Modeling the Sensitivity of Field Surveys for Detection of Environmental DNA (eDNA)

The environmental DNA (eDNA) method is the practice of collecting environmental samples and analyzing them for the presence of a genetic marker specific to a target species. Little is known about the sensitivity of the eDNA method. Sensitivity is the probability that the target marker will be detected if it is present in the water body. Methods and tools are needed to assess the sensitivity of sampling protocols, design eDNA surveys, and interpret survey results. In this study, the sensitivity of the eDNA method is modeled as a function of ambient target marker concentration. The model accounts for five steps of sample collection and analysis, including: 1) collection of a filtered water sample from the source; 2) extraction of DNA from the filter and isolation in a purified elution; 3) removal of aliquots from the elution for use in the polymerase chain reaction (PCR) assay; 4) PCR; and 5) genetic sequencing. The model is applicable to any target species. For demonstration purposes, the model is parameterized for bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix) assuming sampling protocols used in the Chicago Area Waterway System (CAWS). Simulation results show that eDNA surveys have a high false negative rate at low concentrations of the genetic marker. This is attributed to processing of water samples and division of the extraction elution in preparation for the PCR assay. Increases in field survey sensitivity can be achieved by increasing sample volume, sample number, and PCR replicates. Increasing sample volume yields the greatest increase in sensitivity. It is recommended that investigators estimate and communicate the sensitivity of eDNA surveys to help facilitate interpretation of eDNA survey results. In the absence of such information, it is difficult to evaluate the results of surveys in which no water samples test positive for the target marker. It is also recommended that invasive species managers articulate concentration-based sensitivity objectives for eDNA surveys. In the absence of such information, it is difficult to design appropriate sampling protocols. The model provides insights into how sampling protocols can be designed or modified to achieve these sensitivity objectives.

Source 5-4 Water samples have been taken in the field according to Section 2 and filtered according to Section 3. Filters have been shipped to ERDC and personnel assigned to the ERDC eDNA Team have either received or picked-up shipping boxes from the ERDC Receiving Office.     Table 2-1. The specific sampling locations will be chosen by the MWWRG and/or the sampling agencies working in partnership. Specific sampling locations and the rationale for those locations will follow the general protocol given above. Each specific sampling location will be identified using a GPS device and the location and characteristics will be recorded on the logs given in the Exhibits ……………………………………………………………………………… 2-5

Background
Invasive aquatic nuisance species pose a major threat to aquatic ecosystems worldwide. Within Illinois, the manmade Chicago Sanitary & Ship Canal (CSSC), constructed in the early 1900s, provided an unnatural portal for invasive species dispersal between the geologically separated Mississippi River and Great Lakes drainage basins. In 2002, in an effort to curtail the spread of invasive species between the two basins, the U.S. Army Corps of Engineers (USACE), constructed a dispersal barrier system within the CSSC. The primary objective of the barrier system when initiated was to stop the dispersal of the invasive round goby into the Mississippi River basin; however, once the project was completed, it was found that the round goby had already surpassed the barrier. Since then, a new threat to the Great Lakes from the Mississippi River basin has become the primary objective of the dispersal barrier system. Invasive Asian carps, including bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix) have been steadily dispersing upstream through the Mississippi, Illinois, and Des Plaines Rivers. Their potential dispersal through the dispersal barrier system within the CSSC poses a potential threat to the Great Lakes ecosystem. In the past, traditional fishery techniques were used to detect the leading edge of the Asian carp population; however, this method was somewhat ineffective at targeting these species at low densities. The University of Notre Dame, with funding from the USACE, developed a method that detected "environmental" DNA (eDNA) left behind in the aquatic system by the targeted species. Environmental DNA enters the system through a variety of mechanisms, some of which include sloughing of external epidermal cells into the water, sloughing of internal epidermal cells into feces and into the water, and as tissue residues following injury or predation. The detection of eDNA in water samples is based on whole DNA extraction from filtered particulate organic and inorganic matter found in the water and polymerase chain reaction (PCR) assays for species-specific mitochondrial DNA markers. Use of this method is to provide detection of Asian carp at low densities and to serve as an early detection system of the spread of Asian carp into previously uninhabited areas. As USACE takes over the lead on eDNA monitoring from the University of Notre Dame, a Quality Assurance Project Plan detailing the eDNA monitoring process, including methodologies and quality controls, has been requested from the U.S. Army Engineer Research and Development Center (ERDC).

General Requirement
The Chicago District (LRC) requires a Quality Assurance Project Plan (QAPP) for eDNA monitoring. Full-scale eDNA monitoring commenced in September 2010. Based on consultation with experts in processing of litigable DNA evidence (which applies to eDNA monitoring), a final, comprehensive QAPP was not in place within that time frame. Instead a provisional QAPP was used during the first year of eDNA monitoring, to be followed later by a finalized version under 1-2 a different scope of work. This allows for inclusion of any beneficial technical or strategic modifications that become apparent during the first year of effort.

Project History
The University of Notre Dame, Department of Biological Sciences, Center for Aquatic Conservation, prepared a Standard Operating Procedure (SOP) in 2010. The SOP provided details regarding eDNA monitoring protocol and was given to USACE in May 2010 in compliance with Cooperative Ecosystem Study Unit agreement #W912HZ-08-2-0014, modification P00007. On 15 and 16 December 2009, a technical and quality systems audit of the Center for Aquatic Conservation Lodge Laboratory at the University of Notre Dame was conducted by USEPA. The laboratory audit report dated 5 February 2010 was provided to USACE in addition to the eDNA monitoring protocol. These documents served as the basis for this provisional QAPP.

Objective
The objective of this QAPP is to provide detailed procedures for Asian carp eDNA sample collection, sample processing (including filtering, DNA extraction, PCR, biomarker analysis, DNA sequencing), data reporting, and quality control/quality assurance procedures for 2011, to ensure that data are as technically defensible, consistent, and usable as possible. The specific goals and objectives of sampling are currently directed by the Asian Carp Regional Coordinating Committee's (ACRCC) Monitoring and Rapid Response Plan.

Project Personnel
The eDNA monitoring project must have personnel appointed to the following positions: •

Assigned Project Leaders and Specialists:
Others serving on the project may include researchers, technicians, and budgetary personnel. Sampling may employ personnel from other agencies in coordinated efforts. All personnel must meet a minimum standard for training and/or experience before independently conducting any portion of the eDNA monitoring protocol. The supporting agency contacts are given in Appendix A. Minimum personnel training requirements are given below.

Personnel Training Requirements
Minimum training and/or experience requirements for the different major components of the eDNA monitoring protocol are detailed below.
Boat Operator: • Must meet USACE boat operator requirements as a minimum. USACE Boat Operator requirements are listed in Engineer Manual 385-1-1.

Sampling:
• A BA/BS degree or its equivalent in biology or related field of study, or 1-4 • At least 2 years of specialized postsecondary training or an associate degree in applied science or science-related technology, or • A high school diploma or its equivalent and a minimum of 2 years professional experience in biology-related field. • First aid and/or boating safety course.
• Minimum 1 year experience in collecting field samples for biological analyses.

Filtering:
• A BA/BS degree or its equivalent in biology or related field of study, or • At least 2 years of specialized postsecondary training or an associate degree in applied science or science-related technology, or • A high school diploma or its equivalent and a minimum of 2 years professional experience in biology-related field. • First aid training.
• Facility-specific safety training. • Minimum one semester college level laboratory experience, plus eDNA-specific training.

DNA Processing:
• A minimum BA/BS degree or its equivalent in biology or related area and successful completion of college course work (graduate or undergraduate level) covering the subject areas of biochemistry, genetics, and molecular biology (molecular genetics, recombinant DNA technology) or other subjects that provide a basic understanding of the foundation of DNA analysis, as well as course work and/or training in PCR amplification as it applies to eDNA analysis. • A minimum of 6 months of general DNA laboratory experience, including experience with DNA extraction and PCR. Additionally, 2 weeks of training on Asian carp eDNA protocols. • Successful completion of a qualifying test demonstrating effective execution of eDNAtype assays before beginning independent work on the project.

Reporting
All agencies, including USACE offices, need to submit resumes (Curriculum Vitaes) for proposed staff, to be reviewed and approved by LRC and the eDNA Program Manager. Those documents will be kept with the project file. For each sampling event, specific staff members conducting the various activities will be documented (e.g., on the field sampling log). That information will also become part of the project file.

SAMPLE COLLECTION
Prior to any field sampling work, all field employees must review this quality assurance plan and acknowledge the procedures and processes to be followed for every sample and every event.
Field employees will acknowledge their understanding and intent to comply by signing the certification form given as Exhibit 7. Field employees will also review the sampling safety plan (separate document) and participate in a safety briefing.
Prior to any filtering or analysis work, all laboratory employees must review this quality assurance plan and acknowledge the procedures and processes to be followed for every sample and every event. Laboratory employees will acknowledge their understanding and intent to comply by signing the certification form given as Exhibit 7. Laboratory employees will also review the laboratory safety plan (separate document) and participate in a safety briefing.

Purpose
Accurate planning of a general collection site is necessary to effectively manage the time of crews collecting samples, as well as to ensure complete and correct sampling procedures are used. Sampling will occur within the Chicago Area Waterway System (CAWS), which includes but is not limited to the following named waters: Illinois River, Des Plaines River, CSSC, Chicago River, Bubbly Creek, North Branch of the Chicago River, North Shore Channel, Cal-Sag Channel, Little Calumet River North Leg, Little Calumet River South Leg, Grand Calumet River, Lake Calumet, and the Calumet River. Reaches to be sampled within the CAWs have been designated (Figure 2-1) and a general description and location (river mile) of each reach is given in Table 2-1. Overall, the reaches to be sampled and number of samples to be taken depends upon guidance from the Asian Carp Monitoring and Rapid Response Work Group (MRRWG); however, the baseline number of samples to be taken from each reach is 60. Prior to sampling, aerial imagery and a site visit should be used to determine where (generally) samples will be collected and to establish access points and gain any necessary permissions to access the river. Specific site selection will be determined in the field per the following guidelines. Table 2-2 contains boat launch information.

