TITLE: BGISEQ-500 WGS library construction
AUTHORS: Jie Huang, Xinming Liang, Yuankai Xuan, Chunyu Geng, Yuxiang Li, Haorong Lu, Shoufang Qu, Xianglin Mei, Hongbo Chen, Ting Yu, Nan Sun, Junhua Rao, Jiahao Wang, Wenwei Zhang, Ying Chen, Sha Liao, Hui Jiang, Xin Liu, Zhaopeng Yang, Feng Mu, Shangxian Gao

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">BGISEQ-500 is a desktop sequencer developed by BGI. Using DNA nanoball and combinational probe anchor synthesis developed from Complete Genomics™ sequencing technologies, it generates short reads at a large scale. Library construction on the platform includes fragmentation, size selection, end repair and A-tailing, adaptor ligation, PCR amplification, and splint circularization.</div></div>

[STEPS]
?. [Overview]
?. [DNA fragmentation]
1) Input genomic DNA sampleGenomic DNA Sample Recommendation  AB1Nucleic Acid
*High-quality genomic DNA
2Molecular Weight
&gt;23k bp
3Amount
1μg
4Concentration
≥12.5ng/μL
5Purity
OD260/OD280=1.8~2.0
*High-quality genomic DNA should be free of salt or organics. It could run as an intact band with DNA length >23kb during 1% agarose gel electrophoresis2) FragmentationUse the Covaris Focused-ultrasonicator for genomic DNA fragmentation following the instructions of the instrument. Optimisation should be performed on DNA prior to experiment and analyzed with agarose electrophoresis or an Agilent 2100 BioAnalyzer  ABCD1Sequencing with
Input Amount
Reaction Volume
Derived Fragments
2PE 100
1μg
80μL
100-700 bp (main band≈200-300 bp)
3PE 50
1μg
80μL
100-500 bp (main band≈200 bp)
4PE 150
1μg
80μL
100-700 bp (main band≈400 bp)
3) Bead-based CleanupPlace AMPure XP magnetic beads at RT for 30 min, fully thaw before use.PE100:Pipette 48 μL AMPure XP magnetic beads to 80 μL shearing product, and mix well by gently pipetting 10 times, incubate for 5min at room temperature. ( PE50:Pipette 80 μL AMPure XP magnetic beads to 80 μL shearing product, and mix well by gently pipetting 10 times, incubate for 5min at room temperature.PE150:Pipette 44 μL AMPure XP magnetic beads to 80 μL shearing product, and mix well by gently pipetting 10 times, incubate for 5min at room temperature. )After brief centrifugation, place the non-stick tube on the magnet for 2min until the liquid clears, carefully transfer the supernatant to a new non-stick tube with a pipette.PE100: Pipette 16 μL AMPure XP magnetic beads to 128 μL supernatant, mix well by gently pipetting 10 times, and incubate at room-temperature for 5 min. (PE50: Pipette 40 μL AMPure XP magnetic beads to 160 μL supernatant, mix well by gently pipetting 10 times, and incubate at room-temperature for 5 min. PE150: Pipette 12 μL AMPure XP magnetic beads to 124 μL supernatant, mix well by gently pipetting 10 times, and incubate at room-temperature for 5 min.)After brief centrifugation, place the non-stick tube on the magnet for 2min until the liquid clears, remove and discard the supernatant with a pipette.Add 500 μL of fresh 80% ethanol, while the tube remains on the magnet, then, rotate the tubes in the rack by half turns 4 times to wash the beads then carefully remove and discard the supernatant after 1 min.Repeat step 6 once, and remove all liquid from tube without disrupting the beads.4) HomogenizationUse double-strand DNA quantification kit such as Qubit® dsDNA HS Assay Kit or Quant-iT™ PicoGreen® dsDNA Assay Kit, and quantify the sample as per the instructions of the quantification kit.4) Homogenization      2. Remove 50 ng of sample (calculated based on its concentration) to a new 0.2 mL PCR tube, and add NF             water to final volume of 40 μL.
