TITLE: DNA Library Prep for BGISEQ-500/MGISEQ-2000/DNBSEQ-G400/MGISEQ-200/DNBSEQ-G50
AUTHORS: Hongfang Zhang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This DNA library prep protocol is specifically designed for WGS library construction for MGI highthroughput sequencing platforms. It's applicable for samples from all common animals, plants, fungi bacteria, etc., including human, mice, rice, Arabidopsis, yeast, </span><span style = "font-style:italic;">E. coli,</span><span> and Metagenomics samples. </span></div></div>

[STEPS]
?. Fragmentation
?. Transfer genomic DNA to a new 0.2 mL PCR tube. The volume should be less than or equal to . If the volume is less than , add dilution buffer to bring the final volume to
45 µl
45 µl
45 µl
?. Mix Frag Enzyme II by inverting 10 times then centrifuge briefly and place on ice for use. DO NOT vortex Frag Enzyme II. Vortex Frag Buffer II 3 times (3s each) then centrifuge briefly and place on ice for use.
?. Prepare the fragmentation mixture on ice. Pipette up and down at least 10 times to mix thoroughly. (DO NOT vortex)
?. Transfer  of the fragmentation mixture to the 0.2 mL PCR tube from step 1.1. Pipette at least 10 times to mix thoroughly and briefly centrifuge to collect the solution at the bottom of the tube.
15 µl
?. Set and run the following program on the thermocycler (see Table 1.3). Make sure the thermocycler has cooled to .Place the 0.2 mL PCR tube from step 1.4 into the thermocycler and skip the  Hold step to start the reaction at .
4 °C
4 °C
30 °C
?. Briefly centrifuge to collect the solution at the bottom of the tube.
?. Size Selection
?. Take DNA Clean Beads out of the refrigerator and allow  for the solution to warm to room temperature. Vortex and mix thoroughly before use.
?. Transfer  of DNA Clean Beads to the 1.5 mL centrifuge tube containing of fragmentation product from step 1.6. Pipette up and down at least 10 times or vortex to mix thoroughly.
36 µl
60 µl
?. Incubate at room temperature for .
?. Centrifuge briefly and place the tube onto the Magnetic Separation Rack for until the liquid becomes clear. Then, carefully transfer the supernatant to a new 1.5 mL centrifuge tube.
?. Transfer  of DNA Clean Beads to the centrifuge tube with of supernatant. Pipette at least 10 times to mix thoroughly.
12 µl
96 µl
?. Incubate at room temperature for .
?. Centrifuge briefly and place the tube onto a Magnetic Separation Rack for  until the liquid becomes clear. Carefully remove and discard the supernatant with pipette.
?. Keep the centrifuge tube on the Magnetic Separation Rack and add of freshly prepared 80% ethanol to wash the beads and the walls of the tube. Carefully remove and discard the supernatant.
200 µl
?. Repeat step 2.8 once. Remove all of the liquid from the tube without disrupting the beads. You may centrifuge briefly to collect any remaining liquid at the bottom of the centrifuge tube, separate the beads magnetically, and then remove any remaining liquid using a small volume pipette.
?. Keep the centrifuge tube on the Magnetic Separation Rack with the lid open, and air-dry beads at room temperature until no wetness (reflectiveness) is observed but before the pellet begins to crack.
?. Remove the centrifuge tube from the Magnetic Separation Rack and add of TE Buffer to elute the DNA. Pipette up and down at least 10 times to mix thoroughly.
43 µl
?. Incubate at room temperature for .
?. Centrifuge briefly and place the tube onto a Magnetic Separation Rack for  until the liquid becomes clear. Carefully transfer  of supernatant to a new 0.2 mL PCR tube.
41 µl
?. Quantify the Size selection products with dsDNA Fluorescence Assay Kits such as Qubit® dsDNA HS Assay Kit or Quant-iT™ PicoGreen® dsDNA Assay Kit.
?. End Repair and A-tailing
?. Transfer ≤100 ng of fragmentation product to a new 0.2 mL PCR tube. Add TE Buffer for a total volume of .
40 µl
?. Prepare the end repair and A-tailing mixture on ice (see Table 3.1).
?. Transfer  of the end repair and A-tailing mixture to the 0.2 mL PCR tube from step 3.1. Vortex 3 times (3s each) and briefly centrifuge to collect the solution at the bottom of the tube.
10 µl
?. Place the 0.2 mL PCR tube from step 3.3 into the thermocycler and run the program in Table 3.2.
?. Briefly centrifuge to collect the solution at the bottom of the tube.