Pre-trip Planning Procedure
USACE will determine the reach to be sampled, the number of samples to be taken within the reach, and confirm with MRRWG. USACE will provide this information to the sampling agency.
(1) Reaches to be sampled on a monthly basis include the North Shore Channel, Chicago River to South Branch Chicago River, and Little Calumet River North Leg to Lake Calumet. Reaches to be sampled on a periodically include the CSSC upstream and downstream of the barrier, CSSC and Cal-Sag Channel confluence, and the Upper Des Plaines River ( (2) The baseline number of samples to be collected from each reach is 60 with at least 1 cooler blank sample per sample cooler.
(3) LRC will seek input from MRRWG prior to each sampling trip to confirm the number of samples to be taken from a reach and the reach to be sampled. The number of samples and the reach to be sampled will be provided to the sampling agency by LRC.
Use interactive aerial imagery software (i.e., Google Earth) to scope out reaches to be sampled, placement of samples, and unique features that should be targeted during sampling.
(1) Aerial maps will be detailed enough to show unique features (e.g., barge slips, factory, etc.) that can be identified in the field and used as markers for location when sampling. The recommended minimum scale is 1':500'.
(2) Aerial maps must be marked with sample locations and should ensure spatial coverage and overall representativeness of the sample area.
(3) Target specific areas (backwaters, island side channels, pooled areas, below and around structures, confluence of tributaries) as well as integrating transect plots in the sampling plan. Transect plots should be spaced approximately 500 meters apart.
(4) Print map(s) with detailed sample plan.
• Locate access points for boat launch and acquire permission to use if necessary. Table  2-2 provides information on boat launch locations, permission contacts, fees, and reaches that can be accessed by a specified launch. The agency that is conducting the sampling event is responsible for coordinating boat launches or any access or real estate issues associated with the sampling.
• If sampling around locks, or if sampling will require lockage, notify the Lockmaster at least 1 day before sampling. Table 2-3 provides Lockmaster contact information. The agency that is conducting the sampling event is responsible for coordinating with the Lockmaster.
• Coordinate sample plan with sample crew, which will comprise four people at a minimum: one boat operator, one lead sampler, and two sampling assistants. All participants involved in the sampling must have their resume (CV) on file with LRC, prior to the sampling event. All participants involved in the sampling must have read this QAPP and must have a signed certification statement (Exhibit 7) on file with LRC, prior to the sampling event. All participants involved in the sampling must meet the minimum qualifications given for their role in Section 1.3 of this document.
• A field equipment checklist (Exhibit Form 1) and datasheets (Exhibit Form 2) should be printed prior to each sampling trip on Rite-in-the-Rain ® paper or other waterproof paper. Datasheets are printed on front and back.

2-3
• Check river stage and weather forecast.
(1) No eDNA sampling should occur within 5 days after a significant rainfall event (more than 1.5 inches in a 24-hr period), on the rising limb of a hydrograph of the river as it exceeds flood stage, and/or within 2 days of a combined sewer overflow (CSO) event. Weather data and river stage for the area to be sampled can be checked at: http://waterdata.usgs.gov/nwis. The occurrence of a CSO event can be verified by contacting the eDNA Program Manager listed in Appendix A.

Field equipment
(1) Minimum 18-ft boat with specified motor, including trailer and vehicle to pull, provided by the sampling agency.
(2) Personal flotation devices for each crew member, using the type of devices listed in the safety plan. Minimum PFD requirements are Type I within the Safety Zone near the existing electric barrier (on the Chicago Sanitary and Ship Canal) or Type II for the remainder of the CAWS.
(3) 100-qt coolers (each cooler capable of holding 20 2L samples); provided cleaned and filled with bottles by USACE. Sampling agency picks up bottles at 536 S. Clark Street, 10 th Floor, Chicago, Illinois.
(4) 2L sample bottles (label affixed and numbered in the lab), provided cleaned and labeled by USACE in the coolers. (6) Field datasheets (Forms 1 and 2) provided by USACE with the coolers and bottles.
(7) Chain of custody form (Form 3) provided by USACE with the coolers and bottles.
(8) Sharpie ® permanent marker in black provided by the sampling agency.
(9) Powderless nitrile gloves provided by the sampling agency.
(11) Ice provided by the sampling agency.
(12) Drinking water for crew provided by the sampling agency.
(13) Safety plan -USACE plan represents minimum requirements; agency-specific alternative plans are allowable as long as all hazards are addressed and minimum requirements are met. 2-4

Site Selection Procedure (in the field)
Using the sampling plan as guidance, refinement of exact sampling should depend on the following factors: (1) Sampling should occur in a downstream to upstream direction to minimize the potential for surface water disturbance caused by the vessels wake within the sample reach. The only exception where sampling may occur in an upstream to downstream direction would be if the nearest boat launch is upstream of the reach to be sampled. Sample direction should be noted on the Field Collection Summary datasheet (Form 1).
(2) Samples will be collected in two ways -transect and targeted sampling.
(a) Transect: Location of transects will be determined by LRC prior to the start of a sampling event. The first transect will be set across the downstream end of the reach to be sampled with subsequent transects set 500 m apart heading upstream (see exception to protocol above (1)). Transects will run perpendicular to flow, and three 2L samples will be collected along each transect using the following scheme: one collected near the left descending bank (LDB), one in mid-channel (MC), and one near the right descending bank (RDB). Where samples are collected should be recorded in the "Habitat" column of the Sampling datasheet (Form 2). Samples should be collected on the upstream side of the boat or off the bow.
• When collecting samples near the bank, be observant of wake-disturbed surfaces. To compensate for the wake created by a passing boat, samples may need to be collected 2-3 ft off the bank to obtain displaced surface film.
(b) Targeted sampling is collecting samples in the most probable places of eDNA accumulation, such as (but not limited to): •   locations and the rationale for those locations will follow the general protocol given above. Each specific sampling location will be identified using a GPS device and the location and characteristics will be recorded on the logs given in the Exhibits .

Purpose
In order to perform laboratory molecular analyses to detect eDNA, vessels and equipment must be sanitized in accordance with the following protocols to eliminate introduction of outside DNA sources in the sampling regime.
Cautions: Precautions should be made to avoid direct skin contact with hypochlorous acid products; hypochlorous acid solution may also stain clothing or other materials. Be aware of pollutants in the aquatic environment and related health hazards.

Equipment Procedure
(1) A 10% hypochlorous acid solution with deionized (DI) water will be prepared in a 3-gallon hand-held sprayer that is dedicated to the project. The hypochlorous acid solution must be prepared immediately prior to use, and each time disinfection activities will be occurring.
(2) At least 1 day prior to use, sample transport coolers will be sterilized with freshly made 10% hypochlorous acid solution by LRC. Using the hand-held sprayer, the inside surfaces of the cooler will be thoroughly covered with the hypochlorous acid solution. At least 10 min of contact between the solution and the inside surfaces of the cooler will be allowed before rinsing with DI water. Coolers will then be left to air dry.
(3) Sterilized disposable 2L bottles may be used for sample collection or 2L autoclavable reusable bottles sterilized in the following way may be used: (a) Reusable 2L bottles will first be thoroughly rinsed with DI water to remove any material that may be on the outside or inside of the bottle and cap.
(b) After rinsing the bottles, lightly screw on cap (cap will not be able to come off, but will still be able to move).

2-8
(d) Once the autoclave cycle is complete, carefully remove bottles and allow them to cool to room temperature before fully screwing on the caps.
(4) Once all 2L bottles have been sterilized, sample labels will be affixed to the outside of the bottles prior to going into the field. Bottle labels will be provided by LRC. Bottles will be labeled with an appropriate ID that does not indicate location (to allow blind processing).
The numbering scheme will be as follows: YYYYMMDD-SSS, where YYYY is the year (such as 2010), MM is the month (such as 09 for September), DD is the day (such as 04 for the fourth), and SSS is the sample ID number (such as 001, 002, 003, etc). An example for a sampling event scheduled for 11 June 2009 would be: 20090611-001, 20090611-002, etc.
The SSS numbers will be consecutive starting from 001 and increasing to the maximum number of samples taken that day (999). Labels will be printed on Rite-N-Rain ® or some type of waterproof paper and affixed by LRC to the outside of the sample bottles using clear duct tape prior to the initiation of field activities.
(5) Once bottles have been labeled, they will be placed in the sterilized sample coolers in numerical order. Sample bottles will be stored in the sterilized coolers until use, and will be transported only in the coolers. Although the number of the sample is not relevant except for identification purposes, collecting in consecutive order will aid in determining where samples were taken in case of a recording error.
(6) Two samples out of approximately every 25 will serve as control samples or at least one control per cooler. Control samples will be chosen randomly and will be filled with DI water by LRC before coming into the field. The baseline number of control samples needed for each reach is listed in Table 2-1.
(7) Six samples out of every 120 samples collected will serve as duplicate samples. Duplicate samples will be determined prior to sampling and their locations will be identified on the aerial maps. At these designated areas, a duplicate sample will be taken in tandem with a regular sample.

Boat Preparation Procedure
(1) Vessels to be used for eDNA sample collection must be washed with a commercial power washer and detergent prior to the disinfection process. All detergent must be thoroughly rinsed and the boat must be allowed to air dry 1 day prior to disinfection. A 10% hypochlorous acid solution with DI water will be prepared in a 3-gal hand-held sprayer that is dedicated to the project. The hypochlorous acid solution must be prepared immediately prior to use, and each time disinfection activities will be occurring.
(2) The outer surfaces of the sampling vessel (i.e., hull, motor, etc.) as well as the transport trailer will be thoroughly rinsed with the prepared hypochlorous acid solution using a hand-held sprayer. Outer surfaces should be thoroughly covered with the hypochlorous acid solution and left in contact with no rinsing. Three gallons of solution may not be sufficient to rinse the entire boat and trailer, and additional batches of hypochlorous acid solution must be mixed when needed to ensure thorough rinsing of the hull, trailer, 2-9 motor, etc. A minimum of 10 minutes contact time must be used for the boat to allow sufficient time for the hypochlorous acid solution to oxidize organic matter that is present on the boat.

