicon protocol (http://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/16s/) with primers: 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT) (Caporaso et al., 2011). Eukaryotic 18S rRNA genes were amplified using EMP 18S Illumina amplicon protocol (http://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/18s/) with primers Euk_1391f (GTACACACCGCCCGTC) and EukBr (TGATCCTTCTGCAGGTTCACCTAC) (Amaral-Zettler et al. 2009). Amplicon libraries were prepared with Illumina Nextera kit. Next generation sequencing (NGS) was carried out using the Illumina MiSeq platform (Illumina, Inc., San Diego, USA), 2x250 bp with paired reads, and performed according to manufacturer’s directions at the Ramaciotti Centre for Genomics (UNSW Sydney, Australia). The 18S and 16S rRNA gene sequence data were processed using a custom pipeline Greenfield Hybrid Amplicon Pipeline (GHAP) which is based around USEARCH tools (Edgar, 2013).

本研究用于环境DNA（environmental DNA, eDNA）分析的地下水样本采集于2015年6月，距2013年11月正式投用的位于西澳大利亚州珀斯郊区的帕西中心地下水监测系统（Pawsey Centre GWC system）约18个月。DNA提取自经过滤的生产井与监测井的井水样，这些井涵盖帕西中心配套井及西澳大利亚水务公司管辖井。
实验处理流程：地下水样本采用蠕动泵过滤至0.1 µm Durapore®微孔滤膜上。菌体收集于该0.1 µm Durapore®微孔滤膜上，称取滤膜上0.2 g的生物量，采用Power Soil DNA提取试剂盒（Powersoil DNA isolation kit）进行DNA提取；该提取流程采用延长孵育时长的操作步骤，并在最终用100 µL C6洗脱缓冲液（elution buffer）洗脱前，额外增加了一次乙醇洗涤步骤。
DNA样本采用环境微生物组计划（Earth Microbiome Project, EMP）的16S rRNA引物进行扩增，以分析细菌群落；同时采用EMP 18S v4 rRNA引物扩增，用于真核生物群落分析。
细菌与古菌的16S rRNA基因扩增采用标准环境微生物组计划（EMP）16S Illumina扩增子实验方案，具体参照公开方案链接http://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/16s/，所用引物为515F（序列：GTGCCAGCMGCCGCGGTAA）与806R（序列：GGACTACHVGGGTWTCTAAT）（Caporaso等，2011）。
真核生物18S rRNA基因扩增采用EMP 18S Illumina扩增子实验方案，具体参照公开方案链接http://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/18s/，所用引物为Euk_1391f（序列：GTACACACCGCCCGTC）与EukBr（序列：TGATCCTTCTGCAGGTTCACCTAC）（Amaral-Zettler等，2009）。
扩增子文库构建采用Illumina Nextera试剂盒完成。
下一代测序（Next Generation Sequencing, NGS）采用Illumina MiSeq平台（Illumina公司，美国圣地亚哥）进行，测序模式为2×250 bp双端测序，实验操作严格遵循厂商说明书，测序工作由澳大利亚悉尼新南威尔士大学拉马乔蒂基因组学中心（Ramaciotti Centre for Genomics, UNSW Sydney）承接完成。
18S与16S rRNA基因序列数据采用自定义开发的格林菲尔德杂交扩增子分析流程（Greenfield Hybrid Amplicon Pipeline, GHAP）进行处理，该流程基于USEARCH工具集（Edgar, 2013）搭建。