Prokaryote 16S rRNA sequence data from antFOCE biofilms

This metadata record contains an Excel spreadsheet with Operational Taxonomic Units (OTUs) gained from 16S rRNA gene sequencing of prokaryotes sampled from Biofilm slides deployed as part of the antFOCE experiment in the austral summer of 2014/15 at Casey station, East Antarctica. Refer to antFOCE report section 4.5.3 for deployment, sampling and analysis details.https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127Sampling design2 trays of 8 horizontal standard glass microscope slides (72 x 25 mm) per chamber. Four of the glass slides were scored with a diamond pencil approximately 18 mm from the right hand end of the slide and deployed scored side up. The remaining four slides were unmodified. Slides were sampled at:*    Tmid - one tray per chamber / open plot. The sampled try was repopulated with fresh slides and redeployed*    Tend – 2 slides trays per chamber / open plot.Sampling procedureAfter 31 days deployment, 1 slide tray per chamber / open plot was sampled. At Tend both trays in each chamber / open plot were sampled. To minimize disturbance while being raised to the surface, each tray was removed from the tray holder by divers and placed in a seawater filled container with a lid. On the surface, slides were removed from the tray using ethanol sterilized forceps. The four unscoured slides per chamber / open plot were placed in a plastic microscope slide holder with a sealable lid. The scoured slides were placed individually in 70 ml plastic sample jars. Lab procedure - CaseyThe slide holder (4 unscoured slides) from each chamber / open plot was frozen at -20C immediately upon return to the lab. The scoured slides were preserved in sea water containing 1% final concentration glutaraldehyde in separate jars.Preservation Issue: Scoured slides were not refrigerated, either at Casey, during RTA or in Kingston before the 26th Nov 2015, when they were transferred to the 4C Cold Store. antFOCE Background The antFOCE experimental system was deployed in O'Brien Bay, approximately 5 kilometres south of Casey station, East Antarctica, in the austral summer of 2014/15. Surface and sub-surface (in water below the sea ice) infrastructure allowed controlled manipulation of seawater pH levels (reduced by 0.4 pH units below ambient) in 2 chambers placed on the sea floor over natural benthic communities. Two control chambers (no pH manipulation) and two open plots (no chambers, no pH manipulation) were also sampled to compare to the pH manipulated (acidified) treatment chambers. Details of the antFOCE experiment can be found in the report – "antFOCE 2014/15 – Experimental System, Deployment, Sampling and Analysis". This report and a diagram indicating how the various antFOCE data sets relate to each other are available at:https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127High throughput sequencing of the 16S rRNA gene (Shane Powell)Genomic DNA samples were sequenced at the Ramaciotti Centre for Genomics at the University of New South Wales. The V4 region of the 16S rRNA gene was sequenced with the primers 515F – 806R on an Illumina MiSeq with MiSeq v2 reagent kit.Sequences were processed using MOTHUR v 1.36.1 (Schloss et al. 2009) following the suggested protocol for processing MiSeq datasets as described in Kozich et al. (2013) with the following modifications. The make.contigs command was used to join the paired-end reads from the fastaq files. Sequences that were longer than 300 bp or contained more than one ambiguous base were removed with the screen.seqs. Within each sample, exact duplicate sequences were merged with unique.seqs. The sequences were then aligned against the Silva database (downloaded March 1 2016). Sequences with 3 or less nucleotide differences in total were clustered together using pre.cluster. Potentially chimeric sequences were removed with the defaults settings of the MOTHUR implementation of uchime. After removal of chimeric sequences, the remaining sequences were grouped into operational taxonomic units (OTU) using cluster.split with taxlevel=4 (Order). Finally a table of the number of times each OTU appeared in each sample was generated with make.shared with a cut-off of 0.03 and the OTU were classified with classify.otu. As the sample with the fewest sequences contained 63955 sequences, rarefaction was carried out using the sub.sample command to randomly select 63 955 sequences per sample.A total of 4 604 760 sequences remained in the final OTU table. Any OTU that contained less than 500 sequences (less than 0.01%) were removed as potentially spurious or chimeric sequences, especially as these were generally unclassified sequences. Multivariate analyses were carried out using the PRIMER software. Data were standardised (converted to a percentage) prior to any other analysis....Kozich, J.J., Westcott, S.L., Baxter, N.T., Highlander, S.K. and Schloss, P.D., 2013. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and environmental microbiology 79:5112-5120.Schloss, P.D., Westcott, S.L., Ryabin, T., Hall, J.R., Hartmann, M., Hollister, E.B., Lesniewski, R.A., Oakley, B.B., Parks, D.H., Robinson, C.J. and Sahl, J.W., 2009. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Applied and environmental microbiology 75:7537-7541.