Purpose
In order to perform laboratory molecular analyses to detect eDNA, samples must first be collected from the appropriate aquatic environment in accordance with the following protocols.
Cautions: Lifejackets must be worn at all times in transport vessels (boats). Additionally, disposable latex or nitrile gloves must be worn when collecting water samples and measuring water depth and temperature. Be aware of pollutants in the aquatic environment and related health hazards.

Water Collection Procedure
(1) Prior to launch, crew members will have reviewed this QAPP, will have signed the QAPP certification form, and will understand their assigned roles in the sample collection procedure. All sampler identification information and other field data will be recorded in pencil on the Field Collection Summary (Exhibit Form 1).
(2) The transport vessel will be launched from an appropriate area (such as those locations listed in Table 2-2) that allows access to the reaches to be sampled.
(3) Sampling will commence at the first transect located at the downstream end of the reach to be sampled and will proceed in an upstream direction. The only exception to this protocol is when the boat launch is located upstream of the sampling reach. Then sampling will commence at the first transect located at the upstream end of the reach to be sampled and will proceed in an upstream direction. The direction traveled for sampling should be recorded on the Field Collection Summary (Exhibit Form 1).
(4) When arriving at a sample location (within either a transect or targeted area), the lead sampler and sampling assistant 1 will put on sterile exam gloves (powderless latex or nitrile). REMINDER -Gloves must be changed before each new transect is taken to prevent cross contamination. The same gloves may be worn when collecting duplicate or blank samples in tandem with a regular sample in a transect.
(5) Going in consecutive numerical order based on the bottle labels, the lead sampler will remove a labeled 2L sample bottle from the sample cooler.
(6) Just prior to collecting the sample, the lead sampler will unscrew and remove the lid from the sample bottle.
(7) The lead sampler will then reach over the upstream side or the bow of the transport vessel with the 2L sample bottle and fill the bottle by skimming the surface of the field 2-10 water. The sample bottle must not be submerged or dipped beyond the upper 2 inches of the surface water for sample collection, since the intent of the sampling is to collect floatable materials that are in the water surface.
(8) Once the sample bottle is filled (approximately 1 in. of space should be left within the sample bottle), the lead sampler will screw the lid back on to the bottle until it is tight.
The closed bottle will then be returned to the sample cooler from which it was removed.
(9) While the lead sampler is collecting the water sample, sampling assistant 1 will take habitat measurements: water temperature, depth, GPS coordinates in Decimal Degrees, military time of sample, location (e.g., LBD, center, RBD), and relate the information to sampling assistant 2, who will record the information on the datasheet next to the appropriate sample ID.
(10) If the lead sampler pulls a transport blank (2L of DI water filled prior to trip) from the cooler, the sampler will unscrew the lid and remove to expose the bottle contents to the atmosphere for 5 sec, reseal the bottle, fully submerge the bottle in the field water, and return the bottle to the sample cooler from which it was removed. The lead sampler should relay to sampling assistant 2 that the sample was a blank, so that it can be recorded on the data sheet next to the appropriate ID. BLANKS ARE TAKEN IN TANDEM WITH THE NEXT ACTUAL SAMPLE AND DO NOT REPLACE A SAMPLE IN THAT LOCATION. If a blank has been pulled, the boat will remain at the same location and an actual sample will be taken.
(11) Duplicate samples will be taken as part of quality control. Duplicate sample locations will be identified by RED STARS on the aerial location map. Duplicate samples will be collected the same as a regular sample; however, the lead sampler should relay to sampling assistant 2 that the sample was a duplicate, so that it can be recorded on the data sheet next to the appropriate ID. DUPLICATE SAMPLES ARE TO BE TAKEN IN TANDEM WITH THE NEXT REGULAR SAMPLE. If a duplicate sample is designated, this sample should be taken concurrently with the regular sample, side-by-side, to best replicate the regular sample collection. Sampling assistant 1 should assist the lead sampler by holding one of the sample bottles and filling that sample bottle in tandem with the lead sampler filling their own sample bottle. If a blank sample is pulled from the cooler at a designated duplicate location on the aerial map, take the duplicate sample at the NEXT DESIGNATED REGULAR SAMPLE LOCATION.
(12) Steps 1 through 10 will be repeated at each sampling location until sampling has been completed for the targeted reach.
(13) Once sampling is complete, ice will be added to the sample coolers as soon as possible. Enough ice should be added to each cooler to completely surround each sample bottle and maintain an inside temperature of 4.4°C. If at any time during transport the inside temperature of the cooler(s) rises above 4.4°C, additional ice should be added.

2-11
(14) Chain of custody (COC) forms (Exhibit 3) will be completed for every sample. All samples, including blanks, will be logged onto COC forms. The forms will be collected and signed whenever the coolers are transferred between parties.

Purpose
In order to isolate eDNA from water samples collected in the field, particulate matter must be filtered from the sample using a vacuum filtration system. Passing each water sample through the appropriate sterile filter (1.5 micron, 5.5 cm diameter glass fiber filter) will collect particulate matter from the water sample, including sloughed cellular materials containing eDNA, on the filter paper. DNA will later be extracted from the filtered particulates and utilized in subsequent analyses.
Cautions: Wear gloves when filtering samples (a glove change is required for each sample). Be careful to avoid unintentional punctures of gloves when using forceps. Punctured gloves must be changed immediately. Be careful not to touch commonly used items in the laboratory when wearing sample gloves (i.e., writing utensils, stationary lab equipment). If in doubt, change your gloves!

Filtering Procedure
Water samples collected in the field need to be filtered within 12-16 hours after the last field sample is collected.
Equipment needed: • Manifolds • Glass fiber filters • Forceps (microforceps) -at least two pair, labeled with different colors or other identifier • Carboy (3.5 gal or larger), for wastewater generated during filtering • Rubber tubing (1.25 in. inner diameter), double hole stopper that fits carboy opening, and glass connectors for connecting manifold to carboy and manifold to vacuum line • Sterile conical tubes (50mL plastic) with caps and labels • Sterile conical tubes (15mL plastic) with caps and labels • Paper towels • Black permanent markers • Sterile powderless latex or nitrile gloves • Vacuum system capable of −75 kPa vacuum • Hypochlorous acid • Sterile 1L graduated cylinder for measuring reagent grade water • Reagent-grade water • Dedicated lab equipment cleaning sink • Waste water disposal location such as nonspecified-use sink 3-13 • Sterile bench paper • Dedicated water bottles: one for DI water; one for hypochlorous acid solution • Washbin for manifolds, such as a dedicated 10 qt plastic tub

Laboratory Preparation
(1) Wash hands thoroughly prior to starting. Prepare a dedicated plastic wash bottle with 10% hypochlorous acid (14-412-53) solution for wiping down lab tables and manifold surfaces prior to processing samples. Sterilize all equipment needed prior to starting. Collect all supplies needed.
(2) Rinse down each workstation with hypochlorous acid solution prior to beginning the filtration process. Cover each workstation surface with sterile bench paper. Bench paper must be switched out between samples.

Sample Preparation
(1) Put on new sterile powder-free latex or nitrile gloves prior to handling each sample.
(2) Remove first sample from transport cooler and rinse bottle thoroughly under ultrapure water to remove residual biological materials. Place prepared bottle at workstation.
(3) Label one sterile 15mL conical tube and one sterile 50mL conical tube with sample number (YYYYMMDD-SSS); indicate that the filter to be stored in the 15mL tube is the equipment control by labeling this tube with a "C". Ensure gloves that come into contact with labeling marker are not used again for handling other samples.

28150-496) equipped with rubber stopper on manifold and open vacuum line
(3) Take designated forceps for handling clean filter paper (1.5 micron, 5.5 cm diameter glass fiber filter paper (Type 934-AH); #1827-055), remove one filter and place on bottom portion of sterile magnetic filter funnel. Once the filter paper is positioned on the magnetic filter funnel, attach the upper portion of the magnetic filter funnel (i.e., the funnel) to the bottom portion.
(4) A sterile and clean magnetic filter funnel must be used for each sample. The cleaning process is described under the Equipment and Work Area Cleaning section.
(5) Using a sterile 1L graduated cylinder, measure out 1L of DI water (6) Once the magnetic filter funnel top is secured to the bottom portion, pour the 1L of DI water from the graduated cylinder into the magnetic filter funnel top as a control. In order to capture any potential contaminant DNA in the funnel, be sure to pour the DI water quickly so as to immerse all the internal surfaces of the magnetic filter funnel top with DI water. Once the DI water has been poured into the filter funnel top, turn the vacuum on to draw the water down quickly and filter the material as quickly as possible.
(7) Once the 1L of DI water has been filtered through the funnel, remove the filter funnel top. Take the sterile forceps designated for used filter paper and grasp the edge of the filter paper. Roll or fold the filter paper until it is of a size to fit into the 15mL conical tube labeled as a control for the appropriate sample ID.
(8) Place the control filter paper into the 15mL tube, screw on top, and place tube with control sample filter paper into a −20°C frost-free freezer. The freezer should be secured (i.e., locked) if samples are left for any period of time unattended.

Filtering the Sample
(1) Take designated forceps for handling clean filter paper (1.5 micron, 5.5 cm diameter glass fiber filter paper (Type 934-AH); #1827-055), remove one filter and place on bottom portion of sterile magnetic filter funnel. Once the filter paper is positioned on the magnetic filter funnel, attach the upper portion of the magnetic filter funnel (i.e., the funnel) to the bottom portion.
(2) Take the sample bottle and gently shake to distribute the contents within the sample evenly.
(3) Once the magnetic filter funnel top is secured to the bottom portion, turn on the vacuum and then pour approximately one-third of the sample into the magnetic filter funnel top.
(4) DI water from a wash bottle may be used to rinse any particulates attached to the sides of the magnetic filter funnel onto the filter paper. Make sure that DI in wash bottle is fresh and was replaced prior to the filtering process for the entire collection.

