Nov 01, 2024
Protocol of shotgun metagenomics library construction
- Elizabeth Selene Gómez Acata1,
- Braulio Ricardo Perez Alva2,
- Yendi Navarro1
- 1Laboratorio de Interacciones Bióticas, Centro de Investigación en Ciencias Biológicas, Universidad Autónoma de Tlaxcala;
- 2Posgrado en Ciencias Biológicas, Universidad Autónoma de Tlaxcala
Protocol Citation: Elizabeth Selene Gómez Acata, Braulio Ricardo Perez Alva, Yendi Navarro 2024. Protocol of shotgun metagenomics library construction. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzk9dlzp/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
We use this protocol and it's working
Created: September 18, 2024
Last Modified: November 01, 2024
Protocol Integer ID: 107913
Funders Acknowledgement:
CONAHCYT
Grant ID: 2649154
Abstract
This protocol is an optimization for metagenomic library construction from peat bog and arable soil samples. It is suitable for DNA inputs ranging from 20 pg to 10 ng, with a fragment size of 250 bp for Illumina sequencing.
Materials
QIAseq‱ FX DNA Library Core Kit Cat. No. 1120146
QIAseq‱ UDI Y-Adapter Kit B (96) Cat. No. 180314
AMPure XP Beckman CoμLter Cat. No. A63880
FastGene Gel/PCR Extraction Kit Cat. No. FG-91302
Magnetic bead separation DynaMagTM-2 Magnet Life technologiesTM Cat. No. 12321D
Labtop cooler TrueNorth‱
Quant-iT‱ PicoGreen‱ Cat. No. P7589
Ethanol (100% molecular grade) Sigma Aldrich Cat. No. E7023
QX200‱ ddPCR‱ EvaGreen Supermix Bio-Rad Cat. No. 186-4036
Tween 20 Sigma Aldrich Cat No. P1379-25ML
PCR tubes Axygen Cat. No. 321-02-051
qPCR tubes
Filter tips and pipettes
Equipment
Vortex Genie
Microcentrifuge Legend Thermo Scientific
Rotor-Gen Q Qiagen
Mastercycler Nexus Gradient Eppendorf
NanoDrop‱ 3300 Fluorospectrometer
Before start
- The workspace should be kept clean and sterile, using UV radiation for sterilization.
- Library preparation needs to be carried out in a laminar flow hood.
- Ensure pipettes are clean and calibrated.
- A negative control should be included throughout the process to confirm the absence of reagent contamination.
- To prevent batch effects, the same person should conduct the library construction.
- Do not dissolve DNA in 1x TE or any solution containing EDTA.
- Quantify DNA concentration using Quant-iT‱ PicoGreen‱ assay kit before starting the fragmentation process.
- Each DNA sample must be normalized to 1 ng μL-1 using Tris 10 mM or water.
Thermocycler programs
Thermocycler programs
Set up the thermocycler with the following programs:
