TITLE: Fish eDNA: water sampling and filtration through Sterivex filter unit
AUTHORS: Marine  Vautier, Cécile Chardon, Isabelle Domaizon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The  objective of this protocol is the sampling and the filtration of water samples  through 0.45 µm Sterivex™ filter units, and their buffer preservation prior to DNA extraction. This protocol is used upstream to molecular biology analysis (e.g. qPCR, metabarcoding, ddPCR) to specifically target fish eDNA.</div><div class = "text-block">The use of preservation buffer allows sample preservation for up to several hours at room temperature, several days at 4°C and several months at -20°C, and thus the possibility to filter samples on site.</div><div class = "text-block">This protocol allows to filter approximately between 0.7 L and 5 L through a Sterivex™ filter unit depending on the water sample characteristics and the chosen filtration method (syringe or vacuum pump).</div></div>

[STEPS]
?. [WATER SAMPLING]
Put on gloves and open the  bottle without touching the inside of the cap or the neck of the bottle.Collect by hand subsurface water (10-20 cm below the water surface) and close the bottle carefully. If water is collected deeper with a sampling tank, it must also be decontaminated prior to sampling.After water collection, the bottle has to be stored in cleaned containers to avoid contamination. Use a cooler if the samples have to be transported. Collect by hand subsurface water (10-20 cm below the water surface) and close the bottle carefully. If water is collected deeper with a sampling tank, it must also be decontaminated prior to sampling.After water collection, the bottle has to be stored in cleaned containers to avoid contamination. Use a cooler if the samples have to be transported. After water collection, the bottle has to be stored in cleaned containers to avoid contamination. Use a cooler if the samples have to be transported.
?. [WATER FILTRATION]
Proceed rapidly to water filtration in order to minimize any potential DNA damage during transport. Place the water at  in the dark for max. 6 hours until filtration.
4 °C
?. [WATER FILTRATION]
Filtration procedure with a syringe : The set-up is easy and does not necessarily require countertop space (can be done on the field). Get the filtration material ready to use: gloves, Sterivex cartridge, caps and syringe.Put on gloves and remove the syringe and the Sterivex from their blister pack (handle carefully avoiding to touch either end of the filter to prevent contamination)Homogenize gently the water sample & fill the syringe (60mL) with water sample (verify that the neck of the bottle is larger than the syringe’s diameter) Connect the syringe and the Sterivex together (Figure 1)    Filter (slowly) the water sample through the Sterivex unit Detach the syringe from the Sterivex to refill the syringe with water and filter it through the same Sterivex.Continue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final water volume filtered (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, detach the Sterivex from the syringe, and remove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Homogenize gently the water sample & fill the syringe (60mL) with water sample (verify that the neck of the bottle is larger than the syringe’s diameter) Connect the syringe and the Sterivex together (Figure 1)    Filter (slowly) the water sample through the Sterivex unit Detach the syringe from the Sterivex to refill the syringe with water and filter it through the same Sterivex.Continue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final water volume filtered (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, detach the Sterivex from the syringe, and remove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Connect the syringe and the Sterivex together (Figure 1)    Filter (slowly) the water sample through the Sterivex unit Detach the syringe from the Sterivex to refill the syringe with water and filter it through the same Sterivex.Continue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final water volume filtered (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, detach the Sterivex from the syringe, and remove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Filter (slowly) the water sample through the Sterivex unit Detach the syringe from the Sterivex to refill the syringe with water and filter it through the same Sterivex.Continue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final water volume filtered (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, detach the Sterivex from the syringe, and remove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Detach the syringe from the Sterivex to refill the syringe with water and filter it through the same Sterivex.Continue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final water volume filtered (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, detach the Sterivex from the syringe, and remove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Continue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final water volume filtered (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, detach the Sterivex from the syringe, and remove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)At the end of the filtration, detach the Sterivex from the syringe, and remove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)