3-15
(5) Once the one-third portion of the sample has been filtered through the funnel, remove the filter funnel top. Take the forceps designated for used filter paper and grasp the edge of the filter paper. Roll or fold the filter paper until it is of a size to fit into the 50mL conical tube labeled for the appropriate sample ID.
(6) Repeat steps 1-4 at least two more times or until the entire sample has been filtered. All filter paper used to process the sample is placed in a single 50mL conical tube labeled for the appropriate sample ID. Place the sample filter paper into the 50mL conical tube, screw on the top, and place the tube with sample filter paper into a −20°C frost-free freezer. The freezer should be secured (i.e., locked) if samples are left for any period of time unattended.
(7) The number of filters used to process the sample is up to the discretion of personnel processing the sample. If the water sample exhibits an excessively slow filtration rate, multiple filters should be used. Also up to the discretion of the personnel processing the sample is the amount of sample water to run through a single filter. A general rule is to run one-third of a 2L sample through a single filter; however, if the sample water is extremely turbid, for example, less water should be put through a filter. At the other end of the spectrum, if the sample water is extremely clear, more than one-third of a 2L sample may be run through a single filter.
(8) On the field datasheet (Exhibit Form 2) next to the appropriate sample ID, mark the military time of filter completion and the initials of the person that processed the sample.
(9) Change gloves and sterilize the workstation between samples. Repeat steps 1-8 until all samples have been processed.
(10) When filtering, if the water collection carboy becomes full, disconnect the carboy from the vacuum and manifold and dispense water in a sink separate from the one used to clean equipment. Once emptied, reconnect the carboy to the vacuum and manifold and proceed with the filtering process. CAUTION: Be sure to open manifold valve and turn off the vacuum air supply when disconnecting and connecting the carboy so as to prevent explosion of the glass.
(11) If sample is accidentally spoiled during the filtering process (e.g., hypochlorous acid was used to rinse filter funnel instead of DI water, forceps from previous sample used, etc.), immediately throw away ruined samples. If portions of the sample are still viable, place in 50mL conical tube. On the outside of the sample tube, label with the amount of the viable sample (e.g., 2/3 sample). On datasheet, label with the same information (e.g., 2/3 sample) next to appropriate sample ID. Note the reason for the ruined or diminished sample (i.e., spilled bottle, acid solution used for rinsing instead of DI water, suspected cross contamination due to dirty gloves, etc.).

Preparing and Filtering a Positive Equipment Control
(1) In advance of filtering, small aliquots (0.5-1.5ml) of a dilute slurry of DNA material from Asian carp (e.g., scales, slime coat) should be maintained in a freezer. Positive control slurries should be prepared using different equipment than any used for filtering and should be prepared in a different room from that used for filtering.
(2) Following the completion of all other filtering (Sections 3.5-3.6), a DNA slurry aliquot should be dispensed into 2L of sterile water. Opening and dispensing the slurry aliquots should be done, if possible, in a room separate from the filtering lab. If that is not possible, it should occur in an area of the room not used for filtering, preferably within a sink or other area where genetic material is likely to be washed away and/or unlikely to come into contact with equipment or laboratory personnel.
(3) The 2L positive control solution should then be vigorously swirled for 1 min. Shaking should be avoided in order to minimize the chance of spreading DNA contaminants in the work area.
(4) The positive control solution should then be treated and filtered in the same way as other samples were treated and filtered (Sections 3.5-3.6).
(5) The positive control filters will be sent to ERDC for processing along with all other samples from an eDNA sampling event.

Equipment and Work Area Cleaning After Filtering Each Sample
(1) Fill a 500mL glass beaker with 10% hypochlorous acid solution. Forceps designated for used filter paper must be switched out for each sample. Used forceps will be placed in beaker with 10% hypochlorous acid solution for a minimum of 10 min for sterilization. Once sterilized, remove forceps from hypochlorous acid solution and rinse thoroughly with DI water before use.
(2) Fill at least a 10-qt plastic tub (e.g., Rubbermaid ® plastic storage bin) with 10% hypochlorous acid solution. Once a sample has been processed, the filtering apparatus must be dismantled (i.e., the magnetic filter funnel should be separated into the upper and lower parts), rinsed with DI water to remove any particles and/or film, and placed in the plastic tub with the 10% hypochlorous acid solution for a minimum of 10 min for sterilization. Once sterilized, remove the two parts of the magnetic filter funnel and thoroughly rinse with DI water before use. Rinsing should continue until all residues and scent from the hypochlourous acid can no longer be detected.
(3) In between each sample, dispose of bench paper. Wipe down surface with 10% hypochlorous acid solution using wash bottle and paper towels. Cover work station with new bench paper. CHANGE GLOVES! 4-1

Purpose
Samples must be shipped to the ERDC lab in Vicksburg, MS for processing of eDNA within 24 hours of sample preparation (after filtering is complete) or up to one week after filtering is complete if samples are properly stored before shipping (-20°C). The Filtering Leader is responsible for ensuring that samples are properly packed and shipped according to the procedure below.
Cautions: Wear gloves and use caution when working with dry ice.

Shipping Procedure
(1) Corrugated boxes (minimum outer dimensions 12" X 12" X 12") with Styrofoam cooler inserts will be prepared for shipment. The number of boxes to prepare depends upon the number of samples collected (e.g., a 120-sample collection will require more boxes for shipping than a 50-sample collection); however, at least two boxes will be prepared for every collection: one for the controls and one for the samples.
(2) The bottom of the coolers will be lined with dry ice pellets (approximately 1-2 inch thickness). Oven-mitt type gloves must be worn by personnel that are handling dry ice to protect hands.
(3) Remove the 15mL conical tubes with control filter paper from secure (i.e., locked) −20°C freezer and place in clean 1-gal resealable bag (e.g., Ziploc ® ). Multiple bags may be used if the entire sample does not fit in one bag. Seal the opening of all bags used with tamperevident tape (e.g., Evidence Tape -NC9709516). Be sure to remove as much air as possible from all resealable bags used before sealing. Place bags of conical tubes on top of dry ice pellets in cooler. Layer approximately 1 in. of dry ice pellets on top of bag before placing another bag in the cooler. Repeat until only 2 in. of space is left at the top of cooler and fill the space with dry ice pellets. Before closing the Styrofoam cooler, record the inside temperature on the datasheet (Exhibit Form 1). Place the Styrofoam lid on top of the sample contents and seal with tamper-evident tape (e.g., Evidence Tape).
(4) Sign and place the COC form (Exhibit Form 3) in a clean 1-gal resealable bag on top of the cooler before closing the corrugated shipping box and sealing with packing tape. A copy of the COC form should be made and retained by the sender.
(5) Repeat steps 1-4 for 50mL conical tubes and additional boxes. Each box will have a separate signed COC form included to document the samples included therein. (9) Upon receipt of the samples at ERDC, inside temperature of all the coolers must be taken and recorded. Samples that have remained at room temperature (approximately 20°C) for more than 24 hours will be discarded, and the sample names and reason(s) for discarding will be noted in the laboratory log.
(10) Personnel receiving the shipment must immediately sign the COC form.

General Quality Assurance and Chain-of-Custody Considerations
(1) Any change to described DNA handling, storage, or processing procedures must not result in reduction of eDNA sensitivity relative to current values and must be cleared with the Project Leader. The current protocol produces the following results at different concentrations of total genomic DNA 1 Species in sterile water: (2) Each stage of eDNA genetic processing procedures (eDNA sample extraction, PCR setup, and post-PCR processes) should be performed in a separate room in order to minimize the risk of sample cross-contamination.
(3) Every effort should be made to ensure that equipment, work areas, and solutions are free from DNA contamination. As a minimum, all surfaces should be wiped clean with 10% hypochlorous acid solution before and after use.
(4) All centrifuge tubes and glassware must be autoclaved at 121˚C for 20 min before being used.
(5) Good housekeeping policy should be practiced at all times. Reagents that have passed expiration dates should not be used, nor should any reagents that have been kept at incorrect storage temperatures for a significant length of time. All reagents, reaction tubes, etc., must be clearly labeled. Records of batch numbers of all reagents used in individual assays should be made whenever reagents are signed out from the designated freezer. The temperatures of cold storage units must be monitored twice a day, once in the morning and once at end of day, using the form given in Exhibit 5.
(6) Positive and/or negative reactions should be used to test all new batches of critical components prior to or concurrent with their application to eDNA samples.
(7) Standard sterile techniques should be used in the DNA laboratory to prevent the unintended transfer of DNA between surfaces, and to prevent cross-contamination 5-2 between samples. Contamination can adversely affect the outcome of a case; therefore, it is essential that the laboratory have procedures in place to limit, recognize, and address contamination.
(8) Gloves (e.g., sterile nitrile or latex) must be worn throughout sample processing. At a minimum, gloves should be changed at the completion of each step of the process. If gloves become contaminated or if contamination is suspected, discard them and replace them with new ones. For example, gloves should not be worn when using or handling keyboards, notebooks, pens, telephones, etc. and must be replaced immediately before recommencing bench work.
(9) Centrifuge tubes before opening the reagents. Uncap and close tubes carefully to prevent aerosol contamination.
(10) Ensure that centrifuges are always balanced when centrifuging samples.
(11) Ensure that all equipment, including paper, pens, and lab coats, are dedicated for use only in that particular laboratory (e.g., laboratory coat for each stage of procedure rooms. Workbooks that have been in contaminated areas shall not be taken into clean PCR areas. A Project Lab Book should be kept in a room separate from the DNA Extraction Room and DNA PCR Room. Each room (Extraction, PCR, Post-PCR) should have note-taking materials (e.g., loose-leaf paper, networked tablet PCs) that can be transported or viewed for consolidation in the Project Lab Book. Other solutions for preventing contamination of sensitive areas via lab notes may be used following approval by the eDNA Processing Leader. Any changes should be incorporated into a revised QAPP. Laboratory notes/notebooks should: • Be written or printed on tamper-proof paper (e.g., does not exactly photocopy).
• Have lab book identification, with consecutive numbering, dates, and signatures (of the note-taker) on each page. • Be made using permanent ink. Special pens may be required for certain paper types.
• Have any changes to notebooks be dated and initialed by the person who made the change. Any incorrect information should have a single line drawn through it and not be completely obscured. • Contain all data images (e.g., gel photographs, denaturing curves, DNA sequence electropherograms). Images should be permanently affixed to the notebook and signed across both the edge of the insert and the page. • Be kept in a locked drawer or cabinet with restricted access when not in use.
(12) A log of all batches of critical components should be kept. This log should include material safety data sheets (MSDS) and product information sheets. Dates of receipt, opening, testing, and disposal for each component should be recorded in the log. 5-3

Quality Control for Sample Custodian Procedure and Storage
(1) A COC Log Book should be kept for all samples. Tamper-proof paper should be used. The log book should be kept in a locked drawer or cabinet when not in use.
(2) Separate freezers should be designated for storage of (a) filter samples, (b) DNA extracts, (c) PCR and sequencing product, and (d) PCR, cloning, and sequencing kit components. A dedicated refrigerator should also be maintained for any PCR, gel electrophoresis, cloning, and sequencing kit components that require 4˚C storage.
(3) Maps or other designations of the location of samples within freezers should be maintained.
(4) All items in freezers should have indelible ink identifications.
(5) All freezers should have non-universal locks or marine brackets attached that can be used with keyed locks.
(6) All samples removed from freezers should be signed for on freezer log in addition to sample log (see Exhibits 2 and 4). All reaction components should be signed for.