AB1Nucleic Acid
*High-quality genomic DNA
2Molecular Weight
&gt;23k bp
3Amount
1μg
4Concentration
≥12.5ng/μL
5Purity
OD260/OD280=1.8~2.0
ABCD1Sequencing with
Input Amount
Reaction Volume
Derived Fragments
2PE 100
1μg
80μL
100-700 bp (main band≈200-300 bp)
3PE 50
1μg
80μL
100-500 bp (main band≈200 bp)
4PE 150
1μg
80μL
100-700 bp (main band≈400 bp)
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?. [End Repair and Tailing]
1) Prepare the mixture as follows in PCR tube (do not vortex enzymes):  AB1Components
Volume
2DNA
40μL
3ERAT Buffer
7.1μL
4ERAT Enzyme
2.9μL
5Total
50μL
2) Mix well by gently pipetting (Do not mix by vortexing), concentrate the reaction liquid to tube bottom by brief centrifugation.3) Place the PCR tube containing the reaction mixture of above step in a Thermal Cycler, and initiate the reaction as per the following conditions:  AB1Temperature
Time
2Heated lid
On
337℃
30 min
465℃
15 min
54℃
Hold
AB1Components
Volume
2DNA
40μL
3ERAT Buffer
7.1μL
4ERAT Enzyme
2.9μL
5Total
50μL
AB1Temperature
Time
2Heated lid
On
337℃
30 min
465℃
15 min
54℃
Hold
[3）]
?. [Ligate Adapters]
1) Add 5 μL of Adapter Mix to above PCR tube, and mix well by pipetting. Now 16 Adapter Mix are available, 8 libraries in one lane strategy, every sample with 4 different barcodes.2) Prepare the following reaction mixture (Note: Ligation Buffer II is viscous, pipette slowly):  AB1Components
Volume
2Ligation Buffer
23.4 μL
3Ligation Enzyme
1.6 μL
4Total
25 μL
3) Add 25 μL of above reaction mixture to the reaction solution containing adapters from above step.4) Place the tube in a Thermal Cycler, then initiate reaction as per following condition:  AB1Temperature
Time
2Heated lid
On
323℃
30 min
44℃
Hold
5) After ligation, add 20 μL TE to final volume of 100 μL, then transfer entire volume to a non-stick tube containing 50 uL of room temperature AMPure beads and mix by slow pipetting 10 times to avoid bubble formation.
AB1Components
Volume
2Ligation Buffer
23.4 μL
3Ligation Enzyme
1.6 μL
4Total
25 μL
AB1Temperature
Time
2Heated lid
On
323℃
30 min
44℃
Hold
[4）]
?. [Purify Ligated DNA]
1) Incubate at room temperature for 5 min.2) After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, remove and discard the supernatant with a pipette:3) Add 500 μL of fresh 80% ethanol, while the tube remains on the magnet, then, rotate the tubes in the rack by half turns 4 times to wash the beads. Carefully remove and discard the supernatant after 1 min.4) Repeat step 4) once, remove all liquid from tube without disrupting the beads.5) Open the cap of non-stick tube, while the tube remains on the magnet, and dry at room temperature for 3 min.6) Remove the non-stick tube from the magnet, add 46 μL of TE for DNA elution, mix well by pipetting and incubate at room temperature for 5 min.7) After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, transfer all 44 μL of supernatant to a new 0.2 mL PCR tube ready for PCR in next step, or store at -20℃.
