DNA sequence data collected during the SIPEX II voyage of the Aurora Australis, 2012

Purpose of experiments:

Sequence data obtained to determine community structure of pack sea-ice microbial communities and whether it is effected by exposures to elevated CO2 levels.

Summary of Methods:

Cells in sea-ice brines were filtered onto 0.2 micron filters and material extracted using the MoBio Water DNA extraction kit. The DNA was analysed by Research and Testing Laboratories Inc. (Lubbock, Texas, USA) via 454 pyrosequencing. The bacteria were analysed using primers set 10F-519R, which targets 16S rRNA genes. 16S rRNA genes associated with chloroplast and mitochondria are included in this dataset but represent a minority of sequences in most samples. Eukaryotes were analysed using primers set 550F-1055R, which targets 18S rRNA genes. The 454 pyrosequencing analysis with the Titanium GS FLX+ kit used generates on average 3000 reads incorporating custom pyrotags for later stages of the data analysis. The specific steps used for subsequent data analysis are described in the attached PDF file (Data_Analysis_Methodology.PDF). This output was further refined by first determining consensus sequences at the 98% similarity level using Weizhong Li’s online software site CD-HIT (http://weizhongli-lab.org/cd-hit/) Reference: Niu B, Fu L, Sun S, Li W. 2010. Artificial and natural duplicates in pyrosequencing reads of metagenomic data. BMC Bioinformatics 1:187 doi:10.1186/1471-2105-11-187. The consensus sequences were then checked for errors, manually curated, and aligned against closest matching sequences obtained from the NCBI database (www.ncbi.nlm.nih.gov) to finally obtained a list of consensus operational taxonomic entities and the number of reads obtained for each samples analysed.

File: SIPEXII_DNA_Sample_information.xlsx provides sampling and analysis information for the detailed results in the other two files
File: SCIPEXII__sea_ice_bacteria_OTUs.xlsx contains information on the number of 16S rRNA reads in bacteria Phylum/Class and OTUs
File: SCIPEXII_sea_ice_brines_eukaryote_community_OTU_data.xlsx contains information on the number of 16S rRNA reads in eukaryotic microbes: Phylum/Order/Closest taxon and OTUs

实验目的：
本实验旨在获取序列数据，以解析成片海冰微生物群落的群落结构，并探明该群落是否会受到高浓度CO₂暴露的影响。

实验方法总结：
将海冰盐卤中的微生物细胞过滤至0.2μm滤膜上，随后采用MoBio Water DNA提取试剂盒提取核酸物质。提取得到的DNA由美国德克萨斯州拉伯克市的Research and Testing Laboratories Inc.通过454焦磷酸测序（454 pyrosequencing）完成分析。
细菌群落分析采用靶向16S rRNA基因的引物对10F-519R。本数据集包含与叶绿体和线粒体同源的16S rRNA基因序列，但在多数样本中这类序列仅占总序列的少数。真核生物群落分析则采用靶向18S rRNA基因的引物对550F-1055R。
使用Titanium GS FLX+试剂盒开展的454焦磷酸测序分析平均可产生3000条序列读长（reads），并附带定制焦磷酸标签（pyrotags）以供后续数据分析使用。后续数据分析的具体步骤详见附件PDF文件《Data_Analysis_Methodology.PDF》。
该测序结果将通过以下步骤进一步优化：首先借助李为忠开发的在线软件CD-HIT（http://weizhongli-lab.org/cd-hit/）以98%的相似性阈值生成一致性序列。参考文献：Niu B, Fu L, Sun S, Li W. 2010. 宏基因组数据焦磷酸测序读长中的人工与自然重复序列. BMC Bioinformatics 1:187 DOI:10.1186/1471-2105-11-187。
随后将对一致性序列进行错误校验与人工手动校正，并与从美国国家生物技术信息中心（NCBI，www.ncbi.nlm.nih.gov）获取的最相似序列进行比对，最终得到一致性操作分类单元（Operational Taxonomic Unit，OTU）列表以及各分析样本对应的序列读长数量。

文件：
文件《SIPEXII_DNA_Sample_information.xlsx》提供了样本采集与分析信息，用于阐释另外两个文件中的详细实验结果。
文件《SCIPEXII__sea_ice_bacteria_OTUs.xlsx》包含细菌群落按门/纲及操作分类单元（OTU）分类的16S rRNA序列读长数量信息。
文件《SCIPEXII_sea_ice_brines_eukaryote_community_OTU_data.xlsx》包含真核微生物按门/目/最相近分类类群及操作分类单元（OTU）分类的16S rRNA序列读长数量信息。