Nov 04, 2024

Public workspaceDNA Library Prep with MGIEasy UDB Universal Library Prep Set

DOI
dx.doi.org/10.17504/protocols.io.j8nlk9brxv5r/v1
  • 1BGI Research;
  • 2China National GeneBank;
  • 3MGI Tech
Qianyue Ji
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk9brxv5r/v1
Protocol Citation: Qianyue Ji, Guoqiang Mai, Xiaohan Wang, Shanshan Liu, Mo Han 2024. DNA Library Prep with MGIEasy UDB Universal Library Prep Set. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk9brxv5r/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: October 27, 2024
Last Modified: November 04, 2024
Protocol Integer ID: 110973
Abstract
The MGIEasy UDB Universal Library Prep Set(1000022803,MGI Tech) is specifically designed for creating WGS libraries for the MGI high-throughput sequencing platform series. This library prep set is optimized to convert 1-1000ng of fragmented DNA into a customized library. Unique Dual barcode supported double checked barcode split method ensuring sequencing Homogeneity and accuracy.
Materials
Materials
MGIEasy Dual Barcode Circularization Kit (Cat. No.: 1000020570)
Qubit ssDNA Assay Kit
Supplies:
  • Pipette tips (assorted volumes)
  • 5ml round-bottom polystyrene tubes, PCR tube, centrifuge tube
Equipment:
  • Qubit Fluorometer
  • Vortex
  • Micropipettes, various volumes
  • Microcentrifuge
  • Thermocycler
  • Magnetic rack
Sample Preparation
Sample Preparation
20m
20m
Fragmentation
Use Covaris series models to fragment Amount55 µL gDNA sample into size between 300-500bp.
Double size selection
The following sample use a Amount60 µL 1st bead selection and a Amount20 µL 2nd bead selection to target a 280bp size fragement from fragmented DNA( Amount100 µL ).
Transfer all fragmented DNA to a new 1.5mL centrifuge tube. Add TE buffer for a final volume ofAmount100 µL .
Add Amount60 µL of DNA Clean Beads to each sample tube. Mix with a vortexer until all beads are suspended.
Incubate at TemperatureRoom temperature forDuration00:05:00 .
5m
Incubation
Centrifuge the tube(s) briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Then, carefully transfer the supernatant to a new 1.5mL centrifuge tube. Retain the supernatant and dicard the beads.
5m
Centrifigation
Add Amount20 µL of DNA Clean Beads to the centrifuge tube with Amount144 µL supernatant. Mix with a vortexer until all beads are suspended.
Incubate at TemperatureRoom temperature for Duration00:05:00 .
5m
Centrifuge the tube(s) briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Carefully remove and discard all the supernatant.
5m
Centrifigation
While keeping the centrifuge tube on the magnetic rack, add Amount200 µL of 80% ethanol to each tube to wash the beads and tube wall. Wait for Duration00:00:30 . Carefully remove and discard the supernatant.

30s
Repeat step 2.8 and try to remove all of the liquid from the tube.
Keep the tube(s) on the magnetic rack. Open the tube cap and air-dry the beads at TemperatureRoom temperature until no wetness or glossiness is visible on the beads' surface. There should be no visible cracking on the surface of the beads.
Remove the tube(s) from the magnetic rack and add Amount32 µL of TE to elute the DNA. Mix with a vortexer until all beads are suspended.
Incubate the tube(s) at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the tube(s) briefly and place on the magnetic rack for 2 to 5 min until the liquid is clear. Carefully transfer Amount30 µL of supernatant to a new 1.5mL centrifuge tube.
Centrifigation
DNA Quantification
Quantify the purified fragment products with Qubit dsDNA Assay Kit.
End repair and A-tailing
End repair and A-tailing
End repair and A-tailing
Transfer DNA sample to a new 0.2mL PCR tube and add TE buffer for a total volume of Amount39.5 µL .
Prepare the End repair and A-tailing Mixture TemperatureOn ice .
ComponentsVolume
ERAT Buffer6 μL
ERAT Enzyme Mix4.5 μL
Total10.5 μL
End repair and A-tailing Mixture
Transfer Amount10.5 µL of the mixture to the 0.2mL PCR tube from step 2.1. Votex 3 times and centrifuge briefly.
Place the 0.2mL PCR tube into the thermocycler and run the program.
TemperatureTime
Heated lid(105°C)On
20°C30 min
65°C15 min
4°CHold
End repair and A-tailing program

PCR
Brifly centrifuge to collect the solution at the bottom of the tube.
Centrifigation
Adapter ligation
Adapter ligation
Adapter ligation
Add Amount5 µL of UDB Adapter to the corresponding sample tube. Vortex it 3 times (Duration00:00:03 each), centrifuge briefly, and place TemperatureOn ice .

