Freshwater monitoring by nanopore sequencing (Albacore v2.3.1 base calls). Freshwater monitoring by nanopore sequencing (Albacore v2.3.1 base calls)

1. Summary We conducted metagenomic analyses of freshwater samples (400 ml) from nine distinct locations (samples 1-9, with 9.1 and 9.2 as biological replicates from the same site) along a ~12 kilometre trajectory of the River Cam in Cambridge, East Anglia, UK. This was done at three time points in 2018: on April 15th, June 17th, and August 19th. 2. Workflow and Data Samples were filtered through 0.22 µM pore-sized nitrocellulose membranes. We then performed DNA extractions with a modified DNeasy PowerWater protocol (Qiagen, Hilden, Germany). 400 ml of deionised water were subjected to the same workflow as negative controls (N). DNA isolates were barcoded according to their location or negative control ID, amplified for full-length 16S rDNA (~1.5 kb) and complemented by a positive control (P) containing a defined microbial community standard which features eight distinct bacterial species (ZymoBIOMICS D6305, Zymo Research, Irvine, CA, USA). The resulting amplicons were sequenced with one MinION flow cell per time point (LSK109 kit with R9.4 pore chemistry, Oxford Nanopore Technologies). The MinION instrument was run for approximately 48 hours, until no further sequencing reads were obtained. Raw fast5 files were recorded and locally base called using Albacore (v2.3.1); please note that we advise you to use more recent base calls from the Guppy software (v3.1.5) provided here (https://www.ebi.ac.uk/ena/data/view/PRJEB34900). Reads were demultiplexed and adapters trimmed using porechop (v0.2.4). The only non-default parameter was ‘--check_reads’ (set to 50,000 to increase the subset of reads to search for adapter sets). Next, we removed all reads shorter than 1.4 kb and longer than 1.6 kb using filtlong (v0.2.0). Here, we deposit the de-multiplexed and processed reads as one fastq file per time point and location (or control). 3. Analyses and Media We encourage you to read about our findings with this data via bioRxiv (https://www.biorxiv.org/content/10.1101/2020.02.06.936302v2). Please also visit the project website (www.puntseq.co.uk) and Twitter account (https://twitter.com/puntseq) for more details and updates.

1. 研究概述 我们对英国东安格利亚剑桥市剑河（River Cam）约12公里沿岸的9个不同点位采集的400毫升淡水样本开展了宏基因组分析（metagenomic analyses）：样本编号1至9，其中9.1与9.2为同一位点的生物学重复（biological replicates）。采样于2018年的三个时间点进行：4月15日、6月17日与8月19日。
2. 实验流程与数据处理 首先使用孔径0.22微米的硝酸纤维素膜过滤样本。随后采用经改良的DNeasy PowerWater实验方案（Qiagen，德国希尔德）完成DNA提取。我们以400毫升去离子水按照相同流程处理，作为阴性对照（N组）。根据样本采集点位或阴性对照的ID对DNA提取物进行条形码标记，扩增全长16S核糖体DNA（16S rDNA，~1.5 kb），并设置阳性对照（P组）：该对照包含一组明确的微生物群落标准品，涵盖8种不同的细菌（ZymoBIOMICS D6305，美国加州欧文市Zymo Research公司）。
所得扩增子将按时间点分别使用一张MinION测序流通池进行测序，配套LSK109测序试剂盒与R9.4测序孔化学试剂（英国牛津纳米孔科技公司，Oxford Nanopore Technologies），测序时长约48小时，直至无额外测序读段（sequencing reads）产出。原始fast5格式文件（fast5 file）将被记录，并通过Albacore（v2.3.1）完成本地碱基识别（base called）；请注意，我们推荐使用本数据集提供的Guppy软件（v3.1.5）生成的更新版碱基识别结果，相关链接为https://www.ebi.ac.uk/ena/data/view/PRJEB34900。
使用porechop（v0.2.4）对测序读段进行解复用（demultiplexed）并修剪接头序列（adapter），仅使用了一项非默认参数："--check_reads"（设置为50000，以扩大用于搜索接头序列的读段子集）。随后使用filtlong（v0.2.0）移除所有长度短于1.4 kb或长于1.6 kb的测序读段。
本数据集将经过解复用与处理后的测序读段，按每个时间点、每个采集点位（或对照组）分别存储为单个fastq格式文件（fastq file）。
3. 数据分析与相关资源 我们欢迎您通过bioRxiv平台（https://www.biorxiv.org/content/10.1101/2020.02.06.936302v2）查阅使用本数据集得到的研究结果。更多细节与更新信息，也可访问项目官网（www.puntseq.co.uk）及Twitter账号（https://twitter.com/puntseq）获取。