TITLE: Aquatic eDNA sampling and plant community metabarcoding with portable Nanopore Flongle sequencing (v0.0.3)
AUTHORS: Jordan Callahan, Robert Harbert

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Aquatic eDNA sampling, extraction, and plant metabarcoding on Oxford Nanopore's Flongle platform.</div><div class = "text-block">Sampling method adapted from:</div><div class = "text-block">Laramie, M.B., Pilliod, D.S., Goldberg, C.S. and Strickler, K.M., 2015. Environmental DNA sampling protocol-filtering water to capture DNA from aquatic organisms (No. 2-A13). US Geological Survey. https://pubs.er.usgs.gov/publication/tm2A13 </div><div class = "text-block">Extraction protocol nearly identical to Qiagen DNEasy PowerWater kit manual: </div><div class = "text-block">https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=2ahUKEwj4jpSKkqfnAhUjnOAKHdAbDQ8QFjABegQIBBAB&url=https%3A%2F%2Fwww.qiagen.com%2Fus%2Fresources%2Fdownload.aspx%3Fid%3Dbb731482-874b-4241-8cf4-c15054e3a4bf%26lang%3Den&usg=AOvVaw32l8w94T6GAZUI4TAyUyT1</div><div class = "text-block">Amplicon sequencing is a modified version of the PCR barcoding protocol from Oxford Nanopore for kit SQK-PBK004.</div></div>

[STEPS]
?. [Aquatic eDNA sampling]
Assemble hose ends and drill pump and attach to wooden bracket with zip ties. Lock drill driver to pump spindle.
?. [Aquatic eDNA sampling]
Attach fresh, sterile 0.045 μm filter funnel assembly (Fisher Sci catalog #09 740 30K) to hose on the “In” side of the pump and tighten hose clamp. Be sure to leave the filter chamber cover on.
?. [Aquatic eDNA sampling]
Filter water samples with the drill at ¾ to maximum drive (max = 1,700RPM) for  . STOP if the filter becomes clogged and no water is observed leaving the outlet hose. Refill the filter reservoir as needed. The volume of water filtered will depend on the microbial load and turbidity of the water sample.
?. [Aquatic eDNA sampling]
When filtering is complete, switch to clean gloves and change gloves if they contact anything other than the filter assembly or the water being sampled.
?. [Aquatic eDNA sampling]
Remove the upper portion of the filter assembly.
?. [Aquatic eDNA sampling]
Using two sets of sterile forceps, pick up the white filter membrane at opposite edges and roll the filter into a cylinder with the top side facing inwards.
?. [Aquatic eDNA sampling]
Place in fresh 15mL Falcon tube and freeze at -20C (or in 200 proof molecular grade Ethanol) until ready to extract DNA.
?. [Aquatic eDNA sampling]
Label Tubes with location, date, time, and duration of sampling.
?. [Aquatic eDNA sampling]
Record data (Assign locality name, GPS location, water temperature, sampling duration, and any other observations) in the field notebook.
?. [Aquatic eDNA sampling]
Mark sample site with flagging tape if you plan to resample the same location. Label flagging tape with sample date, locality name, and email contact information.
?. [DNA Extraction]
Place all pipettes and tools needed inside of the UV box. Turn on the UV light source to treat tools and workspace. After this time ONLY open tubes with eDNA inside of this box.
?. [DNA Extraction]
Next steps use materials from the DNeasy PowerWater kit. Check for:    Solutions -- PW1, IRS, PW3, PW4, Ethanol, and EB.    Collection  tubes (2mL)    Bead tubes (5mL)    MB Spin columns + tubesWipe down all closed reagent bottles with 50% bleach solution and place in a designated area of UV box.
?. [DNA Extraction]
Using bleach cleaned forceps (spray with 50% bleach and leave for ), insert the filter membrane into a 5mL PowerWater DNA bead tube. (included in kit).
?. [DNA Extraction]
Add  of Solution PW1 to the  PowerWater DNA bead tube
1 ml
?. [DNA Extraction]
Secure tubes to a rack with tape. Secure rack to shaker platform with tubes horizontal.
?. [DNA Extraction]
Shake at >=500rpm for
?. [DNA Extraction]
Centrifuge the tubes ≤ 4000 x g for   at room temperature. (According to the manufacturer: "This centrifugation step is optional if a centrifuge with a 15 ml tube rotor is not available, but will result in minor loss of supernatant").
?. [DNA Extraction]
Transfer the supernatant to a clean 2 ml collection tube (included in kit). Note: placing the pipette tip down into the beads is required. Pipette until you have removed all the supernatant. Expect to recover 600-650 μL of supernatant (DNA is in supernatant).
?. [DNA Extraction]
Centrifuge at 13,000 x g for   at room temperature.
?. [DNA Extraction]
Avoiding the pellet, transfer the supernatant to a clean 2 ml collection tube (included in kit). (DNA is in supernatant).
?. [DNA Extraction]
Add   of Solution IRS (15 ml included in kit) and vortex briefly to mix.
