Microbial Whole Genome Sequencing by NGS Protocol

Collection of Cells by Micromanipulation

1. Apply 2 ml of 1× LB into a sterile, 0.2 ml PCR tube and incubate on ice or a PCR-cooler.
2. Attach a glass capillary of 30-100 mm diameter to micromanipulators. The diameter of the capillary depends on the size of the cells harboring the target bacterial symbiont.
3. Mark the position corresponding to the depth of a 0.2 ml PCR tube on a glass capillary of 15 mm diameter, and attach it to the other micromanipulator.
4. Apply 50 ml of sol U, five to ten times, onto the inner surface of the lid.
5. Collect the host protist cells.
6. Wash the cells more than four times.
7. Apply 50 ml of 1% NP-40 onto the inner side of the lid of a new Petri dish.
8. Collect a single cell of the host protist with a new capillary.
9. Replace the lid of the Petri dish on the stage by the lid with 1% NP-40.
10. Dip the tip of the 15 mm diameter capillary in the 1% NP-40 drop and adjust the pressure inside the capillary by handling the Cell Tram Vario. Be careful not to completely expel 1% NP-40 within the capillary. Bubbles make the phase-contrast image unclear.
11. Dip the tip of the 30–100 mm diameter capillary near the tip of the 15-mm diameter capillary in the 1% NP-40 drop and carefully release the host protist cell.
12. Collect the bacterial cells as many as possible with a capillary of 15 mm diameter.
13. Release the collected cells into LB in the PCR tube.

Whole-Genome Amplification

1. Incubate the collected bacterial cells in 1× LB on ice or a PCR cooler for 10 min.
2. Prepare and perform the negative control reaction in parallel with the sample reaction.
3. Add 1.0 ml NB and 22 ml sample buffer, mix briefly.
4. Prepare reaction mixture: mix 2.5 ml enzyme solution and 22.5 ml reaction buffer by pipetting.
5. Add the reaction mixture to the sample mixture.
6. Incubate at 30°C for 2.5 h.
7. Stop the reaction by incubating at 65°C for 10 min.
8. Check the amplification by agarose gel electrophoresis.
9. Purify DNA by ethanol precipitation.
10. Dissolve the precipitate in 50 ml TE.
11. Measure the DNA concentration.

Checking Purity of Sample

1. Prepare 1:200 fold dilution of the purified WGA sample dissolved in TE and of the negative control reaction without purification. Use 1/10 volume as the template for the following PCR.
2. Perform PCR using specific primer sets to eubacterial, archaeal, and eukaryotic SSU rRNA genes, respectively, and also those specific to protein-coding genes such as hsp60 and gyrB. Always perform the negative control experiment for PCR. PCR conditions: 95°C 30 s, 25 cycles of (95°C 10 s, 50°C 30 s, 72°C 2 min), 72°C 4 min.
3. If there is no PCR amplification from the negative control sample either for WGA or PCR, and only PCRs using bacteria-specific primers generate products from the WGA sample, move to the next step.
4. Perform PCR amplification of eubacterial 16S rRNA genes with a proof-reading DNA polymerase.
5. Clone and sequence the PCR products with standard methods.
6. Check whether the genomes of the target bacterial species predominate in the sample and also evaluate within-species variations of the target bacterium.

Genome Sequencing

After the purity check of the WGA sample, prepare a library for the Illumina sequencer.

Reference:

1. Hongoh Y, Toyoda A. Whole-genome sequencing of unculturable bacterium using whole-genome amplification[M]//High-Throughput Next Generation Sequencing. Humana Press, Totowa, NJ, 2011: 25-33.