The eDNA Extraction Room
• Extraction of DNA must be performed where PCR products and stocks of cloned material are not handled. • A PCR hood with a built-in ultraviolet (UV) light and HEPA filter may be used to further isolate DNA extraction kit solutions and elutes from ambient DNA. • A complete separate set of necessary laboratory equipment, consumables, and laboratory coats should be dedicated for use in DNA extraction.

Pre-PCR Room
• To prevent carry-over of amplified DNA sequences, PCR reactions should be set up in a separate room from that used for post-PCR manipulations. • A completely separate set of necessary laboratory equipment, consumables, and laboratory coats should be dedicated for the specific use of pre-PCR manipulations. • Reagents and supplies should be taken directly from clean storage into the PCR area and should never be taken or shared with areas in which post-PCR analyses are being performed. Equipment such as pipettes should never be taken to the containments area after use with amplified material. • A sign-in log system should be implemented for use of the thermal cyclers (PCR machines), including the run name, plate orientation, and the number of thermal cycler heads. 5-4

Product Analysis Room
This is the post-PCR Room where post-PCR manipulations are performed, including agarose gel electrophoresis of products.
• This room is a contaminated area; therefore, no reagents, equipment, laboratory coats, etc. from this room should be used in any of the other PCR areas. • A biological or PCR-type hood may be used for setting up cloning or sequencing reactions.

Source
Water samples have been taken in the field according to Section 2 and filtered according to Section 3. Filters have been shipped to ERDC and personnel assigned to the ERDC eDNA Team have either received or picked-up shipping boxes from the ERDC Receiving Office.
(1) Upon receipt of samples from the eDNA sample filtering team, the shipped box(es) should be opened and the temperature inside each box recorded. The general condition of the box(es) should also be recorded.
(2) To measure temperature upon opening the box, either (a) place a glass thermometer inside the styrofoam container, replace lid, and leave the thermometer in place for at least 2 min before removing it and immediately recording temperature or (b) immediately aim an infrared laser thermometer at the samples and press the MEASURE button to record the temperature inside the cooler.
(3) Place samples in filter sample storage freezer (-20°C) and log samples on freezer sheet (see Exhibit 4).
(4) Sign and date the COC forms that accompanied the samples. Place them in a designated file; a copy should also be provided to LRC. If the forms were inside sealed bags, slit the bag to remove the COC forms, and then place unbreakable tape across entire slit. Sign and date tape. Note any condition issues (broken tape or seals, damaged containers or bottles, etc.) with the samples on the COC forms. Note any samples that must be discarded due to condition issues and the reason for discard.
(5) Enter sample data into new sample log book, including noting any samples that are being discarded and that should not be analyzed, and create new ERDC COC form for samples. Note any observations about samples such as condition issues.
(6) Alert Sampling and Filtering Leader that samples have been received. Use e-mail addresses with return/receipt requested or directly contact via telephone; team contacts are listed in Appendix A. This reporting must be done within 1 hour of receipt. 5-5

Source
Filters from eDNA sampling have been received by ERDC eDNA Team and logged. Samples should be in designated −20°C freezer.

DNA Extraction Quality Assurance and Chain of Custody
At this stage, a critical component of quality control should be to correctly label all sample extraction processing tubes so that there is no question about the origin of samples.
(1) Bench areas in DNA extraction laboratory and PCR-type hood (if used) should be wiped before and after use with 10% hypochlorous acid. PCR hood should be sterilized using a built-in UV radiation bath capability following use. Validated, commercially available sterilization reagents, such as LookOut® DNA Erase®, may be preferred.
(2) After an item or surface is cleaned with hypochlorous acid, it must be rinsed with purified water or alcohol to prevent the build-up of sodium hypochlorite crystals. Instruments or equipment cleaned with hypochlorous acid must be rinsed to avoid corrosion.
(3) It is common practice for moisture barrier paper towels to be placed on the bench top while processing samples to act as a barrier. The paper barriers must be changed and the bench top cleaned between sample batches.
(4) Centrifuges, thermal cycler, tube racks, pipettes, and any other equipment used for the extraction process should be cleaned before and after each use.
(5) Instruments such as forceps and scissors should be cleaned just prior to use. Sterile disposable equipment should be opened just prior to sample processing and discarded after one use.
(6) When using pipettors, use filtered tips and never allow the liquid in a pipette tip to rise up to the cotton barrier.
• Do not rest the pipette on a dirty surface.
• Avoid cross-contamination by changing pipette tips after each use.
• Watch that the tip --and only the tip --is allowed to go into a bottle of reagent, never the pipette itself.
(7) Record all solution batch numbers used for reactions in lab notes.
(9) No deviations to the DNA extraction protocol are allowed without written approval by the Project Leader. Any errors in processing should be noted in the laboratory log. Samples affected by errors in the extraction protocol should be clearly identified.

5-6
Cautions: As with all components of eDNA processing, quality control and sterilization procedures must be carefully followed in order to avoid contamination of downstream procedures.

Procedure
(1) Remove samples from freezer; note on freezer and sample logs (see Exhibits 2 and 4), as well as COC form.
(3) Positive and negative extraction controls should be added to each eDNA extraction procedure batch.
• Before proceeding with extraction, a positive control filter is prepared by pipetting 25 µl of a mixed slurry of homogenized silver and bighead carp tissue directly onto a sterile filter paper (1.5 micron, 5.5 cm diameter glass fiber filter paper, Type 934-AH; #1827-055). Alternative species (e.g. -sturgeon) may be used as the positive control to reduce risk of sample contamination from carp tissue. Alternative species must have PCR primers that (1) do not cross-react with carp DNA and (2) can be run on the same themocycler settings as carp samples • Additionally, an extraction negative control sample should be prepared by UV-sterilizing a previously unused filter paper (1.5 micron, 5.5 cm diameter glass fiber filter paper, Type 934-AH; #1827-055) and placing it in a new sterile 15mL tube. A batch of extraction blanks can be prepared in advance and kept frozen at −20°C • For every 23 samples processed, conduct DNA extraction (below) on one frozen, sterile extraction negative control filter and one prepared positive control filter.
(4) For all samples and cooler, equipment, and extraction controls, follow the DNA extraction protocol detailed below: This DNA extraction utilizes the PowerWater DNA Isolation Kit (MoBio Laboratories, MoBio Inc.) and the protocol is adapted from the manufacturer's protocol (http://www.mobio.com/images/custom/file/14900.pdf).
(a) Place Solution PW1 in a 55°C water bath for 5-10 min to dissolve any precipitates that have formed at room temperature. Remove Solution PW1 from the water bath immediately prior to use.
(b) Remove the appropriate filter sample from −20°C freezer and transfer the filter(s) to a labeled 5mL PowerWater Bead Tube. If DNA will be extracted from multiple filter samples, continue to remove each filter sample from −20°C freezer immediately prior to filter transfer. Each PowerWater Bead Tube will hold up to three filters per sample. If any filtered water samples required four pieces of filter paper, split the filters into two PowerWater Bead Tubes.

5-7
Note: Change gloves between the transfer of each filter sample to avoid crosscontamination of samples.
(c) Add 1mL of Solution PW1 to the PowerWater Bead Tube and secure the cap tightly. Mount the tube on a vortex adaptor (MoBio Inc.) and vortex on high for 5-10 min, or until the contents of the bead tube appear liquefied. Times can vary depending on the number of filter papers being extracted. No more than four pieces can be extracted per bead tube. If more than four are used for one sample, use multiple bead tubes and combine the supernatants from these tubes in step 9 (below).
(d) Centrifuge the tubes at 4,000 x g for 1 min at room temperature. Ensure centrifuge is balanced before centrifuging. Transfer supernatant using a 1mL pipette to a labeled 2mL collection tube.
(e) Centrifuge tubes at 13,000 x g for 1 min and carefully decant supernatant into a labeled 2mL collection tube.
(f) Add 200μL of Solution PW2, vortex briefly, and incubate at 4°C for 5 min.
(g) Centrifuge the tubes at 13,000 x g for 1 min and carefully decant supernatant into a labeled 2mL Collection Tube.
(h) Add 650μL of Solution PW3 and vortex briefly. Load 650μL of supernatant onto a spin filter and centrifuge at 13,000 x g for 1 min. Discard the flow through and repeat until all the supernatant has been loaded onto the spin filter.
(i) Place the spin filter basket into a labeled 2mL collection tube and add 650μL of Solution PW4.
(j) Centrifuge the tubes at 13,000 x g for 1 min and discard flow through.
(k) Add 650μL of Solution PW5 and centrifuge at 13,000 x g for 1 min. Discard flow through and centrifuge again at 13,000 x g for 2 min.
(l) Place the spin filter basket into a 2mL collection tube labeled with the sample identification number, the date the water sample was collected in the field, and the volume of water collected.
(m) Add 100μL of sterile water (autoclaved, de-ionized) to the center of the white filter membrane and centrifuge at 13,000 x g for 1 min.
(n) Discard the spin filter basket and store the eluted DNA samples at −20°C.
(5) DNA extraction elutes should be placed into a designated freezer or, if immediately used for PCR, kept on ice. Make note of sample addition to freezer log if necessary (see Exhibit 4). Note completion of extraction on sample log. 5-8

Purpose
In order to determine if the DNA of a specific species is present in the filtered water samples taken in the field, the total DNA extracted from the filtered samples must be amplified using species-specific primers.

