Oct 15, 2020
Version 2
BGISEQ-500 WGS library construction V.2
Version 1 is forked from BGISEQ-500 WGS library construction
This protocol is a draft, published without a DOI.
- Jie Huang1,
- Xinming Liang2,
- Yuankai Xuan3,
- Chunyu Geng2,
- Yuxiang Li2,
- Haorong Lu2,
- Shoufang Qu1,
- Xianglin Mei3,
- Hongbo Chen1,
- Ting Yu1,
- Nan Sun1,
- Junhua Rao2,
- Jiahao Wang4,
- Wenwei Zhang2,
- Ying Chen2,
- Sha Liao2,
- Hui Jiang2,
- Xin Liu2,
- Zhaopeng Yang1,
- Feng Mu2,
- Shangxian Gao1
- 1National Institutes for food and drug Control (NIFDC);
- 2BGI-Shenzhen;
- 3State Food and Drug Administration Hubei Center for Medical Equipment Quality Supervision and Testing;
- 4BGI-Qingdao
- BGI
External link: https://doi.org/10.46471/GIGABYTE_SERIES_0004
Protocol Citation: 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 2020. BGISEQ-500 WGS library construction. protocols.io https://protocols.io/view/bgiseq-500-wgs-library-construction-bngwmbxeVersion created by Hongling Zhou
Manuscript citation:
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 15, 2020
Last Modified: October 15, 2020
Protocol Integer ID: 43254
Abstract
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.
Materials
MATERIALS
Fresh 80% ethanolXILONG SCIENTIFIC
ERAT Buffer
ERAT Enzyme
Adapter Mix
Ligation Buffer
Ligation Enzyme
TE bufferAmbion
PCR Enzyme Mix
Splint Buffer
Digestion Buffer
Digestion Enzyme
STEP MATERIALS
Fresh 80% ethanolXILONG SCIENTIFIC
ERAT Buffer
ERAT Enzyme
Adapter Mix
Ligation Buffer
Ligation Enzyme
TE bufferAmbion
PCR Enzyme Mix
Splint Buffer
Digestion Buffer
Digestion Enzyme
Protocol materials
Fresh 80% ethanolXILONG SCIENTIFIC
ERAT Enzyme
ERAT Enzyme
Ligation Buffer
TE bufferAmbion
Digestion Buffer
Digestion Enzyme
ERAT Buffer
Adapter Mix
PCR Enzyme Mix
PCR Enzyme Mix
Ligation Enzyme
TE bufferAmbion
Digestion Buffer
Digestion Enzyme
Adapter Mix
Ligation Enzyme
Splint Buffer
ERAT Buffer
Fresh 80% ethanolXILONG SCIENTIFIC
Ligation Buffer
Splint Buffer
Fresh 80% ethanolXILONG SCIENTIFIC
ERAT Enzyme
ERAT Buffer
Adapter Mix
Ligation Buffer
Ligation Enzyme
TE bufferAmbion
PCR Enzyme Mix
Splint Buffer
Digestion Buffer
Digestion Enzyme
Overview
Overview
DNA fragmentation
DNA fragmentation
1) Input genomic DNA sample
Genomic DNA Sample Recommendation
| Nucleic Acid | *High-quality genomic DNA | |
| Molecular Weight | >23k bp | |
| Amount | 1μg | |
| Concentration | ≥12.5ng/μL | |
| Purity | 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 electrophoresis
2) Fragmentation
Use 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
| Sequencing with | Input Amount | Reaction Volume | Derived Fragments | |
| PE 100 | 1μg | 80μL | 100-700 bp (main band≈200-300 bp) | |
| PE 50 | 1μg | 80μL | 100-500 bp (main band≈200 bp) | |
| PE 150 | 1μg | 80μL | 100-700 bp (main band≈400 bp) |
3) Bead-based Cleanup
- Place 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) Homogenization
- 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. 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.
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Fresh 80% ethanolXILONG SCIENTIFIC
End Repair and Tailing
End Repair and Tailing
1) Prepare the mixture as follows in PCR tube (do not vortex enzymes):
| Components | Volume | |
| DNA | 40μL | |
| ERAT Buffer | 7.1μL | |
| ERAT Enzyme | 2.9μL | |
| Total | 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:
| Temperature | Time | |
| Heated lid | On | |
| 37℃ | 30 min | |
| 65℃ | 15 min | |
| 4℃ | Hold |
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ERAT Buffer
ERAT Enzyme
Ligate Adapters
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):
| Components | Volume | |
| Ligation Buffer | 23.4 μL | |
| Ligation Enzyme | 1.6 μL | |
| Total | 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:
| Temperature | Time | |
| Heated lid | On | |
| 23℃ | 30 min | |
| 4℃ | 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.
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Adapter Mix
Ligation Buffer
Ligation Enzyme
TE bufferAmbion
Purify Ligated DNA
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
PCR
1)
| Components | Volume | |
| DNA | 44 μL | |
| PCR Enzyme Mix | 50 μL | |
| PCR Primer Mix | 6 μL | |
| Total | 100 μL |
2) Place above PCR tube in a Thermal Cycler, and the initiate the reaction as per following conditions:
| Temperature | Time | Cycles | |
| Heated lid | On | ||
| 95℃ | 3 min | ||
| 98℃ | 20 sec | ||
| 60℃ | 15 sec | 8 | |
| 72℃ | 30 sec | ||
| 72℃ | 10 min | ||
| 4℃ | Hold |
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PCR Enzyme Mix
Purify PCR Product
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
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
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:
| Components | Volume | |
| Splint Buffer | 11.6 μL | |
| Ligation Enzyme | 0.2 μL | |
| Total | 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:
| Temperature | Time | |
| Heated lid | on | |
| 37℃ | 30 min | |
| 4℃ | Hold |
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Splint Buffer
Digestion
Digestion
1) Prepare digestion reaction solution on ice as per following system:
| Components | Volume | |
| Digestion Buffer | 1.4 μL | |
| Digestion Enzyme | 2.6 μL | |
| Total | 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:
| Temperature | Time | |
| Heated lid | on | |
| 37℃ | 30 min | |
| 4℃ | 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.
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Digestion Buffer
Digestion Enzyme
Purify Digestion Product
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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