200 µl
?. [DNA Extraction]
Incubate at   for
on ice
?. [DNA Extraction]
Centrifuge the tubes at 13,000 x g for
?. [DNA Extraction]
Avoiding the pellet, transfer the supernatant to a clean 2 ml collection tube (included). (DNA is in supernatant).
?. [DNA Extraction]
Add  of Solution PW3 and vortex briefly to mix. NOTE: If solution PW3 has precipitated, heat at 55 °C for 5-10 minutes to dissolve precipitate.
650 µl
?. [DNA Extraction]
Load   of supernatant onto a MB Spin Column (included in kit). Centrifuge at 13,000 x g for  . Discard the flow-through. (DNA is in Column). Repeat until all the supernatant has been processed.
650 µl
?. [DNA Extraction]
Place the MB Spin Column Filter into a clean 2 ml collection tube (included) (DNA is on filter at this point).
?. [DNA Extraction]
Shake Solution PW4.Add  of Solution PW4 (shake before use) to the MB Spin Column. Centrifuge at 13,000 x g for  .
650 µl
?. [DNA Extraction]
Discard the flow-through and add   of ethanol (included) and centrifuge at 13,000 x g for  . (DNA is on filter).
650 µl
?. [DNA Extraction]
Discard the flow through and centrifuge again at 13,000 x g for  . (DNA is on the filter still, simply washed by ethanol and PW4).
?. [DNA Extraction]
Place the MB Spin Column into a clean 2 ml collection tube (included).
?. [DNA Extraction]
Add   of Solution EB (10mM Tris-HCl pH 8.5) to the center of the white filter membrane. (DNA is released from the filter).
100 µl
?. [DNA Extraction]
Centrifuge at 13,000 x g for  . (DNA is in eluate).
?. [DNA Extraction]
Discard the MB Spin Column. Cap and label collection tubes.The DNA is now ready for downstream applications.
?. [DNA Quantification (OPTIONAL but recommended)]
Qubit Quantification using Qubit Fluorometer and dsDNA HS Assay Kit. Prepare:n = # samples + 2Working solution --  dsDNA HS Buffer +   dsDNA HS ReagentAssay:Mix  and  each DNA sample in separate Qubit tubesMix  and  Standard #1 in 0.5mL TubeMix  and   Standard #2 in 0.5mL TubeWait: Incubate assay reactions Set Standards: Follow Qubit Fluorometer on-screen instructions to calibrate using Standard 1 and then 2Read: Place each sample in Qubit and press "Read", record concentration.
[per n]
[per n]
[working solution]
2 µl
[working solution]
10 µl
[working solution]
10 µl
[at room temperature]
?. [PCR Step 1: Target Amplification]
Prepare on ice:Template DNAForward and Reverse PrimersOneTaq Hot Start 2x Master Mix w/Standard bufferNuclease free water
?. [PCR Step 1: Target Amplification]
Set Up Reactions in 0.5mL PCR LoBind tubes. Add Template DNA Last.Forward Primer                                      of  stockReverse Primer                                       of  stockOneTaq Hot Start Master Mix            Nuclease Free Water                            Template DNA                                       _______________________________________________Total:
0.5 µl
0.5 µl
12.5 µl
9.5 µl
2 µl
25 µl
?. [PCR Step 1: Target Amplification]
PCR Cycling conditions:If using MiniPCR use library:  and protocol "NanoporePCR ampl target"Cycle Step                        Temp.                Time                             # of CyclesInitial Denaturation                       1Step 1                                                                                                         5Denaturation                                       --Annealing                                           --Extension                              --Step 2                                                                                                        30Denaturation                                     --Annealing                                          --Extension                              --Final Extension                                 1*Cool to
94 °C
94 °C
60 °C
65 °C
[per kb]
94 °C
62 °C
65 °C
[per kb]
65 °C
0 Room temperature
?. [PCR Step 1: Target Amplification]
Store at   until ready for PCR Step 2
4 °C
?. [PCR Step 2: Barcode Addition]
Prepare on ice:Template DNA (PCR Product from Step 1)Nanopore PCR Barcoding Kit (SQK-PBK004) primers LWB01-LWB12OneTaq Hot Start 2x Master Mix w/Standard bufferNuclease free water
?. [PCR Step 2: Barcode Addition]
Set Up Reactions in 0.5mL PCR LoBind tubes. Add Template DNA Last.LWB# Primer mix                                  OneTaq Hot Start Master Mix            Nuclease Free Water                            Template DNA                                       _______________________________________________Total:
0.75 µl
12.5 µl
9.75 µl
2 µl
25 µl
?. [PCR Step 2: Barcode Addition]
PCR Cycling conditions:If using MiniPCR use library:  and protocol "NanoporePCR add barcodes"Cycle Step                        Temp.                Time                             # of CyclesInitial Denaturation                           1Amplification                                                                                             35Denaturation                                       --Annealing                                           --Extension                              --Final Extension                                 1*Cool to
94 °C
94 °C
60 °C
65 °C
[per kb]
65 °C
0 Room temperature
?. [PCR Step 2: Barcode Addition]
Store at   until ready to proceed to clean-up section.
