Metagenomic Sequencing Protocol

DNA Extraction

DNA Extraction from Soil or Sediment

1. Weigh 0.5 g of glass beads into a 2 ml Eppendorf tube.
2. Into this tube, weigh 0.5 g of soil or sediment that has previously been homogenized by stirring with a sterile spatula.
3. Add 0.5 ml of 0.1 M sodium phosphate buffer pH 8 (use this to wash the tube with sample).
4. Add 0.5 ml of phenol-chloroform-isoamyl alcohol into the tube.
5. Bead-beat at 8,000 × g for 30 s, put on ice for 30 s, and repeat for another 30 s.
6. Centrifuge for 5 min at 16,000 × g (4°C).
7. Transfer supernatant to a clean 2 ml tube.
8. Add equal volume (~500 ml) of chloroform-isoamyl alcohol and vortex to mix.
9. Centrifuge for 5 min at 16,000 × g, at 4°C.
10. Transfer supernatant to a new 2 ml tube.
11. Add 2× volume of ice-cold 100% ethanol and 1/10 volume sodium acetate and mix. For RNA extraction, use 2.5× the volume of ethanol.
12. Precipitate for at least 1 h in freezer.
13. Centrifuge for 30 min at 16,000 × g.
14. Pipette off and discard liquid, being careful with the pellet.
15. Wash with 200 ml of 70% ethanol, vortex to mix.
16. Centrifuge for 10 min at 16,000 × g.
17. Repeat last two steps and remove remaining ethanol with a pipette. Be careful with the pellet.
18. Air-dry for 10 min. Resuspend pellets in sterile water.

DNA Extraction from Seawater

1. Filter 10-15 L of seawater through a 140 mm diameter, 1.6 mm GF/A filter, to reduce eukaryotic cell abundance and maximize the proportion of prokaryotic cells.
2. Apply filtrate directly to a 0.22 mm Sterivex filter.
3. Following filtration, pump Sterivex cartridge dry and quick-freeze in liquid nitrogen and store at −80°C until DNA isolation.
4. Thaw the Sterivex cartridge on ice.
5. Add 1.6 ml of SET lysis buffer directly onto the top of Sterivex using a 2.5 ml syringe with a 25G 5/8" needle.
6. Add 180 ml of fresh lysozyme and seal the sterivex.
7. Incubate at 37°C for 30 min under continuous rotation in a Hybaid oven.
8. Add 200 ml of SDS (10% w/v) and 55 ml of fresh proteinase K (20 mg/ml).
9. Incubate at 55°C for 2 h with continuous rotation in a Hybaid oven.
10. Withdraw lysate from Sterivex using a 5 ml syringe.
11. Add 1 ml of fresh SET buffer to Sterivex and rotate to rinse.
12. Withdraw SET buffer into the same 5 ml syringe.
13. Add lysate into a 15 ml Maxtract tube containing 2 ml of phenol-chloroform-isoamyl alcohol (25:24:1), pH 8.
14. Shake gently until mixed. Then, centrifuge at 1,500 × g for 5 min.
15. Add an additional 2 ml of phenol-chloroform-isoamyl alcohol (25:24:1). Shake gently until mixed and centrifuge at 1,500 × g for 5 min.
16. Add 2 ml of chloroform-isoamyl alcohol (24:1). Shake gently until mixed and centrifuge at 1,500 × g for 5 min.
17. Decant aqueous phase to a sterile 20 ml centrifuge tube and add 0.5 V of 7.5 M ammonium acetate. Mix briefly and then add 2.5 V of pure ethanol.
18. Mix and leave at −20°C for >1 h (overnight is fine).
19. Centrifuge at 10,000 × g for 30 min at 4°C and decant ethanol.
20. Add 2 ml of 80% ethanol and rinse the tube, then centrifuge at 10,000 × g for 20 min at 4°C and decant ethanol.
21. Repeat above washing step.
22. Decant ethanol and leave inverted for 15 min in fume hood.
23. Suspend invisible pellet in 200 ml of sterile water. Leave on ice for approximately 1 h with frequent finger-tapping to rinse tube walls.
24. DNA can be quantified by visualizing using agarose gel electrophoresis or by using any standard DNA quantification techniques.
25. For pyrosequencing, it is necessary to have ~5 mg of DNA, which this technique will produce in excess. Ideally, the DNA should be at a concentration of approximately 500 ng/ml.

Sequencing

1. The DNA needs to be accurately measured. The ideal amount is 3-5 mg for a fragment library, but it is possible to use less with success. It also needs to be of a reasonable molecular weight to maximize random shearing.
2. The DNA in a volume of 100 ml is mixed with 500 ml of nebulization buffer supplied with the library kit. The DNA is sheared by placing it in a nebulizer vessel and connecting the latter to nitrogen gas at 30 psi for 1 min.
3. The DNA is recovered from the nebulization chamber and cleaned with manufacture's kit following the instructions supplied by the kit, using 2.5 ml of PB buffer and eluting the DNA in 100 ml of elution buffer.
4. At this stage, the small fragments are removed using AMPure beads. A calibrated amount is added to the DNA so that material of less than 300 bp is left in solution. The higher MW DNA binds to the beads, is washed with 70% ethanol, is dried, and is recovered by eluting in 10 mM Tris-HCl pH 7.5.
5. The DNA is checked (1 ml aliquot) for size distribution.
6. Further manipulations are all described in the library kit. The ends of the DNA are polished, the adapters (with or without bar codes) added, fragments containing adapters selected on Dynal beads, and the ends filled. The single-stranded library is recovered by melting from the Dynal beads with NaOH (0.125 N) as per the manufacturer's instructions and cleaned up with a MinElute column.
7. The library is assessed by running on a RNA picochip.
8. A predetermined amount of the library is used to set up an emulsion PCR reaction using either the large- or small-volume kit. This involves binding the DNA to capture beads that are specific for one of the adapters.

Reference:

1. Gilbert J A, Laverock B, Temperton B, et al. Metagenomics[M]//High-Throughput Next Generation Sequencing. Humana Press, Totowa, NJ, 2011: 173-183.