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_Project4127

Sampling design
2 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 procedure
After 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 - Casey
The 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_Project4127

High 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年南极夏季在南极东部凯西站开展的antFOCE实验中，从部署的生物膜载玻片上采集的原核生物16S rRNA基因（16S rRNA gene）测序得到的操作分类单元（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章节。
相关链接：https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127

## 采样设计
每个舱室配备2组载玻片托盘，每组包含8张水平放置的标准玻璃显微镜载玻片（72 × 25 mm）。其中4张载玻片在距玻片右端约18 mm处用金刚石笔刻痕，部署时刻痕面朝上；剩余4张载玻片未做任何修饰。载玻片的采样时间节点分为：
* Tmid：对每个舱室/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：对每个舱室/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时间点，则对每个舱室/开放样区的2组托盘全部采样。为尽量减少上浮过程中的扰动，潜水员将托盘从托盘架取出，放入装有海水的带盖容器中。回到甲板后，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的塑料样品罐内。

## 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℃环境下冷冻保存。刻痕载玻片则保存在添加了终浓度1%戊二醛的海水中，分装于不同的样品罐中。

## 保存问题
2015年11月26日之前，刻痕载玻片在凯西站、RTA转运过程中以及金斯顿存储期间均未进行冷藏处理，直至当日才被转移至4℃冷库。

## antFOCE实验背景
antFOCE实验系统于2014/2015年南极夏季部署在南极东部凯西站以南约5公里的奥布莱恩湾。该系统配备水面及冰下基础设施，可对放置在海底自然底栖群落上方的2个舱室的海水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），用于与酸化处理的实验组舱室进行对比。

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 rRNA基因高通量测序（Shane Powell 负责）
基因组DNA样品在新南威尔士大学拉马科蒂基因组学中心（Ramaciotti Centre for Genomics）完成测序。使用引物515F–806R对16S rRNA基因的V4区域进行扩增，采用Illumina MiSeq测序平台及MiSeq v2试剂试剂盒完成测序。

测序数据使用MOTHUR v1.36.1（Schloss等人，2009）进行处理，参考Kozich等人（2013）中针对MiSeq数据集的推荐分析流程，并做如下修改：使用make.contigs命令合并双端fastq测序读段；通过screen.seqs命令去除长度超过300 bp或包含多于1个模糊碱基的序列；使用unique.seqs命令合并每个样品内的完全重复序列；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；使用MOTHUR内置的uchime工具的默认参数去除潜在嵌合序列；去除嵌合序列后，使用cluster.split命令并设置taxlevel=4（目级分类水平）将剩余序列聚类为操作分类单元（OTUs）；最终通过make.shared命令以0.03的相似度阈值生成每个样品中各OTU的计数表格，并使用classify.otu对OTU进行分类。由于测序量最少的样品包含63955条序列，因此使用sub.sample命令进行抽平，每个样品随机选取63955条序列。

最终的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，此类OTU通常为未分类序列，可能为伪影或嵌合序列。多变量分析使用PRIMER软件完成，所有数据在进行其他分析前先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.