TITLE: Long-read DNA preparation for metagenomic samples
AUTHORS: Christian Brandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">General:</span></div><div class = "text-block">This is an DNA isolation protocol adapted to the properties of sludge samples from biogas and wastewater treatment plants. The protocol reliably retrieves metagenomic DNA of sufficient quality, length and yield. The performance has been validated on 20 different reactor plants. </div><div class = "text-block"><span style = "font-weight:bold;">Observed Performance on 20 ONT runs using this protocol:</span></div><div class = "text-block">It does not negatively impact the overall throughput for Nanopore Sequencing. Sequencing runs were active over 2.5 days, with one refuel step after 18 h.</div><div class = "text-block">N50 Ranges between 4000 and 8000 bp, depending on the sample type. E.g. frozen samples have a lower N50, fresh samples a higher N50. General throughput is between 18-28 Gigabases of a single flowcell (flowcell 106 Rev D, using the LSK-109 Kit).  </div><div class = "text-block"><span style = "font-weight:bold;">Funding:</span></div><div class = "text-block">This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - BR 5692/1-1 and BR 5692/1-2</div></div>

[STEPS]
?. [Sample pre preparation]
Add 1 ml of homogenic sludge/substrate via 10 ml syringe (without needle) to a 2 ml tube
[sludge/substrate]
?. [Sample pre preparation]
Centrifuge for 5 min at 20,000g and remove supernatant
?. [Cell Lysis]
Transfer sample to lysing matrix E tube and add 778 µl sodium phosphate buffer
[sodium phosphate buffer]
?. [Cell Lysis]
Add 122 µl MT buffer, homogenize tube by hand
[MT buffer]
?. [Cell Lysis]
Homogenize via FastPrep®-24 Classic I. for 20 s  at 6 m/s and put directly on ice after lysis.
The device may strongly influence your expected total yield and read lengths. Try different times and strengths first if you are using another device and analyse your DNA length distribution at the end, e.g. by agarose gel or Agilent Bioanalyzer, and determine the best settings for your device.6 m/s corresponds to 3700 rpm, according to the manufacturer
?. [Precipitation and Binding]
Centrifuge for 15 min at 4 °C with 14,000g
4 °C
?. [Precipitation and Binding]
Transfer supernatant to a clean 2 ml tube
?. [Precipitation and Binding]
Add 1 µl RNase A to each sample and incubate for 5 min
[RNase A ]
?. [Precipitation and Binding]
Add  250 µl PPS (protein precipitation solution) and mix carefully but efficiently by inverting at least 10 times
[PPS]
?. [Precipitation and Binding]
Centrifuge at 14,000 g for 10 min at 4 °C
4 °C
?. [Precipitation and Binding]
Transfer supernatant to clean 5 ml tube
?. [Precipitation and Binding]
Resuspend binding matrix suspension and add 1 ml binding matrix suspension to the 5 ml tube
[binding matrix suspension]
?. [Precipitation and Binding]
Invert by hand for 2 min (avoid "matrix pellet" here) Place on a rack for 0.5 to  1 h
[or up to 1 hour]
?. [Precipitation and Binding]
Discard 650 µl supernatant without disturbing the binding matrix
?. [Washing]
Transfer approx. 600 µl of the mixture to a spin filter and centrifuge at 14,000g for 1 min     Empty the catch tube and repeat the steps until all the binding matrix is in the spin filterEmpty the catch tube.Empty the catch tube and repeat the steps until all the binding matrix is in the spin filterEmpty the catch tube.
?. [Washing]
Add 500 µl HA-wash solution and gently resuspend the matrix by stirring using a pipette tipCentrifuge at 14,000 g for 1:00min, empty catch tube
[HA-wash solution]
Slightly stir with the pipette tip, do not overdo resuspending, as well for the next step
?. [Washing]
Add 500 µl SEWS-M and gently resuspend the pellet using a pipette tipCentrifuge at 14,000 g for 1:00 min, replace the catch tube
[SEWS-M]
?. [Washing]
Centrifuge a second time at 14,000 g for 2 min  to dry the membraneDiscard the catch tube and add a new clean 1.5 ml tube
?. [Washing]
Air-dry the spin-filter for 5 min at room temperature
[(room temperature)]
?. [DNA retrival]
Add 90 µl 55 °C warm nuclease-free water to the spin filter and gently stir the binding matrix slightly with a pipette, then incubate for at least 5 min
[55 °C warm nuc. free water]
[at least]
?. [DNA retrival]
Centrifuge at 14,000 g for 1 min  to get the eluate
Store at 4 °C if you want to stop here
?. [Pre Cleaning]
Add 31.5 µl magnetic beads (0.35 volume, e.g. AMPure or HighPrep) to 90 µl of DNA sample, shake gently by hand
[DNA sample]
[magnetic beads]
Around 500 ng/µl DNA is reduced to around 250 ng/µl after cleaning.Alternatively, take less DNA volume and add water if the concentration is over 500 ng/µl
?. [Pre Cleaning]
Incubate for 10 min at room temperature
[room temperature]
?. [Pre Cleaning]
Spin down and bind to magnet until the liquid is clear,
?. [Pre Cleaning]
Stay on the magnet and remove supernatant
?. [Pre Cleaning]
(wash 1/2) Add 150 µl Ethanol, don't disturb pellet
[Ethanol  (80 %)]
?. [Pre Cleaning]
(wash  2/2) Remove supernatant
?. [Pre Cleaning]
[and redo the washing once]
?. [Pre Cleaning]
Spin down, bind to magnet again. Remove the residual liquid with a 10 µl pipette
?. [Pre Cleaning]
Add 30 µl nuclease free water and resuspend gently
[nuclease free water]
?. [Pre Cleaning]
Incubate for 10 min at room temperature
[room temperature]
?. [Pre Cleaning]
Spin down and put on magnetic
?. [Pre Cleaning]
Retrieve the supernatant with your DNA
Determine dsDNA concentration e.g. via Qubit.Determine DNA length via agarose gel or Bioanalyzer.
You should usually lose around half of your total DNA during this process.
?. [Sample pre preparation]
Resuspend in 400 µl nuclease free water
[nuclease free water]