?. Adapter Ligation
?. Add 5 μL of MGIEasy DNA Adapters (diluent) to the PCR tube from step 3.5. Vortex 3 times (3s each) and briefly centrifuge to collect the solution at the bottom of the tube.
?. Prepare the Adapter ligation mixture on ice.
?. Pipette slowly and transfer  of Adapter ligation mixture to the 0.2 mL PCR tube from step 4.1. Vortex 6 times (3s each) and centrifuge briefly to collect the solution at the bottom of the tube.
25 µl
?. Place the PCR tube from step 4.3 into the thermocycler and run the program.
?. Centrifuge briefly to collect the solution at the bottom of the tube.
?. Add  of TE Buffer for a total volume of  and transfer all of the solution to a new 1.5 mL centrifuge tube.
20 µl
100 µl
?. Adapter-Ligated DNA Cleanup
?. Take DNA Clean Beads out of the refrigerator and allow  for the solution to warm to room temperature. Vortex and mix thoroughly before use.
?. Transfer  of DNA Clean Beads to the centrifuge tube from step 4.6. Pipette up and down at least 10 times or vortex to mix thoroughly.
50 µl
?. Incubate at room temperature for .
?. Centrifuge briefly and place the tube onto a Magnetic Separation Rack for  until the liquid becomes clear. Carefully remove and discard the supernatant with a pipette.
?. Keep the tube on the Magnetic Separation Rack and add  freshly prepared 80% ethanol to wash the beads and the walls of the tube. Incubate for 30 seconds, then carefully remove and discard the supernatant.
200 µl
?. Repeat step 5.5 once. Remove all of the liquid from the tube without disrupting the beads. You may centrifuge briefly to collect any remaining liquid at the bottom of the centrifuge tube, separate the beads magnetically and then remove any remaining liquid using a small volume pipette.
?. Keep the centrifuge tube on the Magnetic Separation Rack with the lid open and air-dry beads at room temperature until no wetness (reflectiveness) is observed, but before the pellet begins to crack.
?. Remove the centrifuge tube from the Magnetic Separation Rack and add of TE Buffer to elute the DNA. Pipette up and down at least 10 times to mix thoroughly.
21 µl
?. Incubate at room temperature for .
?. Centrifuge briefly and place the centrifuge tube back onto the Magnetic Separation Rack for  until the liquid becomes clear. Transfer of supernatant to a new 0.2 mL PCR tube.
19 µl
?. PCR Amplification
?. Prepare the PCR amplification mixture on ice.
?. Transfer  of PCR amplification mixture to the PCR tube from step 5.10. Vortex 3 times (3s each) and centrifuge briefly to collect the solution at the bottom of the tube.
31 µl
?. Place the PCR tube from step 6.2 into the thermocycler and run the program in Table 6.2. Please refer to Table 6.2 to adjust the number of cycles with different DNA input.
?. Centrifuge briefly to collect the solution at the bottom of the tube.
?. Transfer all the solution to a new 1.5 mL centrifuge tube.
?. Cleanup of PCR Product
?. Take DNA Clean Beads out of the refrigerator and allow  for the solution to warm to room temperature. Vortex and mix thoroughly before use.
?. Transfer of DNA Clean Beads to the centrifuge tube from step 6.5. Pipette up and down at least 10 times or vortex to mix thoroughly.
50 µl
?. Incubate at room temperature for .
?. Centrifuge briefly and place the tube onto a Magnetic Separation Rack for until the liquid becomes clear. Carefully remove and discard the supernatant with a pipette.
?. Keep the tube on the Magnetic Separation Rack and addof freshly prepared 80% ethanol to wash the beads and the walls of the tube. Incubate for and carefully remove and discard the supernatant.
200 µl
?. Repeat step 7.5 once. Remove all of the liquid from the tube without disrupting the beads. You may centrifuge briefly to collect any remaining liquid at the bottom, separate the beads magnetically, and remove any remaining liquid using a small volume pipette.
?. Keep the centrifuge tube on the Magnetic Separation Rack with the lid open, and air-dry beads at room temperature until no wetness (reflectiveness) is observed but before the pellet begins to crack.
?. Remove the centrifuge tube from the Magnetic Separation Rack and add of TE Buffer to elute the DNA. Pipette up and down at least 10 times to mix thoroughly.
32 µl
?. Incubate at room temperature for .
?. Centrifuge briefly, then place the centrifuge tube back onto the Magnetic Separation Rack for until the liquid becomes clear. Transfer  of supernatant to a new 1.5 mL centrifuge tube.
