gut metagenome
1. Genomic DNA ExtractionExtract the genomic DNA of the sample using the MagPure Soil DNA LQ Kit (Magen, Cat.No. D6356-02).Detect the DNA concentration by agarose gel electrophoresis and NanoDrop2000.2. Fragmentation & End PreparationPrepare the reaction solution:Input DNA: 200 ngNeutralization Buffer: x L (adjust according to the kit instructions)FEA Buffer: 5 LddHO: Add to a total volume of 40 LAdd FEA Enzyme Mix:Add 10 L FEA Enzyme Mix to each sample, mix by pipetting up-and-down or vortexing.Centrifuge to collect the reaction solution to the bottom of the tube and place it in the PCR instrument immediately for reaction.Thermal cycling program:37 for 10 minutes65 for 30 minutes4 Hold3. Adapter LigationPrepare the ligation reaction solution:Products from the previous step: 50 LRapid Ligation buffer: 25 LRapid DNA ligase: 5 LDNA Adapter-S: 5 LddHO: 15 LThermal cycling program:Lid temperature: 10520 for 15 minutes4 Hold4. Ligation Product PurificationWhen the beads are balanced to room temperature, vortex to mix the VAHTS DNA Clean Beads.Add 60 L VAHTS DNA Clean Beads to 100 L Adapter Ligation solution. Mix thoroughly by vortexing or pipetting.Incubate for 5 minutes at room temperature.Briefly centrifuge the PCR tube and place it on the magnetic stand to separate the beads and liquid. After the solution is clear (approximately 5 minutes), carefully remove the supernatant.Always keep the PCR tube on the magnetic stand, add 200 L of freshly prepared 80% ethanol to rinse the beads. Incubate for 30 seconds at room temperature, then carefully remove the supernatant.Repeat the ethanol rinse step twice in total.Air dry the beads on the magnetic stand for 3-5 minutes until there is no ethanol residue.Add 105 L eluent (10 mM Tris-HCl, pH 8.0-8.5) or ddHO for elution, vortex or gently pipette up and down to mix thoroughly and place it at room temperature for 2 minutes.Briefly centrifuge the PCR tube and place it on the magnetic stand. Once the solution is clear (approximately 5 minutes), carefully transfer 100 L supernatant to a new EP tube.5. Two Rounds Beads SelectionAdd 56 L VAHTS DNA Clean Beads to the above 100 L solution. Mix thoroughly by vortexing or pipetting. Incubate for 5 minutes at room temperature.Briefly centrifuge the PCR tube and place it on the magnetic stand to separate the beads and the solution. After the solution is clear (approximately 5 minutes), carefully transfer the supernatant to a new PCR tube and discard the beads.Add 12 L VAHTS DNA Clean Beads to the supernatant. Mix thoroughly by vortexing or pipetting.Incubate for 5 minutes at room temperature.Briefly centrifuge the PCR tube and place it on the magnetic stand to separate the beads and the solution. After the solution is clear (approximately 5 minutes), carefully remove the supernatant.Add 200 L freshly prepared 80% ethanol to rinse the beads. Incubate at room temperature for 30 seconds, then carefully remove the supernatant.Repeat the ethanol rinse step twice in total.Air dry the beads on the magnetic stand for 3-5 minutes until there is no ethanol residue.Remove the PCR tube from the magnetic stand for elution. Add 22.5 L eluent (10 mM Tris-HCl, pH 8.0-8.5) or ddHO (ddHO elution must be used if targeted capture is required later). Vortex or gently pipette up and down to mix thoroughly and place it at room temperature for 2 minutes.Briefly centrifuge the PCR tube and place it on the magnetic stand. Once the solution is clear (approximately 5 minutes), carefully transfer 20 L supernatant to a new EP tube. Do not disturb the magnetic beads.6. Library AmplificationMix gently and centrifuge briefly. Place the PCR tube in the PCR instrument and perform the following reaction:95 for 3 minutes (1 cycle)98 for 20 seconds60 for 15 seconds72 for 30 seconds (repeat for 5 cycles)72 for 5 minutes4 HoldReaction mixture:VAHTS i5 PCR Primer: 2.5 LVAHTS i7 PCR Primer: 2.5 LVAHTS HiFi Amplification Mix: 25 LTotal Volume: 50 L7. Reaction Products Purification and Library Quality ControlPurification:When the beads are balanced to room temperature, vortex to mix the VAHTS DNA Clean Beads.Add 45 L VAHTS DNA Clean Beads to 50 L Library Amplification solution. Mix thoroughly by vortexing or pipetting.Incubate for 5 minutes at room temperature.Briefly centrifuge the PCR tube and place it on the magnetic stand to separate the beads and liquid. After the solution is clear (approximately 5 minutes), carefully remove the supernatant.Add 200 L freshly prepared 80% ethanol to rinse the beads. Incubate at room temperature for 30 seconds, then carefully remove the supernatant.Repeat the ethanol rinse step twice in total.Air dry the beads on the magnetic stand for 5-10 minutes until there is no ethanol residue.Remove the PCR tube from the magnetic stand for elution. Add 25 L eluent (10 mM Tris-HCl, pH 8.0-8.5) or ddHO (ddHO elution must be used if targeted capture is required later). Vortex or gently pipette up and down to mix thoroughly and place it at room temperature for 2 minutes.Briefly centrifuge the PCR tube and place it on the magnetic stand. Once the solution is clear (approximately 5 minutes), carefully transfer 22.5 L supernatant to a new PCR tube.Transfer 18 L to a new EP tube.Quality Control:Quantify library concentration with Qubit 3.0.Use Agilent 2100 Bioanalyzer to check the length of the library.