Klamath River renewal project molecular library

The Klamath River Renewal Project (KRRP) is a large dam removal and river restoration project in California and Oregon, United States. With the removal of four large dams in 2024, restoration of connectivity to 640 river kilometers occurred. We created the KRRP molecular library, an environmental specimen bank, for long-term curation of environmental nucleic acids collected from the restoration project area. The library established sampling stations from 45 main stem and tributary sites, as well as pre-dam removal water samples preserved for environmental nucleic acids (eDNA and eRNA) intended to provide long-term use of both raw and extracted molecular material used to track the ecosystem response to dam removal. On a subset of samples, we conducted DNA metabarcoding using next generation sequencing based on the MiFish-U and modified MiFish-U-F primer sets. We used sequence reads to visualize data and calculate diversity metrics to establish a pre-dam removal baseline and proof of concept that the molecular techniques could resolve changes to fish and other aquatic organisms resulting from short- and long-term changes due to dam removal. These and future sampling efforts should, at a minimum, allow tracking of fish community response to ecosystem restoration.

Methods
Step 1

On the Klamath River, we selected monitoring locations systematically every 2 km along the river and reservoirs and every 1 km along selected tributaries, with additional sites included near long-term monitoring locations important to the restoration project. Monitoring locations spanned approximately 114 km of mainstem and tributary habitat. Sites were selected along navigable streams and only at locations where long-term continued accessibility was assumed. Sites along the river extend from approximately 3.5 km downstream of the lowermost dam upstream to where the river enters the uppermost reservoir to be removed. Tributary sites extended upstream to fish migration barriers, or as far as the project boundary allowed. Additionally, six “reference” sites in the Scott River basin, a large tributary downstream of the project area, were included to represent habitat not impacted by dams. Having a range of non-treatment reaches from which to compare treatment (i.e., dam removal) effects will be more representative of recovered conditions and biological communities.

Step 2

We collected 405 samples from 44 monitoring locations on 17 - 20 July, 2023. Access allowed, we collected three liters of stream water from each bank and the center of the channel at each site, for a total of nine liters. The nine liters of water were combined into a single vessel, agitated to encourage homogenization, and decanted into nine replicate samples via a filtering manifold. Each replicate was filtered in the field through 0.45 µm pore-size PVDF Sterivex filters, which capture eNA from the environment by trapping particles within the filter matrix, using Cole Parmer easy loader II peristaltic pumps and sterile Masterflex silicone tubing for each monitoring site. Pumps were affixed in parallel to allow for simultaneous filtration of three filter replicates and powered using a brushless, cordless drill with a 12.7 mm spade bit attachment. Field crews followed protocols to assess and minimize the risk of contamination, including using sterile single-use filters, caps, and tubing, changing nitrile gloves frequently, and collecting field controls at the start of each sampling day. When field sampling was complete, samples were preserved by pipetting into each filter cartridge 1.5 mL of RNAprotect Tissue Reagent (Qiagen) following practices to maximize the probability of stabilizing genetic material.

Step 3

Site-level water quality measurements (water temperature, dissolved oxygen, and specific conductance) were collected with a Yellowstone Instruments ProQuatro multiparameter meter, and air temperature with a Kestrel rotating-vane thermistor.

Step 4

Total DNA was isolated from each filter and purified to remove non-target cellular and environmental contaminants using the QIAamp DNA mini kit (Qiagen) and following a standard protocol with modifications. First, RNAprotect Tissue Reagent was evacuated from each filter by manually shaking the liquid from the cartridge. The exterior of each filter was sterilized with a PCR clean wipe to avoid cross-contamination. We added 440 μL of the Buffer PBS/Buffer AL/Proteinase K lysis solution to each filter by injecting the solution into the Sterivex cartridge using a filtered pipette tip. The filters were incubated for 5 min at 56 °C, then affixed to a vortex mixer to undergo two ten-minute room temperature vortex sessions. Between sessions, the filters were rotated 180° to ensure full coverage of the filter membrane. The solution was transferred from the Sterivex cartridge to a 1.5 mL microcentrifuge tube, and then QIAamp mini spin columns were used to bind DNA, and the remainder of the DNA purification and elution steps followed the published protocol by Miya et al. (2016). DNA extraction controls, created by adding 880 μL of the lysis solution to a sterile Sterivex filter, were processed in parallel with samples to confirm sample integrity throughout the extraction procedure. All samples and controls were passed through the Zymo OneStep PCR Inhibitor Removal Kit (Zymo Research) following the manufacturer's guidelines. DNA extraction was completed in a separate pre-PCR space using sterilized surfaces and equipment.

