Nov 04, 2024

Public workspaceDNA Library Prep optimized for low DNA inputs with MGIEasy Fast FS DNA Library Prep Set

DOI
dx.doi.org/10.17504/protocols.io.x54v9reo4v3e/v1
  • 1BGI Research;
  • 2China National GeneBank;
  • 3MGI Tech
Qianyue Ji
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DOI: dx.doi.org/10.17504/protocols.io.x54v9reo4v3e/v1
Protocol Citation: Qianyue Ji, Guoqiang Mai, Xiaohan Wang, Shanshan Liu, Mo Han 2024. DNA Library Prep optimized for low DNA inputs with MGIEasy Fast FS DNA Library Prep Set. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9reo4v3e/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: 110970
Abstract
The MGIEasy Fast FS Library Prep Set(1000006987, MGI Tech) is designed to convert genomic DNA (gDNA) into a special library for DNBSEQTM sequencers and combines the fragmentation, end-repair and add A into one step, to simplify the preparation process and significantly shorten the duration of DNA library preparation, reducing the library prep time in 2 hours. 
This library prep set provides to be optimized for low DNA inputs, suitable for microbial sequencing, metagenomics sequencing, WGS.
Materials
Materials
MGIEasy Dual Barcode Circularization Kit (Cat. No.: 1000020570)
Qubit ssDNA Assay Kit
MGIEasy Fast FS Library Prep Set(1000006987, MGI Tech)

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
Preparation
Preparation
Reagent preparation
Prepare the 1x Elute Enhancer according to the following table. Mix it by vortexing, and centrifuge briefly. Store at TemperatureRoom temperature before using. The shelf life of the 1x Elute Enhancer is 7 days.
ReagentVolume
20x Elute Enhancer1 μL
Nuclease-Free Water19 μL
Total20 μL
1x Elute Enhancer
Mix
Prepare the En-TE according to the following table. Mix it by vortexing, and centrifuge briefly. Store at Temperature4 °C before using. The shelf life of the En-TE is 60 days.
ReagentVolume
1x Elute Enhancer3 μL
TE Buffer1497 μL
Total1500 μL
En-TE
Mix
Prepare the En-Beads according to the following table. Mix it by vortexing, and centrifuge briefly. Store atTemperature4 °C before using. The shelf life of the En-Beads is 60 days.
ReagentVolume
1x Elute Enhancer10 μL
DNA Clean Beads990 μL
Total1000 μL
En-Beads
Mix
Samples Preparation

Fragmentation
The extent of fragmentation (size distribution of DNA fragments) is controlled by time and temperature. Therefore, ensure the accuracy of time and temperature during the reaction. Mix the reagents before use and store the remaining reagents immediately after use.
Normalize gDNA.
Refer to the following table. Based on the sample concentration, transfer the appropriate gDNA (recommended 1 ng - 1000 ng) to a new 0.2 mL PCR tube. Add TE Buffer (Ph8.0 ) to make a total volume of Amount45 µL . Place the normalized gDNA TemperatureOn ice .
ComponentsVolume
TE Buffer (pH 8.0)45 - X μL
gDNA (1 ng - 1000 ng)X μL
Total45 μL
Normalization of gDNA dissolved in TE (pH 8.0)
It is recommended that the normalization buffer should be the same as DNA elution buffer.
Enzyme Preparation
Mix the Fast FS Enzyme II by inverting 10 times and flicking the bottom of the tube(s) gently. Ensure that no residual reagent is left at the bottom each time. Centrifuge briefly, and place it TemperatureOn ice until use.
DO NOT vortex the Fast FS Enzyme II. Insufficient mixing will affect the fragmentation process.
Centrifigation
According to the desired reaction number, prepare the fragmentation mixture in a 1.5 mL centrifuge tube TemperatureOn ice . Vortex it 3 times (Duration00:00:03 each), centrifuge briefly, and place TemperatureOn ice .

3s
Add Amount15 µL of fragmentation mixture to each sample tube from step 2.2(Amount45 µL ). Vortex it 3 times (Duration00:00:03 each), centrifuge briefly, and place TemperatureOn ice .

3s
Place the tube(s) into the thermocycler. Skip the first step (4 ℃ Hold) to start the reaction.
TemperatureTime
70 ℃ Heated lidOn
4 ℃Hold
30 ℃10 ng for 18 min; <1ng for 22 min
65 ℃15 min
4 ℃Hold
Fragmentation reaction conditions (Volume: 60 μL)

PCR
After the reaction, centrifuge the tube(s) briefly and immediately proceed to the next step.
DO NOT STOP AT THIS STEP.
Centrifigation
Cleanup of fragmentation product
Cleanup of fragmentation product
Single size selection,the peak size of the single size product is approximately 500 bp - 750 bp.
Check the volume of the fragmentation product. If the volume is less than 60 μL, add En-TE to make a total volume of Amount60 µL .
Mix the En-Beads thoroughly. Add Amount48 µL of En-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 forDuration00:05:00 until the liquid is clear. Carefully remove and discard all the supernatant. If liquid remains on the tube wall, centrifuge the tube(s) briefly and place it on the magnetic rack for separation. Remove all liquid by using a low-volume pipette.

