TITLE: Accurate profiling of diazotrophic communities using unique molecular identifiers with Nanopore sequencing
AUTHORS: Nobuhiko Shigyo

[DESCRIPTION]
This protocol applies the method by Karst et al. (2021), who used UMI-tagged primers to perform highly accurate long-read amplicon analysis on the full-length ribosomal RNA operons, to the nitrogen fixation (nif) gene cluster. A recent study has shown that the functional gene nifH, which is frequently used in community analysis of diazotrophs, detects many false positives (Mise et al. 2021). Therefore, in this protocol, I used newly developed primers targeting the nifD-K gene (approx. 1.8 kbp) to identify the diazotrophic communities. The present protocol is presented using plant litter and mineral soil as examples, but it could potentially be applied in a variety of environmental samples.




[STEPS]
SECTION: DNA extraction
1. Extract total genomic DNA (gDNA) from 500 mgof fresh soil and 100 mg of air-dried litter using the ISOIL for Beads Beating kit (Nippon Gene Corporation, Tokyo, Japan), a CTAB-based DNA extraction kit.
SECTION: DNA extraction
2. Measure the extracted gDNA concentration using a Qubit fluorometer with the Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
SECTION: Tagging nifD-K genes with UMIs (1st PCR)
3. Prepare PCR-master mix in a 0.2 mL PCR tube with the following:
 

SECTION: Clean-up of 1st PCR products
5. Clean up the 1st PCR products using KAPA HyperPure Beads (Kapa Biosystems, Inc., Wilmington, MA, USA)
SECTION: Clean-up of 1st PCR products
5.1. Equilibrate the beads solution at room temperature and allow the beads to resuspend completely.
SECTION: Clean-up of 1st PCR products
5.2. Add 25 µL beads solution to the 50 µL PCR products (0.5× bead solution/sample ratio) and mix by pipetting up and down multiple (10 to 20) times.
SECTION: Clean-up of 1st PCR products
5.3. Incubate the tube at room temperature for 5 min.
SECTION: Clean-up of 1st PCR products
5.4. Place the tube on a magnetic rack (NGS MagnaStand (YS-Model) 8Ch × 0.2 mL PCR tube, Nippon Genetics Co., Ltd., Tokyo, Japan) to capture the beads. Incubate until the liquid is clear (~3 min).
SECTION: Clean-up of 1st PCR products
5.5. Discard the supernatant.
SECTION: Clean-up of 1st PCR products
5.6. Wash beads by adding 200 µL fresh 80% ethanol.
SECTION: Clean-up of 1st PCR products
5.7. Incubate the tube on the magnetic rack at room temperature for 30 sec.
SECTION: Clean-up of 1st PCR products
5.8. Discard the supernatant.
SECTION: Clean-up of 1st PCR products
5.9. Repeat the previous washing steps (4.6-4.8).
SECTION: Clean-up of 1st PCR products
5.10. Dry the beads at room temperature for 3 min.
SECTION: Clean-up of 1st PCR products
5.11. Remove the tube from the magnetic rack.
SECTION: Clean-up of 1st PCR products
5.12. Elute the purified DNA by adding 21 µL of nuclease-free water and mix by pipetting.
SECTION: Clean-up of 1st PCR products
5.13. Incubate the tube at room temperature for 5 min.
SECTION: Clean-up of 1st PCR products
5.14. Place the tube on the magnetic rack to capture the beads. Incubate until the liquid is clear (1 min).
SECTION: Clean-up of 1st PCR products
5.15. Transfer the supernatant to a new PCR tube.
SECTION: Amplification of UMI tagged sequences (2nd PCR)
6. Prepare PCR-master mix in a 0.2 mL PCR tube with the following:
 

SECTION: Tagging nifD-K genes with UMIs (1st PCR)
4. Run the following PCR program:
[98℃ 10 sec → 55℃ 15 sec → 72℃ 30 sec] × 2 → 8℃ ∞
SECTION: Amplification of UMI tagged sequences (2nd PCR)
7. Run the following PCR program:
[98℃ 10 sec → 68℃ 5 sec → 72℃ 30 sec]×30 → 68℃ 5min → 8℃ ∞

SECTION: Clean-up of 2nd PCR products
8. Clean up the 2nd PCR products using KAPA HyperPure Beads (0.5× bead solution/sample ratio) following the same procedure as in Section 5.
SECTION: Quality control
9. Measure the DNA concentration of the purified 2nd PCR products using a Qubit fluorometer with the Qubit dsDNA HS assay kit.
SECTION: Quality control
10. OPTIONAL:
Check the amplified fragments by gel electrophoresis.
   

SECTION: Nanopore sequencing
11. Conduct "DNA repair and end prep", "Adapter ligation and cleanup", and "Priming and loading of SpotOn flow cell" according to the Ligation Sequence Amplicon V14 protocol (ligation-sequencing-amplicons-sqk-lsk114-ACDE_9163_v114_revE_29Jun2022-minion.pdf).