References:

Miya, M. et al. Use of a filter cartridge for the filtration of water samples and the extraction of environmental DNA. J. Vis. Exp. JoVE (2016) doi:10.3791/54741.

Step 5

Purified eNA can be analyzed using a variety of molecular techniques. For this study, we used DNA metabarcoding to assess the community-level composition of fish taxa at each sampling location. Metabarcoding employs next-generation sequencing with universal primers to sequence a diagnostic region of DNA that allows for species identification across taxa. We used a multiplex of the MiFish-U primer set and a modified version of the MiFish-U-F primer (GIQHerp-F), designed to enhance detection of herptile taxa, to sequence a 170 bp region of vertebrate 12S rRNA mitochondrial genome using a three-step PCR approach adapted from previously published library preparation methodologies. The initial PCR was completed using non-indexed primers to enrich subsequent reactions for target DNA. Each sample was amplified in triplicate, in a total reaction volume of 10 μl containing 4 μl extracted eDNA, 0.4 μM of each forward primer (MiFish-U-F: 5’- GTCGGTAAAACTCGTGCCAGC-3’, GIQHerp-F: 5’- GCCGGCTAATCTGGTGCCAGC-3’), 0.8 μM MiFish-U-R (5’- CATAGTGGGGTATCTAATCCCAGTTTG-3’), and 1X Qiagen Plus Multiplex Master Mix. Cycling began with an initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 15 seconds, 5% ramp down to 55 °C for 30 seconds, and 72 °C for 30 seconds. The triplicate PCR products were pooled and then diluted 1:10 prior to starting the Illumina adapter and barcoding processes.

The Illumina hanging tail adapters were incorporated using the MiFish-U and GIQHerp primer multiplex containing the 33 or 34 bp 5’ Illumina hanging tail adaptor sequences to provide a priming site for the addition of dual-indexed barcode sequences. Each reaction consisted of a 12 μl total volume containing 2 μl pooled and diluted product from the previous PCR, 0.3 μM of each Illumina adapter forward primer, 0.6 μM of the Illumina adapter reverse primer, and 6 μl KAPA HiFi HotStart ReadyMix (Roche Diagnostics). The cycling profile was as follows: 95 °C for 5 min, 5 cycles of 98 °C for 20 seconds, 1% ramp down to 65 °C for 15 seconds, and 72 °C for 15 seconds, then 7 cycles of 98 °C for 20 seconds, 5% ramp down to 65 °C for 15 seconds, 72 °C for 15 seconds. PCR products were diluted 1:10 and used as templates in the final PCR step. The paired-end dual indices that allow for sample identification and de-multiplexing were incorporated during the final PCR step. Each PCR was completed in a total volume of 12 μl, composed of 0.3 μM of the forward and reverse index primers, 6 μl 1X KAPA HiFi HotStart ReadyMix, and 1 μl of the diluted product from the previous PCR. Amplification started with 95 °C for 3 min, followed by 10 cycles of 98 °C for 20 seconds, 5% ramp down to 72 °C for 15 seconds, and final extension at 72 °C for 5 min. All PCR steps were completed using Bio-Rad C1000 Touch thermal cyclers (Bio-Rad Laboratories) in a designated PCR space.