5m
Centrifigation
Remove the tube(s) from the magnetic rack and add Amount45 µL of En-TE to elute the DNA. Mix with a vortexer until all beads are suspended and centrifuge briefly.
DO NOT STOP AT THIS STEP.
Adapter ligation
Adapter ligation
Adapter ligation

The UDB Adapter is a universal adapter sequence and does not contain Barcode sequences.
Mix the reagents before using and store the remaining reagents immediately after use.
Adapter ligation
Dilute the UDB Adapter with TE Buffer (Ph8.0 ) based on gDNA input.
gDNA input (N ng)Dilution of UDB AdapterVolume after dilution
105 x5 μL
<150 x5 μL
Recommended adapter usage and dilutions for different amounts of gDNA input

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
According to the desired reaction number, prepare the adapter ligation mixture in a 1.5 mL centrifuge tube TemperatureOn ice . Vortex it 6 times (Duration00:00:03 each), centrifuge briefly, and place TemperatureOn ice .
ReagentVolume per reaction
Fast Ligation Buffer23 μL
Ad Ligase5 μL
Ligation Enhancer2 μL
Total30 μL
Adapter ligation mixture
It is recommended to prepare the adapter ligation mixture while waiting for cleanup of fragmentation product. Place it TemperatureOn ice after preparation, and use it within Duration00:30:00 .

30m 3s
Slowly pipette Amount30 µL 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
Pipetting
Place the PCR tube(s) into the thermocycler. Run the program with the following conditions.
TemperatureTime
30 ℃ Heated lidOn
25 ℃10 min
4 ℃Hold
Adapter ligation reaction conditions (Volume: 80 μL)

PCR
When the program is completed, centrifuge the PCR tube(s) briefly and place TemperatureOn ice .
DO NOT STOP AT THIS STEP.
Cleanup of adapter-ligated product
Cleanup of adapter-ligated product
Cleanup of adapter-ligated product
Add Amount22 µL of En-TE to each sample tube.
Mix the En-Beads thoroughly. Add Amount20 µL of En-Beads to each sample tube. Mix with a vortexer until all beads are suspended.
Incubate the sample tube(s) atTemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Duration00:05:00 Duration00:05:00 Centrifuge the sample 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. 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.

15m
Centrifigation
While keeping the PCR 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 6.5. 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 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 Amount20 µL of En-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 forDuration00:05:00 until the liquid is clear. Carefully transfer Amount19 µL of supernatant to a new 0.2 mL PCR tube.
5m
Centrifigation
After cleanup, the adapter-ligated product(s) can be stored at Temperature-20 °C .
PCR
PCR
PCR Preparation
Mix the reagents before using and store the remaining reagents immediately after use. Barcodes are in the UDB PCR Primer Mix.
ReagentRequirement
PCR Enzyme MixThaw at RT: mix by vortexing; centrifuge briefly; place on ice
UDB PCR Primer MixThaw at RT; mix by vortexing; centrifuge briefly; place at RT
Preparing the reagents

Add Amount25 µL PCR Enzyme Mix to each sample tube.
AddAmount6 µL of the corresponding UDB PCR Primer Mix . Vortex 3 times (Duration00:00:03 each) and centrifuge briefly to collect the solution at the bottom of the tube.
ReagentVolume per reaction
Adapter-ligated product19 μL
PCR Enzyme Mix25 μL
Corresponding UDB PCR Primer Mix6 μL
Total50 μL
PCR mixture


3s
PCR running
PCR
Place the PCR tube(s) into the thermocycler. Run the program with the following conditions.
TemperatureTimeCycles
105 ℃ Heated lidOn-
95 ℃3 min1
98 ℃20 sec10 ng for 8cycles: 1ng for 11cycles; 0.1ng for 13cycles
60 ℃15 sec
72 ℃30 sec
72 ℃10 min1
4 ℃Hold-
PCR cycles required to yield 300 ng of libraries
The number of PCR cycles should be strictly controlled.
PCR
When the program is completed, centrifuge the tube(s) briefly.
Cleanup of PCR product
Cleanup of PCR product
Cleanup of PCR product
Mix the En-Beads thoroughly. Add Amount38 µL of En-Beads to each sample tube. Mix with a vortexer until all beads are suspended.
Incubate the sample tube(s) at TemperatureRoom temperature for Duration00:05:00 .
5m
Incubation
Centrifuge the sample tube(s) briefly and place on the magnetic rack for 2 to 5 min until the liquid is clear. Carefully remove and discard all the supernatant. 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.
Centrifigation
While keeping the PCR 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 10.4. 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.
Centrifigation
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 add Amount32 µL of En-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
Centrifuge the tube(s) briefly and place on the magnetic rack for Duration00:05:00 until the liquid is clear. Carefully transfer Amount30 µL of supernatant to a new 0.2 mL PCR tube.

5m
Centrifigation
After cleanup, the adapter-ligated product(s) can be stored at Temperature-20 °C .
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, add Amount300 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.
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 .
Centrifigation
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 15.4. 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%.