Source
Filters from eDNA sampling have been received by ERDC eDNA Team and DNA has been extracted. DNA elutes from samples should either be located in designated −20°C freezer or carried on ice from DNA extraction room to PCR room.

PCR Quality Assurance and Chain of Custody
This stage of DNA processing is particularly susceptible to contamination and, subsequently, inaccurate results. Carefully follow quality control and COC steps listed below: (1) PCR-type hood bench should be wiped before and after use with 10% hypochlorous acid. PCR hood should be sterilized using a built-in UV radiation bath capability following use. Validated commercially available sterilization reagent such as LookOut ® DNA Erase ® may be preferred.
(2) After an item or surface is cleaned with hypochlorous acid, it must be rinsed with purified water or alcohol to prevent the build-up of sodium hypochlorite crystals. Instruments or equipment cleaned with hypochlorous acid should be rinsed to avoid corrosion.
(3) Centrifuges, thermal cycler, tube racks, pipettes, and any other equipment used for PCR Amplification should be cleaned before and after each use.
(4) Use UV radiation to cross-link 96-well PCR plates and the exteriors of manual pipettors, as well as autoclaved, filtered, or commercially sterile water prior to use for setting up PCR reactions.
(5) Aerosol-resistant pipette tips should be used. Place the sterile tip on the pipette immediately prior to use. If the pipette is set down with the tip on, discard the tip. A new pipette tip must be used for the addition of each reagent to a sample tube.
(7) Only one microcentrifuge tube should be open at a time. Close each tube immediately after labeling and after the addition of sample or reagents to prevent crosscontamination.
(8) Use a tube opener, clean Kimwipe ® , or other suitable barrier, rather than gloved fingers, to open microcentrifuge tubes.

5-9
(9) Record all solution batch numbers used for reactions in lab notes.
(10) PCR reagents should be aliquoted (a portion of the original stock) to avoid excessive freeze-thawing and to protect stock reagents if contamination occurs.
(11) Centrifuge tubes before opening the reagents. Uncap and close tubes carefully to prevent aerosol contamination.
(12) Any revisions to the DNA amplification protocol must be approved by the Project Lead and documented in writing.
Cautions: Wear gloves throughout the DNA amplification and gel electrophoresis procedures. Ethidium bromide, used in DNA gel electrophoresis to visualize DNA, is a known mutagen that affects biological processes.

Procedure
(1) If DNA samples (extraction elutes) are removed from freezer, note on freezer log (see Exhibit 4). Also note on sample log and ERDC CoC form (Exhibits 2 and 3).
(2) Use preprinted 96-well plate map to determine which samples will be pipetted into which wells. Clearly mark plate identification on top right corner of plate. Write plate identification information (number and corresponding samples) in lab notes. Mark left side (technician's left) of the plate with L, right side (technician's right) with R, top (T; 90° clockwise from L side) with T, and bottom (B; 270° clockwise from L side) with B using indelible marker.
(3) Plate identification can be as simple as date, order of processing, and target species. For example, SC011210A might refer to the first (A, B, C...) silver carp PCR assay conducted on 1 December 2010.
(4) Make sure sample map for each plate is attached to lab notes.
(5) Keep samples on ice or in cold block. 2 (6) Follow DNA amplification protocol detailed below.
Primers specific to either Hypophthalmichthys molitrix (silver carp) or H. nobilis (bighead carp) are used to screen eDNA samples and amplify unique DNA sequences in each species potentially present in the eDNA samples by PCR. The PCR programs used to amplify the extracted DNA are specific to the primer set used. The PCR protocol has been optimized to utilize specialized Taq polymerase and buffer from Platinum® Taq DNA polymerase (Invitrogen Corporation, Carlsbad, CA) in the eDNA screening. Eight reactions are set up for 5-10 each sample, in addition to negative (DNA blank) and positive (DNA extracted from tissue 3 (1) Wipe lab bench area with 10% hypochlorous acid, 75% Ethanol, or commercial DNA sterilization wipes. Also wipe down work area with PCR hood. Use built-in UV lamps to radiate bath hood area for 10 min prior to PCR set-up.
) controls for each master mix. The PCR reactions are prepared as follows: (2) UV radiate cross-link water aliquots, manual pipettors, and 96-well plates. This can be done concurrently with Step 1. Electronic pipettors should be wiped down with one of the solutions or wipes listed in Step 1.
(3) Obtain all PCR master mix reagents (using only those that have not expired and that have been tested and found viable). Keep reagents on ice or in cold block: • 10X PCR buffer • 10mM equally mixed dNTP solution (2.5 mM per nucleotide) • 25mM Mg2+ solution • Species-appropriate forward primer • Species-appropriate reverse primer • Taq DNA polymerase • Sterile DI water (commercially sterile or Millipore filtered, autoclaved, and UV crosslinked) (4) Also set aside enough positive control DNA for each species for three PCR reactions each.
(5) Record in lab notebook the lot number of all reagents used. (6) Prepare PCR master mixes. The master mix volume can be adjusted according to the number of samples to be processed. In order to make sure that master mix does not run out prior to supplying all the desired reactions (this may occur as a result of minor errors or variations in pipetting volumes), it is generally helpful to make more than enough master mix than is needed for the desired number of reactions. For example, make enough master mix for 100 reactions when actually preparing for 96 reactions. 3 Extract from tissue using commercial DNA extraction kit (e.g., Qiagen DNeasy Blood & Tissue Kit), and manufacturer protocol. Run test PCR before relying on any DNA extract for eDNA assay positive controls.

5-11
(7) Place a 96-well PCR plate into a prepared cold block, positioned from left to right. Add 24 µL of the master mix into each well on the 96-well PCR plate (except the two empty wells in the 12 th column).
(8) Pipette 1 µL of each sample to be screened into each well of a column, changing the pipette tip between each sample. Each column of eight wells should be filled with the same sample (i.e., eight replicates per sample to be tested). The first 11 columns of the PCR plate can test 11 different samples for one target species. Into the 12 th column, pipette 1 µL of sterile water into the bottom three wells (F, G, H) and pipette 1µL of each of the target species positive control DNA into each of the top three wells (A, B, C). Leave the intervening two wells (D, E) empty.
(9) Place the positive control DNA back in to the appropriate −20°C freezer and change gloves immediately in order to reduce risk of contamination.
(10) Place PCR film over the PCR plate and press firmly to ensure the edges of all wells are sealed. Remove plate from ice block and gently tap a few times on the lab bench to ensure thorough mixing of each reaction.
(11) Place the 96-well PCR plate in the thermal cycler, close and secure lid, and select the appropriate PCR thermal program (thermal cycle programs for silver carp and bighead carp utilize different annealing temperatures). The thermal programs both consist of: • Initial denaturation at 94°C for 10 min Followed by 45 cycles of: • 94°C for 1 min, • 50°C for silver carp program or 52°C for bighead carp for 1 min • 72°C for 1.5 min.
Followed by: • final extension at 72°C for 7 min • 4°C hold temperature until plate removed from thermal cycler.
(12) Record the plate ID, thermal cycler unit or head, plate orientation, and run times for the PCR plate in the PCR log (Exhibit 6).
(13) Place PCR plates and product in designated −20°C freezer for long-term storage, in designated 4°C refrigerator for mid-term storage (1-6 hours) or in ice or in cold block for short-term (less than 60 min) storage.

Purpose
Once amplified, the DNA samples should then be subjected to gel electrophoresis in order to visualize the amplified DNA. This method is useful in determining the presence of DNA from any given species in different aquatic environments.

Source
PCR product following amplification can be taken either from cold storage (see #11 above) or directly from the thermal cycler.

Gel Electrophoresis Assurance and Chain of Custody
This stage of DNA processing is particularly susceptible to pipetting error. It is also highly susceptible to mislabeling and, consequently, confounding of sample results.
(1) Draw or otherwise produce a map of which sample will be electrophoresed on which gel and in which lane of the select gel.
(2) Carefully pipette samples so as to avoid: • Injecting samples to incorrect wells.
• Piercing the bottom of sample wells and losing PCR product.
• Spill over from adjacent wells.
(3) Record all solution batch numbers or name/date identification for stock solutions. Record precast gel batch identification if appropriate.
(4) Centrifuge plates, strip tubes, etc. before removing film or caps in order to prevent aerosol cross-contamination.
(5) Any revisions to the DNA amplification protocol must be approved by the Project Lead (and documented) and incorporated into a revised QAPP.