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?. [PCR]
1)  AB1Components
Volume
2DNA
44 μL
3PCR Enzyme Mix
50 μL
4PCR Primer Mix
6 μL
5Total
100 μL
2) Place above PCR tube in a Thermal Cycler, and the initiate the reaction as per following conditions:  ABC1Temperature
Time
Cycles
2Heated lid
On
&nbsp;
395℃
3 min
&nbsp;
498℃
20 sec
&nbsp;
560℃
15 sec
8
672℃
30 sec
&nbsp;
772℃
10 min
&nbsp;
84℃
Hold
&nbsp;
AB1Components
Volume
2DNA
44 μL
3PCR Enzyme Mix
50 μL
4PCR Primer Mix
6 μL
5Total
100 μL
ABC1Temperature
Time
Cycles
2Heated lid
On
&nbsp;
395℃
3 min
&nbsp;
498℃
20 sec
&nbsp;
560℃
15 sec
8
672℃
30 sec
&nbsp;
772℃
10 min
&nbsp;
84℃
Hold
&nbsp;
[2）]
?. [Purify PCR Product]
1) Place AMPure XP magnetic beads at room temperature 30 min in advance, mix well by vortexing before use.2) Add 100 μL of AMPure XP magnetic beads to 100 μL of PCR product, mix well by gently pipetting 10 times, and incubate at room temperature for 5 min.3) After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, remove and discard the supernatant with a pipette.4) Add 500 μL of fresh 80% ethanol, while the tube remains on the magnet, then, rotate the tubes in the rack by half turns 4 times to wash the beads. Carefully remove and discard the supernatant after 1 min.5) Repeat step 4) once, and try to suck up all liquid from tube bottom.6) Open the cap of non-stick tube, while the tube remains on the magnet, and dry at room temperature for 3 min.7) Remove the non-stick tube from the magnet, add 32 μL of TE water for DNA elution, mix well by pipetting and incubate at room temperature for 5 min.8) After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, transfer the supernatant to a new non-stick tube. Proceed next step reaction or store at -20℃.
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?. [Homogenization]
1）Use double-strand DNA quantification kit such as Qubit® dsDNA HS Assay Kit or Quant-iT™ PicoGreen® dsDNA Assay Kit, and quantify the sample as per the instructions of the quantification kit.2）It is recommended to mix samples of different Barcodes here.3）Add mixed sample (calculated based on its concentration) to a PCR tube, and add NF water to final volume of 48 μL.
?. [Circularization]
1)Denature the homogenized PCR product on a Thermal Cycler at 95℃ for 3 min, then immediately transfer to ice batch.2) Prepare reaction mixture on ice as per following system:  AB1Components
Volume
2Splint Buffer
11.6 μL
3Ligation Enzyme
0.2 μL
4Total
11.8 μL
3) Add 11.8 μL of above reaction mixture to 48 μL of denatured DNA.4) Place above PCR tube in a Thermal Cycler, and initiate the reaction as per following conditions:  AB1Temperature
Time
2Heated lid
on
337℃
30 min
44℃
Hold
AB1Components
Volume
2Splint Buffer
11.6 μL
3Ligation Enzyme
0.2 μL
4Total
11.8 μL
AB1Temperature
Time
2Heated lid
on
337℃
30 min
44℃
Hold
[1）]
[4）]
?. [Digestion]
1) Prepare digestion reaction solution on ice as per following system:  AB1Components
Volume
2Digestion Buffer
1.4 μL
3Digestion Enzyme
2.6 μL
4Total
4 μL
2) After the circularization reaction is finished, directly add 4 μL of digestion reaction solution into circularized DNA solution, mix well and briefly centrifuge, then place the PCR tube in a Thermal Cycler, and initiate the reaction as per following conditions:  AB1Temperature
Time
2Heated lid
on
337℃
30 min
44℃
Hold
3) Add 7.5 μL of Digestion Stop Buffer to each reaction, mix well to terminate the reaction.4) Transfer all the reaction solution to a new non-stick tube, ready for purification.
AB1Components
Volume
2Digestion Buffer
1.4 μL
3Digestion Enzyme
2.6 μL
4Total
4 μL
AB1Temperature
Time
2Heated lid
on
337℃
30 min
44℃
Hold
[2）]
?. [Purify Digestion Product]
1) Place AMPure XP magnetic beads and place at room temperature for 30 min in advance. Mix well by vortexing before use.2) Pipette 168 μL AMPure XP magnetic beads to digestion product, mix well by pipetting 10 times, and incubate at room temperature for 10 min.3) After transient centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, remove and discard the supernatant with a pipette:4) Add 500 μL of fresh 80% ethanol, while the tube remains on the magnet, then, rotate the tubes in the rack by half turns 4 times to wash the beads. Carefully remove and discard the supernatant after 1 min.5) Repeat step 4) once, and try to suck up all liquid from tube bottom.6) Open the cap of non-stick tube, while the tube remains on the magnet, and dry at room temperature for 3 min.7) Remove the non-stick tube from the magnet, add 32 μL of TE for DNA elution, mix well by pipetting and incubate at room temperature for 10 min.8) After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, transfer the supernatant to a new non-stick tube. Store at -20℃, ready for preparation of DNB.
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