4 °C
?. [Clean-Up: Ampure XP Beads]
Prepare   of fresh   ethanol in nuclease-free water per ~10 samples. Keep .
5 ml
on ice
?. [Clean-Up: Ampure XP Beads]
Prepare the AMPure XP beads for use; resuspend by vortexing. Keep
on ice
?. [Clean-Up: Ampure XP Beads]
Prepare Elution Buffer --    Tris-HCl pH 8.0 w/   NaCl as:  Tris-HCl pH 8.0   5M NaClinto a 1.5ml tube and add  of nuclease-free water.
1500 µl
15 µl
15 µl
1470 µl
?. [Clean-Up: Ampure XP Beads]
Transfer the PCR products to 1.5mL Eppendorf LoBind tubes
?. [Clean-Up: Ampure XP Beads]
Add  of resuspended AMPure XP beads to the PCR products and mix by flicking the tube. If using PCR reaction volumes other than  use this equation to determine how much of the beads to dispense: (volume of beads pr rxn) = 1.8 x (reaction volume). See below --Common Sample:Bead volume ratios for 200bp+ size selection:  ABCDE1Volume of PCR product25ul22ul20ul50ul2AMPure XP Beads41ul39.6ul36ul90ul
41 µl
25 µl
ABCDE1Volume of PCR product25ul22ul20ul50ul2AMPure XP Beads41ul39.6ul36ul90ul
?. [Clean-Up: Ampure XP Beads]
Incubate for  at
0 Room temperature
?. [Clean-Up: Ampure XP Beads]
Place samples on magnetic rack for  or until solution becomes clear.
?. [Clean-Up: Ampure XP Beads]
Keep the tubes in the magnetic rack and pipette off the supernatant. (DNA of length > ~200bp is bound to beads)
?. [Clean-Up: Ampure XP Beads]
Keep on magnet, wash beads with   of cold  ethanol without disturbing the pellet and incubate for Remove the ethanol using a pipette and discard.
200 µl
?. [Clean-Up: Ampure XP Beads]
Repeat the previous step for a total of two washes.
?. [Clean-Up: Ampure XP Beads]
Dry tubes (open) while still on the magnetic rack for
?. [Clean-Up: Ampure XP Beads]
Remove the tube from the magnetic rack and resuspend pellet in  of Elution Buffer. Flick tube to mix and incubated for at room temperature.
40 µl
?. [Clean-Up: Ampure XP Beads]
Place tubes back in magnetic rack until the eluate is clear and colorless, or about
?. [Clean-Up: Ampure XP Beads]
Remove and retain   of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
40 µl
?. [Clean-Up: Ampure XP Beads]
Quantify concentration of eluted sample using a Qubit fluorometer.
?. [Nanopore Flongle LIbrary Prep and Sequencing]
Prepare on ice:Amplified and barcoded DNA samples (up to 12)From PCR Barcoding Kit--    RAP    FLT    SQB    FB    LBNuclease Free Water
?. [Nanopore Flongle LIbrary Prep and Sequencing]
Transfer  of each sample into ONE 0.5ml PCR tube (if final concentrations are ). [OR: Pool in equimolar ratios ~50-100 fmol of total DNA into  ]
0.5 µl
5 µl
?. [Nanopore Flongle LIbrary Prep and Sequencing]
Add  RAP. Incubate at  for  .
0.5 µl
0 Room temperature
?. [Nanopore Flongle LIbrary Prep and Sequencing]
Place library
on ice
?. [Nanopore Flongle LIbrary Prep and Sequencing]
Prepare Flush Buffer.Mix  FB with  FLTKeep
117 µl
3 µl
on ice
?. [Nanopore Flongle LIbrary Prep and Sequencing]
Open MinKNOW software, install flowcell in MinION, and run flowcell check per manufacturer's instructions. Ensure that the flowcell has sufficient pores for sequencing and all other checks pass.
?. [Nanopore Flongle LIbrary Prep and Sequencing]
Prepare Library.To the   pooled, RAP treated DNA add: SQB LB (Mix loading beads by pipetting up and down just before measuring this volume)Total library volume should be
5 µl
13.5 µl
11 µl
29.5 µl
?. [Nanopore Flongle LIbrary Prep and Sequencing]
Prime Flowcell.Open the access to the Flongle loading port by peeling back the tape seal. Stick the tape to the lid of the MinION.Measure   of the priming buffer (FB/FLT mix) in a P200 pipette.Position the pipette tip perpendicular to the flowcell and firmly in contact with the open port directly above the window into the pore array. Visually inspect the pipette tip and port for bubbles. Start over with a new tip if  bubbles are observed.Slowly add the priming buffer over 5-10 seconds.
100 µl
?. [Nanopore Flongle LIbrary Prep and Sequencing]
Loading the sampleMix the library by gently flicking the tube.Measure  of the library using a P200 pipette.Slowly add the library via the loading port just as with the priming buffer. Loading should  take 3-5 seconds.
29 µl
?. [Nanopore Flongle LIbrary Prep and Sequencing]
Replace the tape to cover the loading port and seal. Close the MinION lid.
?. [Nanopore Flongle LIbrary Prep and Sequencing]
Start Sequencing Run