Equal volumes of the indexed PCR products were pooled, then size selected (c. 370) using 2% gel electrophoresis and purified using QIAquick Gel Extraction Kit (Qiagen) following the manufacturer's guidelines for next-generation sequencing. Purified libraries were quantified using the Qubit 4 fluorometer and Qubit dsDNA HS assay Kit (Thermo Fisher Scientific), and sequenced on the Illumina Miseq system (Illumina, San Diego, CA, USA) using the v2 300-cycle chemistry. The final loading concentration was 8 pM with a 10% PhiX spike-in added as a sequencing control. A UV-sterilized hood was used to prepare a master mix for all PCR steps and to add extracted DNA during the initial PCR. All intermediate dilution, DNA transfer, and final pooling steps were completed in designated post-PCR spaces using sterilized pipettes and bench tops. No template PCR controls were processed in parallel with samples and sequenced to confirm process integrity.

To determine provisional species identification, the resultant sequencing data were compiled and processed using the MetaWorks pipeline, 12S vertebrate classifier, and default parameters (Porter and Hajibabaei, 2022). We removed any detections with less than 100 sequence reads to screen out potential artifact sequences. Final taxonomic assignment was verified against the NIH National Center for Biotechnology Information reference database using the BLAST algorithm. We used the standard nucleotide BLAST (blastn suite) to compare detected sequences to sequences stored in the core nucleotide database (core_nt). Provisional species with a greater than or equal to 97% sequence similarity to a known species in the reference database were considered a match and successfully identified to species. Sequences with greater than 97% ID match to multiple species that could co-occur within the sampling region were assigned to the taxonomic level that appropriately captured all potential matches (e.g., Cottus spp.). Any detections that could result from anthropogenic inputs (human, cat, dog, cow, chicken, and pig) were removed from analysis.

References:

Porter, T. M. & Hajibabaei, M. MetaWorks: A flexible, scalable bioinformatic pipeline for high-throughput multi-marker biodiversity assessments. PLOS ONE 17, e0274260 (2022).

NCBI Bioproject Accession: PRJNA1236377 ID: 1236377 ID 1236377 - BioProject - NCBI

克拉马斯河复兴项目（Klamath River Renewal Project, KRRP）是美国加利福尼亚州与俄勒冈州的大型大坝拆除与河流修复工程。2024年，随着四座大型大坝的拆除，该项目恢复了总长640公里河道的连通性。本研究构建了KRRP分子库——一处环境标本库，用于长期保存从该修复工程区域采集的环境核酸。该库设置了45个干流与支流采样站点，并留存了大坝拆除前的水样，用于保存环境DNA（environmental DNA, eDNA）与环境RNA（environmental RNA, eRNA），以期长期保留可用于追踪大坝拆除对生态系统影响的原始与提取后的分子材料。针对部分样本，研究团队基于MiFish-U与改良版MiFish-U-F引物组，利用下一代测序技术开展了DNA元条形码（DNA metabarcoding）分析。通过测序读长实现数据可视化与多样性指标计算，以建立大坝拆除前的基线，并验证该分子技术可识别大坝拆除带来的短期与长期变化对鱼类及其他水生生物造成的影响。本研究及后续采样工作至少可实现对鱼类群落响应生态系统修复过程的追踪。

研究方法
步骤1：监测点位布设
在克拉马斯河沿岸，研究团队沿河道与水库以每2公里的间距系统布设监测点位，沿选定支流则以每1公里的间距布设，并在对修复工程具有重要意义的长期监测点位附近增设了额外站点。监测点位覆盖了干流与支流栖息地共约114公里的范围。监测站点均选在可通航河道且预计可长期通行的区域。沿河布设的站点从最下游大坝下游约3.5公里处向上延伸至待拆除的最上游水库入口处；支流站点则向上延伸至鱼类洄游障碍处，或延伸至工程边界允许的最远范围。此外，研究团队在项目区域下游的大型支流斯科特河流域设置了6个“参照”站点，以代表未受大坝影响的栖息地。通过设置一系列非处理河段作为对照，可更准确地对比大坝拆除（即处理措施）的效应，从而更贴合生态系统恢复后的环境与生物群落特征。

