DOI: 10.5061/dryad.0cfxpnwcn
title: Klamath River renewal project molecular library

description: 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.
description: 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
description: # Klamath River renewal project molecular library
 [https://doi.org/10.5061/dryad.0cfxpnwcn](https://doi.org/10.5061/dryad.0cfxpnwcn) ## Description of the data and file structure ### Files and variables #### File: Klamath\_Molecular\_Library\_metadata.xml **Description:** Metadata record of Klamath_ML_Raw.Reads.2023.csv and Klamath_ML_Sample.ID.Metadata.2024.csv following FDGC compliant metadata standards. #### File: Klamath\_ML\_Raw\.Reads.2023.csv **Description:** Comma Separated Value (CSV) file containing data. ##### Variables * MiseqRun: The run identifier code for the DNA next generation sequencing that was conducted on a Illumnina Miseq * Sample.ID: A unique sample identification code for each sampling site * ESVseq: "Exact Sequence Variant", referring to the unique DNA sequence obtained from metabarcoding. * ESVsize: ESVsize is the number of reads of the given ESVseq * CommonName: The common name of the taxon identified from next generation sequencing results. * Klamath.Type: Klamath.type is a taxon-specific which allows tracking of the number of ESVs identified per species. * ReportName: The taxonomic identification from the BLAST results * Type: A broad categorization of the identified taxon into mammal, herpetofauna, bird, or fish groups * sppIDNotes: Notes of interest or explanation regarding the taxonomic identification based on reference database output * Site.Name: Unique site identifying code name * Year: The year the sample was collected #### File: Klamath\_ML\_Sample.ID.Metadata.2024.csv **Description:** Comma Separated Value (CSV) file containing data. ##### Variables * Sample.ID: A unique sample identification code for each sampling site * Replicate.Number: The number of the water sample filter from each site level pooled sample * Sample.Volume: The volume in milliliters that was filtered for a replicate from the pooled water sample at each site location. * Sample.Collection.Method: Identifies whether a replicate was collected from a pooled sample or a deionized water blank serving as a negative control * Filter.Preservation.Method: Description of how each eNA filter was preserved in the field * Sample.Notes: Observations from the field * Filter.Type: The type of filter used to collect the eNA sample * Filter.Pore.Size: The pore size of the filter * Survey.Date.and.Time: The year, month, day, and time of sampling * Site.Name: Unique site identifying code name * Crew: Initials of field staff involved with data collection * Blank.Included: A column flagging whether the site involved collection of a field blank negative control * Site.Notes: Observations from the field * County: County name * State: State Name abbreviation * Year: Year of sampling * Habitat: Aquatic habitat type where sampling occurred * Size: A categorical variable related to the river waterbody size, differentiating mainstem Klamath River and tributary sites and Scott River * Site.Character: Differentiates between sites that could be impacted by dam removal from those that are not expected to be directly impacted by dam removal and and whether those sites are upstream of natural migration barriers to anadromous fish or are accessible to anadromous fish. * Project.Name: The title of the project * Waterbody.Name: Name of river or tributary water body where water samples were collected * Sample.Depth: A description of where in the water column a water sample was collected * Sub.Site.Locations: At each study site, a composite water sample was collected from combinations of left bank, center channel, or right bank locations depending on accessibility * flow.CMS: Model estimated discharge of the river or tributary where the water sample was collected based on USGS gage data (CMS) * Air.Temperature: Temperature of the air during the time of sampling * Water.Temperature: Temperature of the water during the time of sampling * Dissolved.Oxygen: Dissolved oxygen of water sampling location based on multiprobe measurement (mg/l) * Specific.Conductance: Specific conductance of water sampling location based on multiprobe measurement (ms/cm) * pH: pH of water sampling location based on multiprobe measurement * X.Coordinate: Longitude coordinate in decimal degrees * Y.Coordinate: Latitude coordinate in decimal degrees * Extraction.Status: Designates whether the environmental sample has been extracted and the type of extraction * Location.Storage.Method: Description of how the sample has been stored. The location and type of freezer where the sample is reposited. * DNA.Volume.ul.remaining: The amount of extracted DNA sample volume remaining in the original sample * RNA.Volume.ul.remaining: The amount extracted of RNA sample volume remaining in the original sample #### File: Klamath\_ML\_metaworks\_output.csv **Description:** Comma Separated Value (CSV) file containing data. ##### Variables * MiseqRun: The run identifier code for the DNA next generation sequencing that was conducted on a Illumnina Miseq - Sample.ID: A unique sample identification code for each sampling site * ESVseq: "Exact Sequence Variant", referring to the unique DNA sequence obtained from metabarcoding - ESVsize: ESVsize is the number of reads of the given ESVseq * Strand: Direction of the DNA strand (usually "+" or "-" if relevant) * Root: Usually the topmost taxonomic rank (often just "Root") * RootRank: The rank name ("root") * rBP: Bootstrap support for the Root assignment * SuperKingdom: The super kingdom of the sequence * SuperKingdomRank: Rank of the super kingdom * skBP: Bootstrap support for the super kingdom assignment * Kingdom: Kingdom * KingdomRank: Kingdom rank ("kingdom") * kBP: Bootstrap support for the Kingdom assignment * Phylum: Phylum * PhylumRank: Phylum rank ("phylum") * pBP: Bootstrap support for the Phylum assignment * Class: Class * ClassRank: Class rank ("class") * cBP: Bootstrap support for the Class assignment * Order: Order * OrderRank: Order rank ("order") * oBP: Bootstrap support for the Order assignment * Family: Family * FamilyRank: Family rank ("family") * fBP: Bootstrap support for the Family assignment * Genus: Genus * GenusRank: Genus rank ("genus") * gBP: Bootstrap support for the Genus assignment * Species: Species * SpeciesRank: Species rank ("species") * sBP: Bootstrap support for the Species assignment ## Access information Other publicly accessible locations of the data: * There are no other publicly accessible locations of the data. Data was derived from the following sources: * Data were derived as part of an original research study. Data set is considered complete for the information presented, as described in the abstract. Users are advised to read the rest of the metadata record carefully for additional details.