TITLE: Sample collection and DNA purification from virus-enriched tangential flow filtration retentates
AUTHORS: Paul  Den Uyl, Ed Delong

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To collect and concentrate viral particles, seawater samples were collected and prefiltered to remove bacterioplankton cells. The resulting virus-enriched filtrate was concentrated via tangential flow filtration (TFF) before DNA extraction. For each sample, 90 - 110 L of seawater was collected using a Niskin bottle rosette attached to a conductivity-‌temperature-‌‌‌‌depth (CTD) package. The seawater was pre-filtered by peristatic pumping through either a 0.22 um filter (Sterivex GV) for the 25 m (higher biomass) sample, or a 0.1 uM Supor cartridge filter (Acropak 500, Pall, USA) for the 117 m and 250 m (lower biomass) samples. The resulting virus-enriched filtrate was concentrated by tangential flow filtration (TFF) over a 30 kDa filter (Biomax 30 kDa membrane, catalogue #: P3B030D01, Millipore). Subsequently, the retentate was reduced to a volume of ~200 mL, the tangential flow filter was backflushed with 100 mL of permeate to release virus particles trapped in the filter, and the concentrated viruses were recovered in a final retentate volume of ~300 mL. The virus-containing retentates were stored at 4°C until final processing and DNA extraction.</div></div>

[STEPS]
?. [Pre-Cleaning]
Recirculate 0.1N Sodium Hydroxide at  feed speed for .
Centrifuge: 330 33
?. [Pre-Cleaning]
Flush with  MilliQ.
30 L
?. [Pre-Cleaning]
Drain the system (see sample recovery).
?. [Concentrating]
Fill retentate reservoir.
?. [Concentrating]
Attach retentate reservoir.
?. [Concentrating]
Connect feed line.
?. [Concentrating]
Attach permeate collection bottle.
?. [Concentrating]
Place sample tubing into external sample bottle.
Make sure this tubing extends to the bottom of the bottle. Attaching a serological pipet to the end of the tubing can help with this.
?. [Concentrating]
Connect retentate line.
?. [Concentrating]
Connect permeate line.
?. [Concentrating]
Place permate over-flow line into external collection/waste bottle.
?. [Concentrating]
Start the feed pump at around .
Centrifuge: 50 33
Make sure air is only coming out of the small retentate line inside the retentate bottle. This will ensure the tubing is connected properly, and that the feed line extends to the bottom of the bottle.
?. [Concentrating]
Gradually increase the feed pump to approximately . Gradually increasing the speed will help prevent high pressures while the tubing and filters are clearing themselves of air. Once at , additional air can be cleared by gently and quickly pinching the permeate and retentate lines (make sure not to exceed pressures of 5 psi on either the permeate/retentate gauges
Centrifuge: 330 33
Centrifuge: 330 33
?. [Concentrating]
Lower feed pump to .
Centrifuge: 100 33
?. [Concentrating]
Clamp permeate line into permeate pump.
?. [Concentrating]
Slowly increase feed pump until permeate backpressure guage reads 2-3 psi.
Now, as you continue to slowly increase the feed pump, also simultaneously increase the permeate pump (starting at  ), so that the permeate backpressure remains at 2-3 psi while increasing the feed pump. The goal is to increase the feed pump back up to 330 psi, while maintaining 2-3 psi of permeate backpressure. This back pressure will help prevent clogging while filtering. Make final adjustments so that feed pump is at approximately  , while permeate backpressure is at 3 psi (approximately  -  on the permeate pump). While filtering, the permeate backpressure may change. Adjust the permeate pump accordingly to maintain 3 psi.
?. [Filtration Completion]
Continue filtering until you feel you can no longer safely filter without accidentally introducing air bubbles.
Beware: Sometimes the rententate line in the retentate bottle can force air bubbles into the path of the feed line. Be prepared to stop the filtration ahead of time. BOTH pumps will need to be turned off simultaneously.
?. [Filtration Completion]
Once filtration has stopped, begin back flushing.
?. [Back flushing]
Simultaneously turn off both feed and permeate pumps.
OPTIONAL: Disconnect the permeate overflow tubing from the permeate reservoir (this is the tubing that carries permeate overflow from the permeate reservoir to either a collection bottle or a waste container). Depending on the setup, disconnecting this tubing can prevent introducing contaminants into the permeate reservoir during the back flushing procedure. Make sure to also partially unscrew the permeate reservoir cap to allow air displacement, preventing permeate from flowing onto the benchtop.
?. [Back flushing]
Reduce feed pump to  to allow mild circulation of the sample.
Centrifuge: 100 33
?. [Back flushing]
Start feed pump.
?. [Back flushing]
Reduce the permeate pump to the lowest setting (). Switch the direction of the permeate pump, so that permeate is flowing back into the TFF filter.
10
?. [Back flushing]
Start the pump.
?. [Back flushing]
Increase the speed of the permeate pump, but do not exceed 5 psi backpressure.
?. [Back flushing]
Continue backflushing for a few hundred milliliters, which will be seen as an increase in the retentate volume.
?. [Back flushing]
Turn off both pumps.
?. [Back flushing]
Return the pumps to their previous settings for filtration (also make sure to switch the permeate pump direction back to normal).
?. [Back flushing]
Turn both pumps on simultaneously, and filter sample to further concentrate.
?. [Back flushing]
Stop the filtration (both pumps simultaneously) before completely filtered, and repeat the backflushing protocol.
?. [Back flushing]
Perform the backflusing protocol for a total of 3 times, and after the third time, recover the sample.
On the 3rd backflush, you can backflush with less fluid (e.g.  - ) to achieve a smaller recovery volume.
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Turn both pumps OFF.
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Disconnect the permeate tubing at the joint between the downstream end of the pump head and the permeate reservoir.
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Drain the System:Release and remove the feed tubing from the feed pump head, to allow fluid to freely flow.
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Switch the direction of the permeate pump so that fluid is flowing back into the TFF filter. Set the speed to .
50
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Turn ON permeate pump.
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Monitor the permeate backpressure gauge. When the needle approaches 1 psi backpressure, turn the pump OFF.
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Release the permeate tube from the permeate pump head, so that fluid/air can flow freely.
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Reconnect the feed tubing to the feed pump head. If the pump can only filter in one direction, make sure to orient the tubing so that it is pumping in the reverse direction (out of the TFF filter)—otherwise, simply switch the direction of the pump.
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Set the feed pump to .
Centrifuge: 50 33
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Turn on the feed pump, and tilt the TFF filter as necessary to assist pumping out all the remaining fluid from the filter.
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
As soon as all the sample has been pumped out, turn the feed pump OFF.
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Detach the retentate bottle and store the concentrate.
?. [Sample Recovery (recovery volume typically between 250-400 mL)]
Proceed with the post-cleaning.
?. [Post-Cleaning]
Flush filter with MilliQ (up to  feed pump speed).
Centrifuge: 330 33
?. [Post-Cleaning]
Recirculate 0.1N Sodium Hydroxide at  feed speed for .
Centrifuge: 330 33
?. [Post-Cleaning]
Flush filter with MilliQ (up to  feed pump speed).
Centrifuge: 330 33
?. [Post-Cleaning]
Drain system (see sample recovery).
?. [Post-Cleaning]
Store filters in 0.05N Sodium Hydroxide solution.