Option A Procedure
(1) Prepare 1% agarose gels with SB (sodium hydroxide and boric acid) buffer and ethidium bromide stain (5μg/mL of gel) and allow the gel to polymerize for a minimum of 25-30 min prior to loading samples. Gels can be prepared at any time prior to PCR or immediately after PCR.
(2) To prepare PCR samples for gel electrophoresis, transfer 10µl from each well of the 96well plate to new wells in an identically labeled 96-well plate. Add 2µl of 6X loading dye to each well with PCR product.
(3) Place the 1% agarose gel in the electrophoresis chamber that contains SB Buffer and remove the gel combs. In the first well of each row on the 1% agarose gel, load 100bp DNA adder/loading dye mix. Next, load 10μL of each sample mixture (i.e., each PCR reaction and loading dye), positive controls, and negative controls into the remaining wells. An aliquot (∼ 500 ng) of an appropriate DNA ladder (distinct bands between 0-500 bp) is loaded at one end of each row and a positive control PCR product at the other end. Run electrophoresis at ~100V for ~45 min depending on migration times through the gel. Times and voltages required to run each gel are approximate.
• Prepare DNA samples in a total sample volume of 15 with deionized water or 6X loading buffer.
(2) Selecting Program on E-Base ™ • Connect the Daughter E-Base ™ to a Mother E-Base ™ or another Daughter E-Base ™ if running multiple gels. • Select program EG by pressing and releasing the pwr/prg (power/program) button on the base.
(3) Loading E-Gel ® 48 Gels • Load each gel within 30 min of removing gel from the pouch and run within 15 min of loading. • Remove gel from the pouch. Remove comb from the gel.
• Slide gel into the two electrode connections on the Mother E-Base ™ or Daughter E-Base ™ . If gel is properly inserted, a fan in the base begins to run, a red light illuminates, and digital display shows 20 min. • Load 15μL prepared DNA sample into each well of an E-Gel ® 48 gel. Keep all sample volumes uniform. Load with a multichannel pipettor. • Load appropriate DNA ladder (distinct bands between 0-500 bp) into one marker well and positive control PCR product into the other marker well. Ensure the marker salt concentration is similar to that of adjacent samples (2% gel uses 100 bp DNA Ladder). • Load 15μL of sample buffer containing the same salt concentration as the sample into any empty wells.
(4) Run Conditions • To begin electrophoresis, press and release the pwr/prg button on the Mother E-Base ™ and Daughter E-Base ™ . The red light changes to green. • At the end of the run (signaled with a flashing red light and rapid beeping), press and release the pwr/prg button on the base to stop the beeping and flashing red light. • Remove gel cassette from the base and analyze results.

Purpose
Once eDNA gels have been visualized, the results must be documented, interpreted (i.e., scored), and recorded. In some cases, very light bands may be visible, making scoring difficult. Documentation and storage are critical for later quality control review.

Source
Following electrophoresis, agarose gels should be immediately documented.

Gel Documentation and Storage Assurance and Chain of Custody
Because of the difficult nature of scoring some results, careful records must be kept of all gels and results. These results must be maintained so as to minimize the risk of tampering or data loss.
(1) Gel image quality should be assessed at the time images are obtained. Images should exhibit all bands on gels as clearly as possible. All gel digital image files should be saved and should be archived at the end of each working day. All gel image data are referenced to the samples batch receiving date to make sure the consistency of the sample custody.
(2) Gel score data should be entered and stored in the appropriate database in an Excel file and in the spreadsheet manually in a designated binder. The binder should be stored in a locked drawer.
(3) All reports should reviewed by the eDNA Processing Leader before being reported.
(4) A paper copy of the report should be held in the files for 5 years.
(5) Electronic copies of all reports should be held for 5 years or longer, as space permits.
(6) Any substantive revisions to the DNA amplification protocol must be approved by the Project eDNA Processing Leader and approved by the Project Leader. Any such changes must be incorporated into a revised QAPP.

Procedure
(1) After electrophoresis is complete, remove casting tray with gel from the electrophoresis chamber and place the gel onto the gel scanner (BioRad Molecular imager FX), select DNA ethidium bromide stain gel, set up scanning area, and then select 100 micrometer to start scanning the gel.
(2) Alternatively, place the gel on a UV transilluminator equipped with a digital camera, such as the Alpha red Imager (Cell Biosciences, Inc.), and capture a digital photograph of the gel.

5-15
(3) After the gel scanning is done, properly label file name and save the file on the hard drive immediately.
(4) Print out a picture of the gel image and insert into lab book. A copy should be kept with the Project Lab Book.

Purpose
Once a gel is visualized, the quality of the results and presence of potential positive bands must be assessed in order to determine which samples need to be further assayed.

Source
Immediately following the cessation of electrophoresis, the agarose gel containing the eDNA PCR products should have been visualized on either the UV-based imager or the laser-based imager. In both cases, the gel image should be captured (saved to hard disk) immediately.

Gel Interpretation Quality Assurance and Chain of Custody
(1) The three positive controls should have bright bands at the appropriate migration distance (number of base pairs), indicating a positive reaction.
(2) No bands at the targeted sizes (~200bp silver, ~300bp bighead) should be observed in the negative controls.
(3) If any of the initial PCR reactions are positive (i.e., a visible band at the appropriate migration distance), the initial sample is designated a "presumptive positive".
(4) Record the number of presumptive positive reactions for each sample both in the gel electrophoresis notebook and the personal laboratory notebook of the researcher.
(5) Presumptive positive results will initiate a series of results confirmation mechanisms (see below). These mechanisms include screening the transport and equipment controls, and DNA sequencing.

Purpose
These confirmation mechanisms are initiated if a sample returns as a positive for the PCR test (any number of the eight reactions, e.g., one of eight up to eight of the eight PCR reactions).

Source
Positive results for Asian carp eDNA require that those positive samples be further assayed. The original DNA elutes from samples should be located in designated −20°C freezer.

Gel Interpretation Assurance and Chain of Custody
Positive assays (PCR reactions) are validated through DNA sequencing and testing of additional control samples.
Any revisions to the DNA QA/QC amplification protocol must be approved by the eDNA Processing Leader and approved by the assigned LRD senior executive. Any such changes should be incorporated into a revised QAPP.

Procedure
(1) Conduct PCR assays of the paired equipment control for each presumptive positive. DNA extraction, amplification, documentation, and interpretation following protocols detailed above (Sections 5. 6-5.9).
(2) Ensure that the transport blanks (see 2.2.2 (6) and 2.3.2 (10)) have been tested for that sample group (i.e., from the same cooler in which the presumptive positive sample was transported).
(3) If the equipment control and transport blanks test negative, the sample is designated a "confirmed positive." (4) All positive samples and for samples testing positive from ecologically sensitive areas (to be determined by Sampling Lead), bidirectional sequencing confirmation is performed. This is done by using a commercially available gel extraction kit (e.g., Qiagen Qiaquick Gel Extraction kit) per the manufacturer's recommendations on the positive PCR reactions, or E-Gel® CloneWell Agarose Gels (Invitrogen) per manufacturer's recommendation. Samples in Hi-Dye can be stored at 4°C overnight, but may not be left any longer than 12 hours.
(7) To sequence: • Denature samples for 5 min at 95°C • Place immediately on ice • Load into sequencer plate and onto sequencer.
Resulting sequencing reactions that are successful are screened in GenBank (http://www.ncbi.nlm.nih.gov/blast) using the BLAST (Basic Local Alignment Search Tool) algorithm. If the resulting sequence is a positive match to the targeted species of Asian carp, the sample is designated a "confirmed positive -sequenced".

Purpose
To convey to LRD designated personnel the progress and results of eDNA assays.

Source
ERDC keeps a record of each samples progress through the EDNA assay procedure. These records are summarized for each batch for reporting to USACE.

Quality Control
The ERDC eDNA Team designates one of its personnel to be the Progress & Results Reporter (PRR). The PRR should provide updates and reports to the LRD designated Point of Contact for eDNA results. At this time, Matt Carr is the ERDC eDNA Team PRR designee. Kelly Baerwaldt is the LRD POC designee. Any permanent or temporary changes to either position should be communicated immediately to the eDNA Processing Leader and the assigned LRD senior executive. Any permanent changes should be incorporated into a revised QAPP.
Any revisions to the reporting procedures must be approved by the eDNA Processing Leader and approved by the assigned LRD senior executive. Any such changes should be incorporated into a revised QAPP.

Procedure
The ERDC PRR should, on every Friday during the period over which the ERDC eDNA Team is processing samples, provide updates on all sample batches to the LRD POC no later than 1200 CST/CDT. Reports should be organized by batch and should consist of spreadsheets showing the stage of processing for each sample. Additionally, following approval by the eDNA Processing Leader, the ERDC PRR should convey final results for each batch (= all samples confirmed as positive or negative for AC eDNA) within 18 hours of completion of processing for the last sample within a batch.
The LRD POC may request updates from the PRR at any point. The ERDC PRR is expected to respond as soon as possible during normal working hours.

INTERNAL QUALITY CONTROL CHECKS
Details on quality control are found within each of the various protocol sections. In summary, however, quality control relative to sample contamination is covered by the transport (or cooler), equipment, DNA extraction, PCR, and sequencing blanks. Quality control for efficacy of methodology, solutions, etc. is covered by positive DNA controls for each sample handling step (sampling, filtering, extraction, PCR, and sequencing) of the eDNA protocol. Furthermore, each new solution or kit to be used in eDNA processing will be tested with positive and negative controls before use.
The ERDC eDNA processing facilities and protocols will be reviewed by an EPA audit shortly after completion of the QAPP and full deployment of Asian carp eDNA dedicated equipment.

Laboratory Quality Control Evaluation Criteria
Quality control is measured in two ways: • If transport, filtering, extraction, PCR, and DNA sequencing negative controls show product (e.g., bands in PCR or DNA sequence), the associated data are negated and, when possible, samples are reprocessed. Contamination of DNA extract will require that samples be removed from consideration. • Positive controls are currently employed for extraction, PCR, and sequencing. If the positive controls fail to behave as expected, any sample showing an apparent lack of results will be rerun at the same time or following rerunning of the positive controls. This will be done until all positive controls produce the expected results. • We incorporate two types of positive controls during sequencing. One positive control PCR products from positive control reactions and several one sequencing reactions with a standard DNA sequencing template (pGem) provided by the manufacturer are sequenced with every set of 16 eDNA samples that are sequenced. In the case that any of these fail, any samples that fail to produce sequence data that were run at the same time will be rerun at the same time as positive controls are rerun. • In cases where fewer than 16 eDNA samples are sequenced, both types of positive sequence controls are still run. During each filtering event, two positive control samples, consisting of dilute solutions of carp scales and slime coat, will be pipetted into 2L aliquots of clean water and filtered according to filtering protocols. Filters will be sent to ERDC for processing. ERDC will report results of PCR reactions from filtering positive controls.

