Dec 07, 2022
Accurate profiling of diazotrophic communities using unique molecular identifiers with Nanopore sequencing
- 1Department of Forest Soils, Forestry and Forest Products Research Institute
Protocol Citation: Nobuhiko Shigyo 2022. Accurate profiling of diazotrophic communities using unique molecular identifiers with Nanopore sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4embgmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
I use this protocol and it's working
Created: September 14, 2022
Last Modified: December 07, 2022
Protocol Integer ID: 69982
Keywords: Oxford Nanopore MinION, Nitrogen fixation, nifD-K, Forest soil, Leaf litter
Funders Acknowledgements:
A research grant of the Forestry and Forest Products Research Institute
Grant ID: #202206
JSPS Grants-in-Aid for Early-Career Scientists
Grant ID: 20K20003
Abstract
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.
Materials
Materials and Reagents
- 0.2 mL PCR tubes
- 1.5 mL tubes
- DNA extraction kit for soil (e.g., ISOIL for Beads Beating kit, Nippon Gene Co., Ltd., Tokyo, Japan)
- Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, United States)
- Qubit Assay Tubes (Thermo Fisher Scientific)
- 1st PCR primers (PAGE purification) (Integrated DNA Technologies, Coralville, IA)
- Forward primer (nifD229f_UMI): 5′-CAAGCAGAAGACGGCATACGAGAT NNNYRNNNYRNNNYRNNN TGGGGNCCVRTCAAGGAYAT-3′
- Reverse primer (nifK476r_UMI): 5′-AATGATACGGCGACCACCGAGATC NNNYRNNNYRNNNYRNNN CCRATSACYTCBGCCATRCA-3′
- 2nd PCR primers (PAGE purification) (Karst et al. 2021)
- Forward primer (lu_pcr_i1_fw_v7): 5′-ACGAGACTGATT CAAGCAGAAGACGGCATACGAGAT-3′
- Reverse primer (lu_pcr_i1_rv_v7): 5′-TACAGCGCATAC AATGATACGGCGACCACCGAGATC-3′
- PrimeSTAR Max Polymerase (Takara Bio Inc., Shiga, Japan)
- Nuclease-free water
- KAPA HyperPure Beads (Kapa Biosystems, Inc., Wilmington, MA, USA)
- 80% ethanol (molecular grade)
- Ligation sequencing kit V14 (SQK-LSK114) (Oxford Nanopore Technologies, Oxford, United Kingdom)
- R10.4.1 flow cell (FLO-MIN114) (Oxford Nanopore Technologies)
- NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (New England BioLabs, Beverly, MA, USA)
- Bovine Serum Albumin (BSA) (Invitrogen, Carlsbad, CA, USA)
Equipment
- Thermal cycler (e.g., TaKaRa PCR Thermal Cycler Dice Touch, Takara Bio Inc.)
- Micropipette (P1000, P200, P100, P20, P10, and P2)
- Vortex mixer
- Microfuge
- Magnetic stand (e.g., NGS MagnaStand (YS-Model) 8Ch × 0.2 mL PCR tube, Nippon Genetics Co., Ltd., Tokyo, Japan)
- Qubit fluorometer (Qubit 4 fluorometer, Thermo Fisher Scientific)
- Nanopore sequencer (e.g., MinION Mk1C, Oxford Nanopore Technologies)
DNA extraction
DNA extraction
2h 10m
2h 10m
Extract total genomic DNA (gDNA) from 500 mg of 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.
2h
Measure the extracted gDNA concentration using a Qubit fluorometer with the Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
10m
Tagging nifD-K genes with UMIs (1st PCR)
Tagging nifD-K genes with UMIs (1st PCR)
15m
15m
Prepare PCR-master mix in a 0.2 mL PCR tube with the following:
| Component | Volume (total 50 µL) | |
| Nuclease-free water | X µL | |
| Forward primer (nifDK229f_UMI, 10µM) | 2.5 µL | |
| Reverse primer (nifK476r_UMI, 10 µM) | 2.5 µL | |
| PrimeSTAR Max Premix | 25 µL | |
| Template DNA | Y µL (50 ng) |
5m
Run the following PCR program:
[98℃ 10 sec → 55℃ 15 sec → 72℃ 30 sec] × 2 → 8℃ ∞
10m
Clean-up of 1st PCR products
Clean-up of 1st PCR products
30m
30m
Clean up the 1st PCR products using KAPA HyperPure Beads (Kapa Biosystems, Inc., Wilmington, MA, USA)
Equilibrate the beads solution at room temperature and allow the beads to resuspend completely.
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.
Incubate the tube at room temperature for 5 min.
5m
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).
Discard the supernatant.
Wash beads by adding 200 µL fresh 80% ethanol.
Incubate the tube on the magnetic rack at room temperature for 30 sec.
30s
Discard the supernatant.
Repeat the previous washing steps (4.6-4.8).
Dry the beads at room temperature for 3 min.
3m
Remove the tube from the magnetic rack.
Elute the purified DNA by adding 21 µL of nuclease-free water and mix by pipetting.
Incubate the tube at room temperature for 5 min.
5m
Place the tube on the magnetic rack to capture the beads. Incubate until the liquid is clear (1 min).
1m
Transfer the supernatant to a new PCR tube.
Amplification of UMI tagged sequences (2nd PCR)
Amplification of UMI tagged sequences (2nd PCR)
2h 10m
2h 10m
Prepare PCR-master mix in a 0.2 mL PCR tube with the following:
| Component | Total volume (100 µL) | |
| Nuclease-free water | 20 µL | |
| Forward primer (lu_pcr_i1_fw_v7) | 5 µL (0.5 µM) | |
| Reverse primer (lu_pcr_i1_rv_v7) | 5 µL (0.5 µM) | |
| PrimeSTAR Max Premix | 50 µL | |
| Template DNA (1st PCR products) | 20 µL |
10m
Run the following PCR program:
[98℃ 10 sec → 68℃ 5 sec → 72℃ 30 sec]×30 → 68℃ 5min → 8℃ ∞
2h
Clean-up of 2nd PCR products
Clean-up of 2nd PCR products
30m
30m
Clean up the 2nd PCR products using KAPA HyperPure Beads (0.5× bead solution/sample ratio) following the same procedure as in Section 5.
30m
Quality control
Quality control
10m
10m
Measure the DNA concentration of the purified 2nd PCR products using a Qubit fluorometer with the Qubit dsDNA HS assay kit.
10m
OPTIONAL:
Check the amplified fragments by gel electrophoresis.
Lanes:
1. 1kb DNA ladder
2. Purified 2nd PCR products
Nanopore sequencing
Nanopore sequencing
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).