30 µl
?. Quality Control of PCR Product
?. Quantify the purified PCR products with dsDNA Fluorescence Assay Kits such as Qubit® dsDNA HS Assay Kit or Quant-iT™ PicoGreen® dsDNA Assay Kit. The required yield for PCR products is ≥ 1 pmol. Please refer to Formula 1 to calculate the amount of DNA needed. For example, for 384 bp PCR product the yield should reach . For pooled sequencing, please follow instructions provided by MGIEasy DNA Adapters User Manual. Detailed information shows how to plan your sample pooling. Quantify your Adapter-ligated samples before pooling. The total yield after pooling should be 1 pmol, with a total volume ≤ . Formula 1 Conversion between 1 pmol of dsDNA sample and Mass in ng Mass (ng) corresponding to 1 pmol PCR Products= DNA Fragment Size (bp) /1000 bp ×660 ng
250 ng
48 µl
?. Assess the fragment size distribution of purified PCR products with electrophoresis-based equipment such as Bioanalyzer, Tapestation (Agilent Technologies), LabChip® GX, GXII, GX Touch (PerkinElmer), or Fragment Analyzer (Advanced Analytical).
?. Denaturation
?. According to the PCR product size, transfer 1 pmol of PCR product to a new 0.2 mL PCR tube. Add TE Buffer for a total volume of .
48 µl
?. Place the 0.2 mL PCR tube from step 9.1 into the thermocycler and run the program in Table 9.1.
?. After the reaction is completed, immediately place the 0.2 mL PCR tube on ice for , then centrifuge briefly.
?. Single Strand Circularization
?. Prepare the single strand circularization mixture on ice (see Table 10.1).
?. Transfer 12.1 μL single strand circularization mixture to the 0.2 mL PCR tube from step 9.3. Vortex 3 times (3s each) and centrifuge briefly to collect the solution at the bottom of the tube.
?. Place the PCR tube into the thermocycler and run the program in Table 10.2.
?. After the reaction is complete, immediately place the tube on ice for the next reaction.
?. Enzymatic Digestion
?. Prepare the following enzymatic digestion mixture (see Table 11.1) on ice during the reaction in step 10.3.
?. Transfer  of enzymatic digestion mixture into the PCR tube from step 10.4. Vortex 3 times (3s each) and centrifuge briefly to collect the solution at the bottom of the tube.
4 µl
?. Place the PCR tube from step 11.2 into the thermocycler and run the program in Table 11.2.
?. Centrifuge briefly to collect the solution at the bottom of the tube.
?. Add Digestion Stop Buffer to the 0.2 mL PCR tube. Vortex 3 times (3s each) and centrifuge briefly to collect the solution at the bottom of the tube. Transfer all of the solution into a new 1.5 mL centrifuge tube.
7.5 µl
?. Enzymatic Digestion Product Cleanup
?. Take DNA Clean Beads out of the refrigerator and allow for the solution to warm to room temperature. Vortex and mix thoroughly before use.
?. Transfer  of DNA Clean Beads to the Enzymatic Digestion product from step 11.5. Gently pipette at least 10 times or vortex to mix thoroughly.
170 µl
?. Incubate at room temperature for .
?. Centrifuge briefly and place the 1.5 mL tube on the Magnetic Separation Rack for  until the liquid becomes clear. Carefully remove and discard the supernatant using a pipette.
?. With the 1.5 mL tube on the Magnetic Separation Rack, add  of freshly prepared 80% ethanol to the tube without disturbing the beads. Incubate for . Carefully remove and discard the supernatant.
500 µl
?. Repeat step 12.5 once. Remove all of the liquid from the tube without disrupting the beads. You may centrifuge briefly to collect any remaining liquid at the bottom, separate the beads magnetically, then remove any remaining liquid using a small volume pipette.
?. Keep the 1.5 mL centrifuge tube on the Magnetic Separation Rack with the lid open, and air-dry beads at room temperature until no wetness (reflectiveness) is observed but before the pellet begins to crack.
?. Remove the 1.5 mL centrifuge tube from the Magnetic Separation Rack and add  of TE Buffer to elute the DNA. Gently pipette up and down at least 10 times to mix thoroughly or until the beads are fully resuspended.
32 µl
?. Incubate at room temperature for .
?. Centrifuge briefly and place the centrifuge tube back onto the Magnetic Separation Rack for until the liquid becomes clear. Transfer of supernatant to a new 1.5 mL centrifuge tube.
30 µl
?. Quality Control of Enzymatic Digestion Product Quantitate the purified Enzymatic Digestion product with Qubit® ssDNA Assay Kit. The final Enzymatic Digestion products (ssDNA, ng) / input products of PCR (dsDNA, ng) should be ≥ 7%. For example, for 384 bp PCR product the final Enzymatic Digestion products should reach 17.5 ng.