3s
Prepare the adapter ligation mixture. Vortex it 6 times (Duration00:00:03 each), centrifuge briefly, and place TemperatureOn ice .
ComponentsVolume
DNA Ligase4 μL
Ligation Buffer21 μL
Total25 μL
adapter ligation mixture


3s
Slowly pipette Amount25 µL of adapter ligation mixture to each sample tube. Vortex it 6 times (Duration00:00:03 each), centrifuge briefly, and place TemperatureOn ice .
The adapter ligation mixture is highly viscous. Pipette slowly and carefully.
3s
Place the PCR tube(s) into the thermocycler. Run the program with the following conditions.
TemperatureTime
Heated lid(50°C)On
23°C30 min
4°CHold
adapter ligation program

PCR
When the program is completed, centrifuge the PCR tube(s) briefly and place TemperatureOn ice .
Add Amount20 µL TE buffer, for a total volume of Amount100 µL and transfer to a new 1.5mL centrifuge tube.
Cleanup of Adapter-ligated DNA
Cleanup of Adapter-ligated DNA
5m
5m
Cleanup of Adapter-ligated DNA

Add Amount50 µL of DNA Clean Beads to each sample tube. Mix with a vortexer until all beads are suspended.
Incubate the sample tube at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the sample tube briefly and place on the magnetic rack for 2 to 5 min until the liquid is clear. Carefully remove and discard all the supernatant.
Centrifigation
Keep the tube on the magnetic rack, add Amount200 µL of 80% ethanol to each tube to wash the beads and tube wall. Wait forDuration00:00:30 . Carefully remove and discard the supernatant.

30s
Repeat step 4.4 once. Try to remove all liquid from the tube. If some liquid remains on the tube wall, centrifuge the tube briefly and place it on the magnetic rack for separation.Remove all liquid by using a low-volume pipette.
Keep the tube on the magnetic rack. Open the tube cap and air-dry the beads at TemperatureRoom temperature until no wetness or glossiness is visible on the beads' surface. There should be no visible cracking on the surface of the beads.
Remove the tube from the magnetic rack and add Amount40 µL of TE buffer to elute the DNA. Mix with a vortexer until all beads are suspended.
Incubate the tube(s) at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the tube briefly and place on the magnetic rack for 2 to 5 min until the liquid is clear. Carefully transfer Amount38 µL of supernatant to a new 0.2 mL PCR tube.
Centrifigation
PCR Amplification
PCR Amplification
PCR Amplification
Add Amount50 µL PCR Enzyme Mix to each sample tube.
Add Amount12 µL of UDB PCR Primer Mix to each sample tube . Vortex 3 times (Duration00:00:03 each) and centrifuge briefly to collect the solution at the bottom of the tube.
ComponentsVolume
Ligation product38 μL
PCR Enzyme Mix50 μL
UDB PCR Primer mix12 μL
Total100 μL
PCR Mixture

3s
Place the PCR tube into the thermocycler. Run the program with the following conditions.
TemperatureTimeCycles
105 ℃ Heated lidon
95 ℃3min1
98 ℃20s
60 ℃15s
72 ℃30s
72 ℃10min1
4 ℃Hold1
PCR program


PCR
When the program is completed, centrifuge the tube(s) briefly and transfer to a new 1.5mL centrifuge tube.
Cleanup of PCR Product
Cleanup of PCR Product
Cleanup of PCR Product
Add Amount100 µL of DNA Clean Beads to each sample tube from 5.4. Mix with a vortexer until all beads are suspended.
Incubate the sample tube at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the sample tube briefly and place on the magnetic rack for 2 to 5 min until the liquid is clear. Carefully remove and discard all the supernatant.
Centrifigation
Keep the tube on the magnetic rack, add Amount200 µL of 80% ethanol to each tube to wash the beads and tube wall. Wait for Duration00:00:30 . Carefully remove and discard the supernatant.
30s
Repeat step 6.4 once. Try to remove all liquid from the tube. If some liquid remains on the tube wall, centrifuge the tube briefly and place it on the magnetic rack for separation. Remove all liquid by using a low-volume pipette.
Keep the tube on the magnetic rack. Open the tube cap and air-dry the beads at TemperatureRoom temperature until no wetness or glossiness is visible on the beads' surface. There should be no visible cracking on the surface of the beads.
Remove the tube from the magnetic rack and add Amount32 µL of TE buffer to elute the DNA. Mix with a vortexer until all beads are suspended.
Incubate the tube(s) at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the tube briefly and place on the magnetic rack for 2 to 5 min until the liquid is clear. Carefully transfer Amount30 µL of supernatant to a new 1.5mL centrifuge tube.
Centrifigation
Quantify the dsDNA with Qubit dsDNA-HS Assay Kit. Yeild for PCR products:>1 pmol.
Assess the size range of purified PCR products with Agilent 2100 Bioanalyzer.
Denaturation and single-stranded circularization
Denaturation and single-stranded circularization
20m 30s
20m 30s
Combined with MGIEasy Dual Barcode Circularization Kit (Cat. No.: 1000020570) for ssCir preparation and further for DNB preparation.
Denaturation
Based on the PCR products concentration, addAmount300 ng of PCR products into a new 0.2 mL PCR tube. If the volume is less than Amount48 µL , add TE Buffer to make a total volume Amount48 µL .
Place the PCR tube(s) into the thermocycler. Run the program with the following conditions.
TemperatureTime
100 ℃ Heated lidOn
95 ℃3 min
4 ℃10 min
Denaturation reaction conditions (Volume: 48 μL)