步骤2：样本采集与保存
2023年7月17日至20日，研究团队在44个监测点位采集了405份样本。在通行条件允许的情况下，每个站点分别从河道两岸与河道中心采集3升河水，总计9升。将9升河水混合至同一容器中，搅拌均匀后通过过滤歧管分装为9份平行样本。每份平行样本均在野外通过孔径0.45μm的PVDF材质Sterivex过滤器进行过滤——该过滤器可通过截留过滤基质内的颗粒物捕获环境核酸（environmental nucleic acids, eNA），采集时使用Cole Parmer Easy Loader II蠕动泵与无菌Masterflex硅胶管，每个监测站点配备独立管路。泵体采用并联方式安装，可同时完成3份过滤平行样的操作，动力来源为搭载12.7mm扁钻附件的无刷充电式电钻。野外采样团队严格遵循污染防控规程，包括使用无菌一次性过滤器、瓶盖与管路，频繁更换丁腈手套，并在每个采样日开始时采集野外空白对照。野外采样结束后，研究人员通过移液枪向每个过滤柱中加入1.5mL RNAprotect组织保存液（Qiagen），以最大化稳定遗传物质的概率。

步骤3：环境参数测定
使用Yellowstone Instruments ProQuatro多参数水质分析仪采集点位级别的水质数据，包括水温、溶解氧与比电导率；使用Kestrel旋转叶片热敏电阻测量空气温度。

步骤4：总DNA提取与纯化
从每份过滤器中提取总DNA，并通过QIAamp DNA迷你试剂盒（Qiagen）并遵循经改良的标准规程进行纯化，以去除非目标细胞与环境污染物。首先，手动晃动过滤柱以排出其中的RNAprotect组织保存液。使用PCR清洁湿巾擦拭过滤柱外壁，避免交叉污染。通过带过滤器的移液枪头向Sterivex过滤柱中注入440μL PBS缓冲液/AL缓冲液/蛋白酶K裂解液。将过滤柱置于56℃下孵育5分钟，随后固定于涡旋混合器上，以室温条件进行两轮各10分钟的涡旋处理，每轮处理后将过滤柱旋转180°，以确保过滤膜全面覆盖裂解液。将裂解液从Sterivex过滤柱转移至1.5mL微量离心管中，随后使用QIAamp迷你离心柱结合DNA，后续的DNA纯化与洗脱步骤均遵循Miya等人（2016）发表的规程。设置DNA提取空白对照：向无菌Sterivex过滤柱中加入880μL裂解液，与样本同步处理，以验证整个提取过程中样本的完整性。所有样本与对照均按照制造商指南，通过Zymo OneStep PCR抑制剂去除试剂盒（Zymo Research）进行处理。DNA提取操作在独立的PCR前区域完成，使用灭菌的工作台面与设备。

步骤5：文库构建、测序与数据分析
纯化后的环境核酸可通过多种分子技术进行分析。本研究采用DNA元条形码技术评估每个采样点位鱼类类群的群落组成。元条形码技术利用通用引物的下一代测序技术，对可实现跨类群物种鉴定的DNA诊断区域进行测序。研究团队采用MiFish-U引物组与改良版MiFish-U-F引物（GIQHerp-F）的多重PCR体系——该改良引物旨在提升对爬行动物与两栖动物类群（herptile）的检测效率——以靶向扩增脊椎动物12S rRNA线粒体基因组的170bp区域，实验方案采用基于已发表的文库制备方法优化的三步PCR流程。第一轮PCR使用未带索引的引物，以富集后续反应中的目标DNA。每份样本设置3次生物学重复，总反应体系为10μL，包含4μL提取得到的环境DNA、0.4μM的每条正向引物（MiFish-U-F：5’-GTCGGTAAAACTCGTGCCAGC-3’，GIQHerp-F：5’-GCCGGCTAATCTGGTGCCAGC-3’）、0.8μM MiFish-U-R（5’-CATAGTGGGGTATCTAATCCCAGTTTG-3’）与1×Qiagen Plus Multiplex Master Mix。PCR循环程序为：95℃初始变性5分钟，随后35个循环：95℃变性15秒，以5%的降温速率降至55℃退火30秒，72℃延伸30秒。将3次重复的PCR产物混合后以1:10比例稀释，用于后续的Illumina接头与条形码标记流程。