?. [WATER FILTRATION]
Filtration procedure with a vacuum pump : The set-up does not necessarily require countertop space, but is  easier to set up in a laboratory. Get the filtration material ready to use: vacuum pump, tubing, filter ramp adapted to the size of the Sterivex, gloves, Sterivex cartridge, caps and syringePut on gloves and remove the Sterivex from their blister pack (handle carefully avoiding to touch either end of the filter to prevent contamination)Insert the Male Luer-lock Outlet tip of the Sterivex (Figure 2) into the clean silicone filter support holder (Figure 3).  Insert the clean silicone filter support holder onto the filtration ramp (Figure 3)Remove the 50 mL syringe from its blister pack and remove the plunger from the syringe to use the body of the syringe as a funnel (keep the plunger in its blister pack to use it at the end of the filtration process).Connect the body of the syringe to the Female Luer-lock Inlet tip of the Sterivex (Figure 2 & 3).Homogenize gently the water sampleFill slowly the syringe with the water directly from the bottle as if it were a funnel (Figure 3) Switch on the pump and open the valves (the pressure must not exceed 400mBa (40kPa)Fill the syringe body (funnel) to ensure that there is always water in itContinue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final volume of filtered water (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, disconnect the Sterivex from silicone filter support holderRemove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Insert the Male Luer-lock Outlet tip of the Sterivex (Figure 2) into the clean silicone filter support holder (Figure 3).  Insert the clean silicone filter support holder onto the filtration ramp (Figure 3)Remove the 50 mL syringe from its blister pack and remove the plunger from the syringe to use the body of the syringe as a funnel (keep the plunger in its blister pack to use it at the end of the filtration process).Connect the body of the syringe to the Female Luer-lock Inlet tip of the Sterivex (Figure 2 & 3).Homogenize gently the water sampleFill slowly the syringe with the water directly from the bottle as if it were a funnel (Figure 3) Switch on the pump and open the valves (the pressure must not exceed 400mBa (40kPa)Fill the syringe body (funnel) to ensure that there is always water in itContinue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final volume of filtered water (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, disconnect the Sterivex from silicone filter support holderRemove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Insert the clean silicone filter support holder onto the filtration ramp (Figure 3)Remove the 50 mL syringe from its blister pack and remove the plunger from the syringe to use the body of the syringe as a funnel (keep the plunger in its blister pack to use it at the end of the filtration process).Connect the body of the syringe to the Female Luer-lock Inlet tip of the Sterivex (Figure 2 & 3).Homogenize gently the water sampleFill slowly the syringe with the water directly from the bottle as if it were a funnel (Figure 3) Switch on the pump and open the valves (the pressure must not exceed 400mBa (40kPa)Fill the syringe body (funnel) to ensure that there is always water in itContinue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final volume of filtered water (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, disconnect the Sterivex from silicone filter support holderRemove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Remove the 50 mL syringe from its blister pack and remove the plunger from the syringe to use the body of the syringe as a funnel (keep the plunger in its blister pack to use it at the end of the filtration process).Connect the body of the syringe to the Female Luer-lock Inlet tip of the Sterivex (Figure 2 & 3).Homogenize gently the water sampleFill slowly the syringe with the water directly from the bottle as if it were a funnel (Figure 3) Switch on the pump and open the valves (the pressure must not exceed 400mBa (40kPa)Fill the syringe body (funnel) to ensure that there is always water in itContinue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final volume of filtered water (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, disconnect the Sterivex from silicone filter support holderRemove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Connect the body of the syringe to the Female Luer-lock Inlet tip of the Sterivex (Figure 2 & 3).Homogenize gently the water sampleFill slowly the syringe with the water directly from the bottle as if it were a funnel (Figure 3) Switch on the pump and open the valves (the pressure must not exceed 400mBa (40kPa)Fill the syringe body (funnel) to ensure that there is always water in itContinue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final volume of filtered water (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, disconnect the Sterivex from silicone filter support holderRemove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Homogenize gently the water sampleFill slowly the syringe with the water directly from the bottle as if it were a funnel (Figure 3) Switch on the pump and open the valves (the pressure must not exceed 400mBa (40kPa)Fill the syringe body (funnel) to ensure that there is always water in itContinue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final volume of filtered water (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, disconnect the Sterivex from silicone filter support holderRemove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Fill slowly the syringe with the water directly from the bottle as if it were a funnel (Figure 3) Switch on the pump and open the valves (the pressure must not exceed 400mBa (40kPa)Fill the syringe body (funnel) to ensure that there is always water in itContinue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final volume of filtered water (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, disconnect the Sterivex from silicone filter support holderRemove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Switch on the pump and open the valves (the pressure must not exceed 400mBa (40kPa)Fill the syringe body (funnel) to ensure that there is always water in itContinue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final volume of filtered water (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, disconnect the Sterivex from silicone filter support holderRemove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Fill the syringe body (funnel) to ensure that there is always water in itContinue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final volume of filtered water (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, disconnect the Sterivex from silicone filter support holderRemove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Continue the filtration (with the same syringe and sterivex) to filter a maximum water volume and note the final volume of filtered water (by subtracting the remaining volume from the initial volume of water, for example). The filtration process should not take longer than 1 hour. Typical volumes filtered are from 0.5L to 5L : the volume of water filtered will depend on the microbial load and turbidity of the water sample.At the end of the filtration, disconnect the Sterivex from silicone filter support holderRemove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)At the end of the filtration, disconnect the Sterivex from silicone filter support holderRemove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)Remove as much of the remaining water as possible by inserting the plunger of the syringe and passing air through the SterivexProceed to sample preservation (step 3)
?. [Sample preservation]
Identical preservation method for both filtration systems : Get the preservation material ready to use: buffer, 1000µL pipette or 5 mL seringe,  indelible marker or pre-printed label, sterile bag (or tube) to store the Sterivex after filtration, field sheet and pen.Place the outlet cap on the Sterivex (Figure 2). Add immediately 2mL of buffer using a pipette (tip 1000µL) or a 5 mL seringue. Be careful to well insert the tip into the Sterivex to ensure the buffer does not overflow the cartridge (Figure 4). Caution: it is important not to put more than 2 mL of buffer into the Sterivex because reagents will be added during the DNA extraction step  Place the Inlet cap on the Sterivex (Figure 2).Place the labelled Sterivex in a clean bag/tube.Sterivex buffered can be stored at room temperature for several hours , or in a fridge or a cooler for few days. If a longer storage is needed, store it at -20°C.Complete the field sheet Add immediately 2mL of buffer using a pipette (tip 1000µL) or a 5 mL seringue. Be careful to well insert the tip into the Sterivex to ensure the buffer does not overflow the cartridge (Figure 4). Caution: it is important not to put more than 2 mL of buffer into the Sterivex because reagents will be added during the DNA extraction step  Place the Inlet cap on the Sterivex (Figure 2).Place the labelled Sterivex in a clean bag/tube.Sterivex buffered can be stored at room temperature for several hours , or in a fridge or a cooler for few days. If a longer storage is needed, store it at -20°C.Complete the field sheet Place the Inlet cap on the Sterivex (Figure 2).Place the labelled Sterivex in a clean bag/tube.Sterivex buffered can be stored at room temperature for several hours , or in a fridge or a cooler for few days. If a longer storage is needed, store it at -20°C.Complete the field sheet Place the labelled Sterivex in a clean bag/tube.Sterivex buffered can be stored at room temperature for several hours , or in a fridge or a cooler for few days. If a longer storage is needed, store it at -20°C.Complete the field sheet Sterivex buffered can be stored at room temperature for several hours , or in a fridge or a cooler for few days. If a longer storage is needed, store it at -20°C.Complete the field sheet