TITLE: Extraction of high molecular weight DNA from nasal lining fluid
AUTHORS: Samuel Montgomery

[DESCRIPTION]
Assessing the microbiome in respiratory samples is often difficult due to the low biomass of microbes often present in these samples. While there are published methods for efficient extraction of DNA from samples such as bronchoalveolar lavage fluid for 16s rRNA sequencing or metagenomic sequencing (Saladie et al., 2020), these methodologies are usually optimised for traditional short-read based sequencing technologies. With the advent of accessible and affordable long-read sequencing technologies for full length 16s rRNA sequencing (PacBio) and whole metagenomic sequencing (Oxford Nanopore), there is increased importance in extracting high quality, high molecular weight fragments of DNA from metagenomic samples. This protocol can be used to extract DNA from both low biomass (nasal lining fluid, bronchoalveolar lavage, nasal swabs) and high biomass (pure bacterial culture) samples.

[BEFORE_START]
This protocol extracts DNA from samples stored in 45μL of PBS - if the sample is in a larger volume, the volumes of all following reagents can be scaled up, ensuring consistent ratios throughout the protocol. Otherwise, centrifuge sample at high speed for ~5 minutes and remove supernatant, resuspending pellet in 45uL of PBS




[GUIDELINES]
This protocol can be used to extract DNA from both low biomass (nasal lining fluid, BALf, nasal swab) and high biomass samples (bacterial culture)

[STEPS]
SECTION: Preparation of reagents
1. MetaPolyzyme is recieved as a lypholysed powder. Reconstitute following manufacturers instructions to a final concentration of 5 mg/mL, aliquot into 0.6mL microtubes, and store at -20ºC until use

SECTION: Preparation of reagents
2. Create a 70 % (v/v) solution of molecular grade ethanol with UltraPure water (approx. 1 mL per sample)

SECTION: DNA extraction
4. Add 5uL of MetaPolyzyme (5 mg/mL) to 45 µL of sample in PBS in a 1.5mL microtube

SECTION: DNA extraction
5. Incubate for 60 min at 35 °C  using a Thermomixer at  



SECTION: DNA extraction
6. Add sample (~50uL) to the prepared 2.0mL screw cap microtube containing beads
SECTION: DNA extraction
7. Add 250 µL of Cell Lysis Solution containing 0.6 % (v/v) Proteinase K and 0.6 % (v/v) RNase A solution to the sample

SECTION: DNA extraction
8. Bead beat sample for 30 s in the Precellys24 bead beater

SECTION: Preparation of reagents
3. Add a small volume (up to the line of the conical portion on the tube) of 0.1mm silica/zirconia beads to a 2.0mL screw cap microtube suitable for bead beating (one per sample)
SECTION: DNA extraction
9. Place sample on ice for 1 min 

SECTION: DNA extraction
10. Bead beat sample for 30 s  in the Precellys24 bead beater

SECTION: DNA extraction
11. Centrifuge the sample after bead beating at 10000 x g for 1 min 

SECTION: DNA extraction
12. Remove supernatant (~300uL) and place in a new 1.5mL microtube
SECTION: DNA extraction
13. Incubate for 30 min at 37 °C in a thermomixer at   

SECTION: DNA extraction
14. Increase the temperature to 56 °C  and incubate for 60 min in a thermomixer at   

SECTION: DNA extraction
15. Briefly place samples on ice to cool down to room temperature
SECTION: DNA extraction
16. Add 100 µL of protein precipitation solution containing 1 % (v/v) GenElute linear polyacrylamide to the sample and mix thoroughly by inverting the tube 25 times

SECTION: DNA extraction
17. Incubate samples on ice for 5 min 

SECTION: DNA extraction
18. Centrifuge tube at 16000 x g for 3 min 

SECTION: DNA extraction
19. Add supernatant to a new 1.5mL LoBind tube, careful not to disturb the protein pellet
SECTION: DNA precipitation
20. For low biomass samples where expected DNA recovery is low
SECTION: DNA precipitation
20.1. Add 1.5 volumes of PEG solution to the sample and incubate in the dark at Room temperature for 120 min 

SECTION: DNA precipitation
20.2. Centrifuge at 14000 x g for 30 min at 24 °C 

SECTION: DNA precipitation
21. For high biomass samples where expected DNA recovery is high
SECTION: DNA precipitation
21.1. Add 1 volume of isopropanol to the sample and incubate at Room temperature for 5 min 

SECTION: DNA precipitation
21.2. Centrifuge at 16000 x g for 5 min at Room temperature 

SECTION: DNA precipitation
22. Discard the supernatant by slowly drawing with a pipette at the air-liquid interface to avoid disturbing the pellet 
 

SECTION: DNA precipitation
23. Add 400 µL of 70 % (v/v) ethanol to the sample and invert several times to wash the pellet

SECTION: DNA precipitation
24. Centrifuge at 16000 x g for 1 min 

SECTION: DNA precipitation
25. Discard the supernatant and add 400 µL of 70%  ethanol to the sample and invert several times to wash the pellet

SECTION: DNA precipitation
26. Centrifuge at 16000 x g for 1 min 

SECTION: DNA precipitation
27. Discard the supernatant, and allow the pellet to air dry for 5 min 
SECTION: DNA precipitation
28. Add 30 µL of DNA hydration solution or nuclease free water to the pellet

SECTION: DNA precipitation
29. Incubate in the fridge at 4 °C    

SECTION: DNA precipitation
30. After this step, DNA is ready to be quantified and used downstream. DNA resuspended in water should be stored at -20 °C, while DNA resuspended in DNA hydration solution can be stored for up to one month at 4 °C