通过包含33或34bp 5’端Illumina悬挂接头序列的MiFish-U与GIQHerp引物多重体系，加入Illumina悬挂尾接头，为后续添加双索引条形码序列提供引物结合位点。该步反应总体系为12μL，包含2μL上一轮PCR混合并稀释后的产物、0.3μM的每条Illumina接头正向引物、0.6μM的Illumina接头反向引物与6μL KAPA HiFi HotStart ReadyMix（Roche Diagnostics）。PCR循环程序为：95℃变性5分钟，5个循环：98℃变性20秒，以1%的降温速率降至65℃退火15秒，72℃延伸15秒；随后7个循环：98℃变性20秒，以5%的降温速率降至65℃退火15秒，72℃延伸15秒。将PCR产物以1:10比例稀释后作为模板，用于最终的PCR步骤。最终PCR步骤中加入了可实现样本鉴定与双端解复用的配对末端双索引序列。每份反应总体系为12μL，包含0.3μM的正向与反向索引引物、6μL 1×KAPA HiFi HotStart ReadyMix与1μL上一轮PCR稀释后的产物。扩增程序为：95℃预变性3分钟，随后10个循环：98℃变性20秒，以5%的降温速率降至72℃退火15秒，最终72℃延伸5分钟。所有PCR步骤均在指定的PCR区域内，使用Bio-Rad C1000 Touch热循环仪（Bio-Rad Laboratories）完成。

将带索引的PCR产物按等体积混合，随后通过2%凝胶电泳完成大小筛选（目标片段约370bp），并按照制造商指南使用QIAquick凝胶提取试剂盒（Qiagen）进行纯化。纯化后的文库通过Qubit 4荧光计与Qubit dsDNA HS检测试剂盒（Thermo Fisher Scientific）进行定量，随后使用Illumina MiSeq测序系统（Illumina，美国加利福尼亚州圣地亚哥），采用v2 300循环化学进行测序。最终加载浓度为8pM，并加入10%的PhiX作为测序对照。使用紫外线灭菌的超净台完成所有PCR步骤的母液配制与初始PCR中提取DNA的添加。所有中间稀释、DNA转移与最终混合步骤均在指定的PCR后区域内，使用无菌移液管与工作台面完成。设置无模板PCR空白对照，与样本同步处理并测序，以验证整个实验流程的完整性。

为确定暂定物种鉴定结果，研究团队使用MetaWorks分析流程、12S脊椎动物分类器与默认参数对测序数据进行整理与处理（Porter与Hajibabaei，2022）。移除序列读长少于100的检测结果，以筛选掉潜在的人工序列。最终的分类学鉴定通过BLAST算法与美国国立卫生研究院国家生物技术信息中心（National Center for Biotechnology Information, NCBI）参考数据库进行验证。研究使用标准核苷酸BLAST（blastn套件）将检测到的序列与核心核苷酸数据库（core_nt）中的序列进行比对。与参考数据库中已知物种序列相似度≥97%的暂定物种被视为匹配成功，可鉴定至物种水平。若序列与采样区域内可共存的多个物种相似度均≥97%，则将其分类至可涵盖所有潜在匹配的分类阶元（例如Cottus spp.，杜父鱼属）。所有可能来自人为输入的检测结果（人类、猫、狗、牛、鸡与猪）均从分析中移除。

参考文献
Miya, M. 等. 利用过滤柱过滤水样并提取环境DNA. J. Vis. Exp. JoVE (2016) doi:10.3791/54741.

Porter, T. M. & Hajibabaei, M. MetaWorks: 一款适用于高通量多标记生物多样性评估的灵活可扩展生物信息学流程. PLOS ONE 17, e0274260 (2022).

NCBI生物项目登录号：PRJNA1236377，ID：1236377