Laboratory Data
Every 3 months, a dilution series of Asian carp DNA will be processed from extraction through sequencing to ensure that current practice, instruments, and personnel are maintaining the same level of sensitivity and accuracy. A brief report will be provided by the DNA Processing Lead to the eDNA Program Manager. 8-1

SECTION 8 8. CORRECTIVE ACTIONS
Corrective actions may be required for two classes of problems: analytical/equipment problems and noncompliance problems. Analytical and equipment-related problems may develop during sampling and sample handling, sample preparation, laboratory instrumental analysis, and data review. Noncompliance issues arise when eDNA sampling, filtering, or processing execution deviates from procedures described in the QAPP.
In the case of analytical/equipment problems or deviations from set procedures (as outlined in QAPP), the responsible lead will determine if the problem or deviation will impact the accuracy of the resulting data. If it is determined that the problem or deviation does impact data accuracy, two courses of action may be followed: (1) If possible, the procedure is repeated until it is performed without any problem or deviation, or (2) The sample or samples are removed and not processed any further.
In either case, a corrective action report must be completed. Careful notes of any corrective actions and what incident led to them, as well as the resolution or preventative measure(s) identified will be carefully noted in the corrective action report, which must be provided electronically to all Leaders (Project Leader, eDNA Program Manager, etc) as an after action report. The paper copy of the corrective action report will be maintained in the project file as a long-term record.
In the case that the responsible lead determines that data accuracy is not affected by the analytical/equipment problem or deviation from procedure, the sample or samples may continue to be processed. The responsible lead will make careful note of the incident in project records and include the rationale for continuing processing.

Field Equipment/Instruments
Hand-held sonar: Batteries will be changed at least once a month (if not required sooner) to ensure accurate readings of the instrument. In addition, reading accuracy should be checked once a month. Depth readings may be checked by filling a container of a known depth with water and submerging the instrumentation. Temperature readings of the sonar may be checked against a thermometer.
GPS equipment: Batteries will be changed at least once a month (if not required sooner) to ensure accurate readings of the instrument. In addition, coordinate accuracy will be checked against known benchmarks.
Plastic 2L sample bottles: After autoclaving, bottles will be inspected for dents and/or warping of the material. Any bottle failing inspection will be disposed of and replaced.
Forceps: Forceps will be inspected monthly, and those exhibiting large amounts of rust will be disposed of and replaced.
Carboys: Carboys will be inspected monthly for cracks in the glass that could pose a safety hazard to filtering personnel. Any carboy failing inspection will be disposed of and replaced.
Plastic tubing: Plastic tubing used to connect the carboy to the manifold will be inspected monthly for cracks in the plastic. Any plastic tubing failing inspection will be disposed of and replaced.
All other laboratory equipment will be inspected monthly and undergo proper maintenance to maintain their ideal working condition. Any equipment not performing accurately or to established standards will be disposed of and replaced.

Laboratory Instruments
Pipettes: Annually all pipettes will be inspected, calibrated, and certified. Any pipette failing inspection and certification will be disposed and replaced.
Any thermal-cycler head that fails the manufacturers self-test upon instrument startup will be removed and replaced with the manufacturer's certified replacement part.
Equipment maintenance contracts, with annual maintenance check-ups, will be used for any appropriate equipment (i.e., DNA sequencer).

Field Audits
Internal audits of field crew performance and quality controls may be made semi-annually by the EDNA Program Manager to make sure that all procedures in the sample collection portions of the QAPP are being followed. A brief report will be made to the eDNA Program Manager of audit findings, including a signed checklist of audited procedures.

Laboratory Audits
Internal audits of USACE laboratory performance and quality controls will be made semi-annually by the Filtering Leader to make sure that all procedures in the sample filtering portions of the QAPP are being followed. A brief report will be made to the eDNA Program Manager of audit findings, including a signed checklist of audited procedures.
Internal audits of ERDC laboratory performance and quality controls will be made semi-annually by the DNA Processing Lead to make sure that all procedures in the DNA processing portions of the QAPP are being followed. A brief report will be made to the eDNA Program Manager of audit findings, including a signed checklist of audited procedures. Every 2 years an external review of ERDC eDNA processing will be Refrigerator Temperature Log   To date, no relationship between the number of positive detections and Asian carp population abundance has been established, therefore eDNA results should be interpreted with caution. Additional research on the calibration of the eDNA method has been occurring since 2010; however, the full results of this multi-agency study will not be known until 2013. Until completion of this additional research to calibrate eDNA results and assess potential alternative sources of DNA in the waterway, the MRRWG views positive eDNA results as an indicator of the possible presence of live Asian carp. When viewed over the long term (e.g., multiple positive hits on consecutive sample dates at the same location), these data will be used to guide decisions on the location and timing of targeted rapid response removal actions.
Methods: Standard operating procedures have been outlined in the eDNA Quality Assurance Project Plan (USACE 2011) and were reviewed and agreed upon by all partnering agencies (e.g. USACE, USFWS, and IDNR). In general, IDNR and USFWS will collect 60 water samples on a bi-monthly basis from a specified reach on Monday or Tuesday. Samples will be transferred to USACE biologists at the USEPA laboratory in Chicago where they will be filtered and preserved in a -20ºC freezer. Preserved samples will be shipped overnight to the ERDC laboratory for analysis. Results will be posted on a USACE web site after analysis of each sampling event is complete (approximately 14 days). A general description of the eDNA sample collection method is given below. Detailed field, laboratory, and reporting protocols are available in the eDNA Quality Assurance Project Plan (USACE 2011).
Locations-Samples will be collected every two weeks from late May through October (weather permitting) such that Lake Calumet and each partial barrier to Lake Michigan are sampled once every 30 days (N= 114 samples and 6 cooler blanks bi-monthly; Figure 4). Sample locations were selected based on habitat thought to be preferred by Asian carp (Lake Calumet) and entry points to Lake Michigan (North Shore Channel downstream from Wilmette Pumping Station, Chicago River downstream from Chicago Lock, and Little Calumet River downstream from T. J. O"Brien Lock and Dam. Sampling is complementary to fixed site sampling conducted with conventional gears in the locations listed below. North Shore Channel (60 samples) and South Branch Chicago River to the Chicago Lock (60 samples) Little Calumet River downstream of O"Brien Lock (60 samples) and Lake Calumet (60 samples) Paired sampling stations will be sampled in the same day once every other week so that all four stations will be sampled every 30 days. An additional 480 samples will be available for the 2012 field season. These samples will be used for sampling associated with rapid response actions or to monitor the Upper Des Plaines River, CSSC and Calumet-Sag Channel confluence, the CSSC upstream and downstream of the Dispersal Barrier, or other locations determined to be strategically important (e.g., re-sampling a site with previous positive detections for Asian carp DNA). The USACE will provide aerial site maps with specific sampling locations for each sample 1-2 weeks prior to each sampling event. In addition, sampling maps will be provided with the coolers upon pick-up from the USEPA laboratory, as well as a box of nitrile gloves, datasheets, COC form, handheld depth sonar, and sprayer with 10% bleach solution. The proposed strategy allows for eDNA sampling to take place in support of conventional gear or rotenone rapid response actions or other evaluations that might occur at locations other than those identified above. Highest sampling priority has been set for barriers to Lake Michigan (e.g. Wilmette Control Works, Chicago Lock and O"Brien Lock) and Lake Calumet. Sampling priority for the additional 480 samples that are not part of regular fixed site sampling will be directed by the MRRWG, and may include: 1) Rapid Response Action sites 2) CSSC upstream and downstream of the Dispersal Barrier; 3) CSSC and Calumet-Sag Channel confluence; and 4) upper Des Plaines River. A minimum of 60 samples is recommended for each eDNA sampling event to reduce the probability of obtaining false negative results. Changes to the sampling frequency and/or procedures may be made by the MRRWG, as needed to minimize the risk of Asian carp entering the CAWS upstream of the barrier.
eDNA Sample Collection Protocol. 1) Sampling will be cancelled or postponed due to contamination concerns if a combined sewer overflow (CSO) occurs two days prior to sampling and/or if observed precipitation exceeds 1.5 inches in 24 hours five days prior to sampling. Sample crews will be notified as soon as possible of a cancellation. 2) The sampling boat and transport trailer must be disinfected prior to launching by spraying the outer surfaces (i.e. hull, motor, etc.) with a hand-held sprayer containing a prepared 10% bleach/water solution. 3) Prior to launch, crew members will be given their specific duties for the sampling trip.
One crew member will be designated as the boat operator and will be in charge of driving the vessel to sample locations. A second crew member will be designated as the lead sampler and will be in charge of collecting all water samples and measuring water depth and temperature. A third crew member will record GPS location (decimal degrees) and habitat measurements for each water sample on a datasheet. 4) Sampling will begin at the first transect located at the DOWNSTREAM end of the reach to be sampled and will proceed in an UPSTREAM direction. 5) When arriving at a sample site, the lead sampler will put on sterile exam gloves (powderless latex or nitrile). 6) Going in consecutive order, the lead sampler will remove a labeled 2L sample bottle from the sample cooler. 7) Just prior to collecting the sample, the lead sampler will unscrew and remove the lid from the sample bottle. 8) The lead sampler will then reach over the upstream side or the bow of the boat with the 2L sample bottle and fill the bottle by skimming the water surface. The sample bottle should not be submerged or dipped beyond the upper 2 inches of the surface water for sample collection. 9) Once the sample bottle is completely filled (approximately 1 inch of space should be left within the sample bottle) the lead sampler will screw the lid back on to the bottle until it is tight. The closed bottle should then be returned to the sample cooler from which it was removed. 10) The lead sampler will take a surface water temperature and depth measurement at the sample site. The data recorder will record the bottle ID number, GPS location (decimal degrees), time of sample, water temperature, and water depth on the data sheet. 11) If the lead sampler pulls a transport blank (2L of DI water filled prior to trip) from the cooler, the sampler will unscrew and remove the lid to expose the bottles contents to the atmosphere for 5 seconds, reseal the bottle, fully submerge the bottle in the field water, and return the bottle to the cooler from which it was removed. The lead sampler should