本元数据记录包含一份Excel表格，其中收录了2014/2015年南极夏季于东南极洲凯西站开展的antFOCE实验中，从部署的生物膜载玻片上采集的原核生物16S核糖体RNA（16S rRNA）基因测序所得的操作分类单元（Operational Taxonomic Units, OTUs）。有关部署、采样与分析的详细信息，请参阅antFOCE报告第4.5.3节。相关链接：https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127

采样设计：每个舱室设置2组载玻片托盘，每组托盘含8张水平放置的标准玻璃显微镜载玻片（72×25 mm）。其中4张载玻片于距右端约18 mm处用金刚石笔刻痕，并以刻痕面朝上的方式部署；剩余4张载玻片未做任何处理。载玻片采样节点如下：
* Tmid：每个舱室/开放样区采样1组托盘，采样后的托盘将更换为全新载玻片后重新部署
* Tend：每个舱室/开放样区采样2组托盘

采样流程：载玻片部署31天后，对每个舱室/开放样区的1组托盘进行采样；在Tend节点时，则对每个舱室/开放样区的2组托盘全部采样。为避免被提升至海面过程中受到干扰，潜水员将托盘从托盘架中取出，放入装有海水的带盖容器中。抵达海面后，使用经乙醇灭菌的镊子取出载玻片。将每个舱室/开放样区的4张未刻痕载玻片放入带密封盖的塑料载玻片架中，刻痕载玻片则分别装入70 ml的塑料样品瓶内。

凯西站实验室流程：每个舱室/开放样区的载玻片架（含4张未刻痕载玻片）在返回实验室后立即置于-20℃环境下冷冻。刻痕载玻片则分别保存在装有终浓度1%戊二醛的海水的样品瓶中。

保存问题：2015年11月26日前，刻痕载玻片在凯西站、RTA期间以及金斯顿均未进行冷藏，直至2015年11月26日被转移至4℃冷藏库。

antFOCE实验背景：antFOCE实验系统于2014/2015年南极夏季部署在东南极洲凯西站以南约5公里的奥布莱恩湾（O'Brien Bay）。该系统的海面及海冰下水下基础设施可对放置于海底自然底栖生物群落上方的2个舱室内的海水pH值进行可控调控（较环境pH降低0.4个单位）。同时设置了2个对照舱室（未进行pH调控）与2个开放样区（无舱室、未进行pH调控），用于与酸化处理舱室进行对照比较。有关antFOCE实验的详细信息可参阅报告"antFOCE 2014/15 – Experimental System, Deployment, Sampling and Analysis"。该报告以及展示各antFOCE数据集相互关联关系的示意图可通过以下链接获取：https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127

16S核糖体RNA基因高通量测序（Shane Powell）：基因组DNA样本在新南威尔士大学拉马科蒂基因组学中心完成测序。采用引物515F–806R对16S rRNA基因的V4区进行扩增测序，测序平台为Illumina MiSeq，使用MiSeq v2试剂盒。

序列处理与分析流程：使用MOTHUR v1.36.1（Schloss等人，2009）进行序列处理，遵循Kozich等人（2013）描述的MiSeq数据集处理建议方案，并做如下修改：使用make.contigs命令拼接双端测序的fastq文件；通过screen.seqs命令移除长度超过300 bp或包含1个以上模糊碱基的序列；在每个样本中，使用unique.seqs命令合并完全重复的序列；随后将序列与Silva数据库（2016年3月1日下载）进行比对；使用pre.cluster命令将总核苷酸差异≤3的序列聚类；通过uchime的MOTHUR实现默认设置移除潜在嵌合序列。移除嵌合序列后，使用cluster.split命令（taxlevel=4，即目级分类水平）将剩余序列聚类为操作分类单元（OTU）；最终通过make.shared命令生成各样本中每个OTU的计数表格，聚类阈值为0.03，并使用classify.otu对OTU进行分类。由于序列最少的样本仍包含63955条序列，因此使用sub.sample命令进行稀疏化分析，每个样本随机选取63955条序列。最终OTU表格中共保留4604760条序列。将序列数少于500条（占比<0.01%）的OTU移除，这类OTU通常为未分类序列，可能属于伪影或嵌合序列。多变量分析使用PRIMER软件完成。在进行其他分析前，先将数据标准化为百分比形式。

参考文献：
1. Kozich, J.J., Westcott, S.L., Baxter, N.T., Highlander, S.K. and Schloss, P.D., 2013. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. *Applied and environmental microbiology* 79:5112-5120.
2. Schloss, P.D., Westcott, S.L., Ryabin, T., Hall, J.R., Hartmann, M., Hollister, E.B., Lesniewski, R.A., Oakley, B.B., Parks, D.H., Robinson, C.J. and Sahl, J.W., 2009. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. *Applied and environmental microbiology* 75:7537-7541.