TITLE: Custom eDNA High Throughput Sequencing with MinION (Oxford Nanopore Technologies)
AUTHORS: Luca Mirimin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is a custom workflow to carry out High Throughput Sequencing (HTS) on environmental DNA samples using a MinION portable sequencer (Oxford Nanopore Technologies). The current protocol was tested to target fish-specific amplicons (340 and 1200 bp) in eDNA samples from both marine and freshwater environments.</div></div>

[STEPS]
?. [MATERIALS REQUIRED]
The enzyme and PCR mix used can be changed as long as the enzyme used is efficient and/or recommended for environmental samples. In this case we used Platinum™ II Taq Hot-Start DNA Polymerase (Invitrogen™)
?. [1-step PCR (amplicons and barcodes)]
[Includes experimental design planning, sample prep and PCR]
?. [PCR check, clean up and quantification]
This section aims at verifying that amplicons + barcoding libraries have been successfully generated and cleaned prior to the next section.
?. [INTRODUCTION]
This document provides instructions on carrying out the "Four-primer PCR protocol" for amplicon sequencing from eDNA samples using the PCR barcoding kit SQK-PBK004.The official version of this protocol is "Version: FFP_9038_v108_revP_14Aug2019" and can be found here (note that to access this protocol you will need to register for a Nanopore Community account).The general workflow of this protocol is shown below:Plan your experiment/run based on the goals of the study, keeping in mind the following:- This protocol is recommended (by the manufacturer) for 30ng of starting DNA per reaction, but it has worked for approx. 10ng. Thus, it can work with eDNA samples that contain low amounts of template DNA.- The barcoding kit used below is for 12 distinct libraries, but there are strategies to increase the throughput (e.g. wash and re-run the flow cell; Incorporate custom barcodes in amplicons; "multiplexing" multiple genes per sample).- Related to the above, keep in mind that reagents supplied by the manufacturer are meant for 12 samples/libraries with some excess), so this must be kept in mind when considering to increase throughput
?. [PCR check, clean up and quantification]
SUCCESSFUL AMPLICON/LIBRARY CHECKOnce the PCR has completed, load  of PCR product on a  Agarose Gel to confirm presence of bands in the expected size range. NOTE that the size of amplicons must take into account the addition of both ONT adapters and barcoding indexes, which are incorporated in the PCR step (e.g. add approx. 100bp to the original target size).
10 µl
?. [1-step PCR (amplicons and barcodes)]
Once an experimental plan has been established, prepare the following PCR mix. NOTE that the "Mastermix" recommended is for 3 replicate PCR per sample/library.Reagent name and starting concentrationAmount (µL) in each individual reactionMastermix to enable 3 PCR replicates per sample (3.5 X)Final concentration in each reactionWater,
  nuclease-freeup to 31.6110.6&nbsp;-5X Platinum™ II PCR Buffer10351X10 mM dNTP mix13.50.2 mM each10 µM forward primer0.06250.218750.0125 nM10 µM reverse primer0.06250.218750.0125 nM10 µM forward primer with ONT1 adapter0.18750.656250.0375 nM10 µM reverse primer ONT2 adapter0.18750.656250.0375 nMBPXX (SQK-PBK004)1.55.25 Platinum™ II Taq Hot-Start DNA Polymerase0.41.40.04 U/µLTemplate DNAup to 5 Up to 30 ng per reaction    Total volume50
Reagent name and starting concentrationAmount (µL) in each individual reactionMastermix to enable 3 PCR replicates per sample (3.5 X)Final concentration in each reactionWater,
  nuclease-freeup to 31.6110.6&nbsp;-5X Platinum™ II PCR Buffer10351X10 mM dNTP mix13.50.2 mM each10 µM forward primer0.06250.218750.0125 nM10 µM reverse primer0.06250.218750.0125 nM10 µM forward primer with ONT1 adapter0.18750.656250.0375 nM10 µM reverse primer ONT2 adapter0.18750.656250.0375 nMBPXX (SQK-PBK004)1.55.25 Platinum™ II Taq Hot-Start DNA Polymerase0.41.40.04 U/µLTemplate DNAup to 5 Up to 30 ng per reaction    Total volume50
?. [1-step PCR (amplicons and barcodes)]
THERMAL CYCLING CONDITIONSThe following conditions were adopted when using  and targeting 2 amplicon sizes (340bp and 1200bp).1X - 7x35x1x
94 °C
94 °C
52 °C
68 °C
94 °C
62 °C
68 °C
68 °C
?. [PCR check, clean up and quantification]
PCR CLEAN-UPOnce amplicons of expected size have been confirmed, pool  from each of the three PCR replicates (per library) and clean the mixture with  as follows:Transfer the  pooled PCR into a 1.5 ml DNA LoBind Eppendorf tube and add  of resuspended AMPure XP beads to the reaction and mix by flicking the tube.Incubate on a Hula mixer (rotator mixer) for  at room temperature.Briefly spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.Keep the tube on the magnet and wash the beads with  of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.Repeat the previous step. Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol and briefly allow to dry.Remove the tube from the magnetic rack and resuspend pellet in 10 µl of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl.Incubate for  at room temperature. Pellet beads on magnet until the eluate is clear and colourless.Remove and retain  of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
33.3 µl
100 µl
180 µl
300 µl
10 µl
?. [PCR check, clean up and quantification]
QUANTIFICATION AND NORMALIZATIONQuantify 1uL of purified PCR product using  in a Qubit Fluorometer 3.0.Using 10 mM Tris.HCl pH 8.0 with 50 mM NaCl as a solvent solution, dilute each PCR product to obtain a desired concentration before pooling all libraries in a clean 1.5 ml Eppendorf DNA LoBind tube and aiming for a final volume of 10uL and a total concentration of 100 fmol of PCR product to .
10 µl
At this stage purified PCR products can be stored at   overnight prior to continuing with the next step.
?. [PCR check, clean up and quantification]
ATTACHMENT OF RAPID 1D SEQUENCING ADAPTERSAdd  RAP reagent to the  amplified DNA library. Mix gently by flicking the tube, and briefly spin down.Incubate the reaction for 5 minutes at room temperature and keep on ice prior to flow cell loading.
This step must be done just prior to priming and loading the flow cell.
1 µl
10 µl
?. [PCR check, clean up and quantification]
Priming and loading the SpotON flow cellFollow the steps as detailed from Page 11 in the Nanopore Protocol Four-primer PCR (SQK-PSK004 or SQK-PBK004; Version: FFP_9038_v108_revP_14Aug2019.NOTE that it is recommended to run a flow cell check prior to carrying out the loading of the flow cell.