PCR
After the reaction, centrifuge the tube briefly and place TemperatureOn ice .
Single-stranded circularization
According to the desired reaction number, prepare the circularization reaction mixture in a new 0.2 mL PCR tube TemperatureOn ice . Vortex it 3 times (Duration00:00:03 each), centrifuge briefly, and placeTemperatureOn ice .
ReagentVolume per reaction
Dual Barcode Splint Buffer11.6 μL
DNA Rapid Ligase0.5 μL
Total12.1 μL
Circularization reaction mixture


3s
Add Amount12.1 µL of circularization reaction mixture to each sample tube. Vortex it 3 times (Duration00:00:03 each), centrifuge briefly, and place TemperatureOn ice .

3s
Place the PCR tube(s) into the thermocycler. Run the program with the following conditions.
TemperatureTime
42 ℃ Heated lidOn
37 ℃10 min
4 ℃Hold
Single-stranded DNA circularization reaction conditions (Volume: 60 μL)

PCR
When the program is completed, place the PCR tube(s) TemperatureOn ice , centrifuge briefly, and immediately proceed to the next step.
Digestion
According to the desired reaction number, prepare the digestion mixture in a 0.2 mL PCR tube TemperatureOn ice . Vortex it 3 times (Duration00:00:03 each), centrifuge briefly, and place TemperatureOn ice .

3s
Add Amount4 µL of digestion mixture to each sample tube. Vortex it 3 times (Duration00:00:03 each), centrifuge briefly, and then place TemperatureOn ice .

3s
Place the PCR tube(s) into the thermocycler. Run the program with the following conditions.
TemperatureTime
42 ℃ Heated lidOn
37 ℃10 min
4 ℃Hold
Digestion reaction conditions (Volume: 64 μL)

PCR
When the program is completed, centrifuge the tube briefly and immediately add Amount7.5 µL of Digestion Stop Buffer to each sample tube. Vortex it 3 times (Duration00:00:03 each), centrifuge briefly, and place TemperatureOn ice .

3s
Cleanup of digestion product
Mix the DNA Clean Beads thoroughly. Add Amount130 µL of DNA Clean Beads to each sample tube. Mix with a vortexer until all beads are suspended.
Incubate at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the tube(s) briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Carefully remove and discard the supernatant.
5m
Centrifigation
While keeping the tube(s) on the magnetic rack, add Amount160 µL of 80% ethanol to each tube to wash the beads and tube wall. Wait for Duration00:00:30 . Carefully remove and discard the supernatant.
30s
Repeat step 12.4. Try to remove all liquid from the tube. If some liquid remains on the tube wall, centrifuge the tube briefly and place it on the magnetic rack for separation. Remove all liquid by using a low-volume pipette.
Keep the tube(s) on the magnetic rack. Open the tube cap and air-dry the beads at room temperature until no wetness or glossiness is visible on the beads' surface. There should be no visible cracking on the surface of the beads.
Remove the tube(s) from the magnetic rack and addAmount25 µL of TE Buffer to elute the DNA. Mix with a vortexer until all beads are suspended.
Incubate at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the tube briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Carefully transfer Amount24 µL of supernatant to a new 1.5 mL centrifuge tube.
5m
Centrifigation
QC of digestion product
Quantify the ssCir with Qubit ssDNA Assay Kit. The final Enzymatic Digestion products (ssDNA,
ng) / input products of PCR (dsDNA, 300 ng) should be ≥ 7%.