Sequencing reads used in a metatranscriptomic analysis of changing dynamics in the plankton communities adjacent to aquaculture leases in southern Tasmania, Australia

We hypothesised that the metatranscriptome could be used to monitor changing phytoplankton physiology near leases. To test this hypothesis, opportunistic samples were collected from one oceanic location in winter and one estuarine location in spring and analysed via RNASeq. Transcriptomes from different locations were found to have little overlap, due to different community compositions in the oceanic and estuarine locations. Transcript function was similar at each location. Proximity to the salmon pen had little influence over the transcriptome at the estuarine location. In the oceanic environment, diatom-based production decreased near pens and dinoflagellate-based production increased as demonstrated through the abundance of carbon fixation and nitrogen-acquisition-related transcripts. Our initial results suggest that the use of the metatranscriptome in monitoring is promising.\nLineage: Water samples were filtered through a 5 µm, 47-mm Nuclepore filter, and immediately flash frozen in liquid nitrogen. To extract RNA, cells were homogenized in a custom guanadidium thiocyanate buffer in a bead beater using MoBio lysing matrix E and vortexed for 15 s, then RNA was extracted from the lysate using a TRIzol procedure followed by a Qiagen RNeasy column clean up. RNA was checked for quality using a Fisher nanodrop (minimum quantity = 25 ng RNA per µl), and quantity using an Agilent bioanalyzer with a minimum RIN of 7 (lowered to accommodate the multiple taxa within the extraction and the low yield). Samples were prepared for sequencing at the Ramaciotti Centre for gene function analysis using Illumina’s TruSeq stranded mRNA kit with extended PCR to compensate for the low RNA yield. Samples were run on Illumina’s NextSeq 500 1X75bp using a high output protocol.

我们提出假说：宏转录组（metatranscriptome）可用于监测水产养殖租赁海域附近浮游植物（phytoplankton）生理状态的动态变化。为验证该假说，我们于冬季在一处海洋点位、春季在一处河口点位采集偶遇样本，并通过RNA测序（RNASeq）开展分析。结果显示，不同点位的转录组（transcriptome）重叠度极低，这源于海洋与河口点位的群落组成差异显著；但各点位的转录本功能整体较为相似。河口点位的转录组特征受三文鱼养殖网箱（salmon pen）的空间距离影响极小。在海洋环境中，养殖网箱附近以硅藻（diatom）为优势类群的初级生产力下降，而以甲藻（dinoflagellate）为优势类群的初级生产力上升，该现象可通过固碳与氮获取相关转录本的丰度变化得到验证。本研究初步结果表明，宏转录组应用于近海养殖环境监测的前景可观。

样本处理与测序流程：水样经5 µm、47 mm孔径的Nuclepore滤膜（Nuclepore filter）过滤后，立即置于液氮中快速冷冻。RNA提取环节：将滤膜截留的细胞置于定制的硫氰酸胍缓冲液中，使用MoBio裂解基质E（MoBio lysing matrix E）在珠磨式均质器中进行均质处理，随后涡旋振荡15秒；裂解液经TRIzol试剂法提取总RNA后，再通过Qiagen RNeasy柱式纯化试剂盒完成RNA纯化。使用Fisher NanoDrop（Fisher nanodrop）检测RNA浓度，要求最低浓度不低于25 ng/μL；使用Agilent生物分析仪（Agilent bioanalyzer）评估RNA完整性，要求RNA完整性指数（RNA Integrity Number, RIN）不低于7（该阈值适当下调，以适配提取样本中多类群共存且RNA得率较低的实际情况）。样本建库环节：于Ramaciotti基因功能分析中心（Ramaciotti Centre for gene function analysis）使用Illumina TruSeq链特异性mRNA建库试剂盒进行建库，为补偿低RNA得率，适当延长了PCR扩增循环次数。测序环节：使用Illumina NextSeq 500测序平台，采用高通量输出模式，完成1×75 bp单端测序。