Table 1. Fragmentation reaction program
| Step | Temperature | Time | |
| Pre-chilled | 4ºC | 1 min | |
| Fragmentation | 32ºC | 24 min* | |
| Enzyme inactivation | 65ºC | 30 min | |
| Hold | 4ºC | Hold |
*20 pg of input DNA, we used 14 min
Table 2. Adapter ligation reaction program
| Step | Temperature | Time | |
| Ligation | 20ºC | 30 min | |
| Hold | 4ºC | Hold |
Table 3. Library amplification program
| Step | Temperature | Time | Cycles | |
| Initial denaturation | 98ºC | 2 min | 1 | |
| Denaturation | 98ºC | 20 s | ||
| Annealing | 60ºC | 30 s | 10 | |
| Extension | 72ºC | 30 s | ||
| Final extension | 72ºC | 1 min | 1 | |
| Hold | 4ºC | Hold |
* 20 pg of input DNA, we used 16 cycles
Fragmentation
Fragmentation
1. Thaw the reagents on ice: Fx buffer 10x, FX Enzyme Mix and Fx Enhancer from QIAseq‱ FX DNA Library Core Kit.
2. Keep the reactions on ice during setup.
3. Prepare the following reaction mix for 10 ng or 20 pg of input DNA.
Table 4. Fragmentation reaction mix for samples containing 10 ng of input DNA
| Component | Volume | |
| Fx buffer 10x | 5.0 μL | |
| DNA (1 ng/μL)* | 10.0 μL | |
| Nuclease free-water | 25.0 μL | |
| Total | 40.0 μL |
*DNA dissolved in Tris 10 mM or water, do not contain EDTA
Table 5. Fragmentation reaction mix for samples containing 20 pg of input DNA
| Component | Volume | |
| Fx buffer 10x | 5.0 μL | |
| DNA (20 pg)* | 20.0 μL | |
| Nuclease free-water | 12.5 μL | |
| Fx Enhancer | 2.5 μL | |
| Total | 40.0 μL |
*DNA dissolved in Tris 10 mM or water, do not contain EDTA
4. Add 10 μL of FX Enzyme Mix to each reaction tube and mix thoroughly by pipetting up and down.
5. Place de PCR tubes into a pre-chilled thermocycler set to 4 ºC.
6. Use the thermocycler program from Table 1, ensuring the fragmentation time matches the input DNA.
7. Proceed directly with adapter ligation.
Adapter ligation
Adapter ligation
1. Prepare the adapter's dilutions according to Table 6.
Table 6. Adapter ligation factor
| DNA amount | Adapter dilution | |
| 10 ng | 1:15 | |
| 20 pg | 1:300 |
2. Add 5 μL of diluted adapters to each fragmentation tube.
3. Add 45 μL of adapter ligation mix.
Table 7. Adapter ligation mix
| Component | Volume | |
| DNA ligase buffer 5x | 20 μL | |
| DNA ligase | 10 μL | |
| Nuclease free-water | 15 μL | |
| Total | 45 μL |
4. Mix by pipetting up and down.
5. Incubate 20 ºC for 30 min (thermocycler program Table 2).
Note: do not use thermocycler with heated lid
6. Purify the adapter ligation tube immediately.
Adapter ligation cleanup
1. Use FastGene Gel/PCR Extraction Kit following the manufacturer instructions.
2. Elute the DNA with 40 μL of elution buffer.
Library amplification
Library amplification
1. Thaw reagents on ice: HiFi PCR master mix 2x and Primer mix from QIAseq‱ FX DNA Library Core Kit.
2. Use the following library amplification mix.
Table 8. Library amplification mix
| Component | Volume | |
| HiFi PCR master mix 2x | 25.0 μL | |
| Primer mix (10 μM) | 1.5 μL | |
| Library purified | 23.5 μL | |
| Total | 50.0 μL |
3. Mix all the components by pipetting up and down.
4. Use the library amplification program from table 3.
Library cleanup
Take 50 μL of the amplicon library and cleanup with FastGene Gel/PCR Extraction Kit following the manufacturer instructions.
Library quantification with qPCR
Library quantification with qPCR
1. Prepare a 1:4000 dilution of the samples that used adapter 1:15, and a 1:500 dilution for those with adapter 1:300, using Tween 20 (0.1%).
Note: To prepare Tween 20 (0.1%), mix 10 mL of H2O with 10 μL of Tween-20.
2. Thaw the reagents on ice: QX200‱ ddPCR‱ EvaGreen Supermix.
3. Use the following library amplification mix.
Table 9. Library quantification mix
| Component | Volume | |
| QX200™ ddPCR™ EvaGreen Supermix 2x | 4.50 μL | |
| Primer forward (10 μM) | 0.18 μL | |
| Primer reverse (10 μM) | 0.18 μL | |
| Diluted library | 1.00 μL | |
| Nuclease free-water | 4.14 μL | |
| Total | 10.00 μL |
4. Mix all components thoroughly by pipetting up and down
5. Open the lid of the Rotor-Gen Q, place the samples, and follow the instructions in the equipment's operation manual to start the quantification process.
Library pooling
Library pooling
1. Normalize each sample to 4 ng μL-1 use the data obtained from qPCR.
2. Add 4 μL of each sample (4 ng μL-1) to a tube and proceed with cleanup with AMPure XP Beckman Coulter beads.
Library pool cleanup
Library pool cleanup
1. Add 80 µL of AMPure XP Beckman Coulter beads to each 75 µL of pooled library and mix by pipetting.
2. Incubate for 5 minutes at room temperature.
3. Place the tube on a DynaMag‱-2 Magnetic Separator (Life Technologies‱) for a few minutes until a brown pellet forms.
4. Carefully remove the supernatant with a micropipette, ensuring the magnetic bead pellet is not disturbed.
5. Wash the beads with 200 µL of 80% ethanol and remove the supernatant.
6. Repeat the 80% ethanol wash once more.
7. Incubate the beads on the DynaMagTM-2 for 5-10 min, or until dry. NOTE: Do not overdry the beads, as this reduces DNA recovery efficiency.
8. Remove the tube from the DynaMagTM-2 . Add 37.5 µL of elution buffer (1x TE) and mix using the micropipette.
9. Place the tube back on the DynaMagTM-2 to separate the magnetic beads from the solution.
10. Transfer the eluate to a new sterile tube.