```markdown
# Goal/Experiment:
Construct a Whole Genome Sequencing (WGS) Library using BGI's BGISEQ-500.

# BGISEQ-500 WGS Library Construction

**Authors:** Jie Huang, Xinming Liang, Yuankai Xuan, Chunyv 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

## 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
- Fresh 80% ethanol by [XILONG SCIENTIFIC](https://www.xilong.cn/)
- ERAT Buffer by Contributed by users
- ERAT Enzyme by Contributed by users
- Adapter Mix by Contributed by users
- Ligation Buffer by Contributed by users
- Ligation Enzyme by Contributed by users
- TE buffer by [Ambion](https://www.thermofisher.com/)
- PCR Enzyme Mix by Contributed by users
- Splint Buffer by Contributed by users
- Digestion Buffer by Contributed by users
- Digestion Enzyme by Contributed by users

## Protocol

### Overview
Step 1.

![Flow Chart](images/flow_chart.png)

### DNA Fragmentation
**Step 2.**

#### 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=1.82.0 |

> 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. Optimization should be performed on DNA prior to the experiment and analyzed with agarose electrophoresis or an Agilent 2100 BioAnalyzer.

**Sequencing with Input Amount Reaction Volume Derived Fragments**

|                        | PE 100 | PE 50  | PE 150 |
|------------------------|--------|--------|--------|
| Input Amount           | 1µg    | 1µg    | 1µg    |
| Reaction Volume        | 80µL   | 80µL   | 80µL   |
| Derived Fragments      | 100-700 bp (main band≈200-300 bp) | 100-500 bp (main band≈200 bp) | 100-700 bp (main band≈400 bp) |

#### 3) Bead-based Cleanup
1. Place AMPure XP magnetic beads at room temperature (RT) for 30 min, fully thaw before use.
2. **PE100:** Pipette 48µL AMPure XP magnetic beads to 80µL shearing product, and mix well by gently pipetting 10 times, incubate for 5 min at room temperature *(PE50: Pipette 40µL AMPure XP magnetic beads to 80µL shearing product, and mix well by gently pipetting 10 times, incubate for 5 min at room temperature. PE150: Pipette 44µL AMPure XP magnetic beads to 80µL shearing product, and mix well by gently pipetting 10 times, incubate for 5 min at room temperature.)”
3. After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, carefully transfer the supernatant to a new non-stick tube with a pipette.
4. **PE100:** Pipette 16µL AMPure XP magnetic beads to 128µL supernatant, mix well by gently pipetting 10 times, and incubate at room temperature for 5 min. *(PE50: Pipette 40µL AMPure XP magnetic beads to 160µL supernatant, mix well by gently pipetting 10 times, and incubate at room temperature for 5 min. PE150: Pipette 12µL AMPure XP magnetic beads to 124µL supernatant, mix well by gently pipetting 10 times, and incubate at room temperature for 5 min.)*
5. 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.
6. Add 500µL 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.
7. Repeat step 6 once, and remove all liquid from the tube without disrupting the beads.

#### 4) Homogenization
1. Use a 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 50ng of sample (calculated based on its concentration) to a new 0.2mL PCR tube, and add NF water to the final volume of 40µL.

### End Repair and Tailing
**Step 3.**

1. Prepare the mixture as follows in PCR tube (do not vortex enzymes):

    **Components**

    | DNA | 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 the above step in a Thermal Cycler, and initiate the reaction as per the following conditions:

    | Temperature | Time |  |
    |-------------|------|--|
    | Heated lid  | On     |  |
    | 37°C | 30 min |  |
    | 65°C | 15 min |  |
    | 4°C | Hold |  |

### Ligate Adapters
**Step 4.**

1. Add 5µL 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 | 23.4µL |
    | Ligation Enzyme | 1.6 µL |
    | **Total** | **25µL** |

3. Add 25µL of the 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°C | 30 min |  |
    | 4°C | Hold |  |

5. After ligation, add 20µL TE to the final volume of 100µL, then transfer the entire volume to a non-stick tube containing 50µL of room temperature AMPure beads and mix by slow pipetting 10 times to avoid bubble formation.

### Purify Ligated DNA
**Step 5.**

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 500µL 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 46µL 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 44µL of supernatant to a new 0.2mL PCR tube ready for PCR in the next step or store at -20°C.

### PCR
**Step 6.**

1. Prepare the PCR reaction mixture as follows:

    **Components**

    | | Volume |
    |----------------|----|
    | DNA | 44µL |
    | PCR Enzyme Mix | 50µL |
    | PCR Primer Mix | 6µL |
    | **Total** | 100µL |

2. Place the above PCR tube in a Thermal Cycler and initiate the reaction as per the following conditions:

    | Temperature | Time | Cycles |
    |-------------|------|--------|
    | Heated lid    | On     |          |
    | 95°C         | 3 min  |          |
    | 98°C         | 20 sec |          |
    | 60°C         | 15 sec | 8       |
    | 72°C         | 30 sec |          |
    | 72°C         | 10 min |          |
    | 4°C          | Hold   |          |

### Purify PCR Product
**Step 7.**

1. Place AMPure XP magnetic beads at room temperature 30 min in advance, mix well by vortexing before use.
2. Add 100µL of AMPure XP magnetic beads to 100µL 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 500µL 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, try to suck up all liquid from the tube's bottom.
6. Open the cap of the 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 32µL 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 to the next step of the reaction or store at -20°C.

### Homogenization
**Step 8.**

1. Use double strrand 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 the final volume of 48µL.

### Circularization
**Step 9.**

1. Denature the homogenized PCR product on a Thermal Cycler at 95°C for 3 min, then immediately transfer to an ice bath.
2. Prepare reaction mixture on ice as per the following system:

    **Components**

    | | Volume |
    |-----------------|--|
    | Splint Buffer     | 11.6µL |
    | Ligation Enzyme | 0.2 µL |
    | **Total** | 11.8µL |

3. Add 11.8µL of the above reaction mixture to 48µL of denatured DNA.
4. Place the above PCR tube in a Thermal Cycler and initiate the reaction as per the following conditions:

    | Temperature | Time |  |
    |-------------|------|--|
    | Heated lid  | On   |  |
    | 37°C        | 30 min |  |
    | 4°C         | Hold |  |

### Digestion
**Step 10.**

1. Prepare digestion reaction solution on ice as per the following system:

    **Components**

    | | Volume |
    |----------------|----|
    | Digestion Buffer | 1.4µL |
    | Digestion Enzyme | 2.6 µL |
    | **Total** | 4µL |

2. After the circularization reaction is finished, directly add 4µL of digestion reaction solution into the circularized DNA solution, mix well and briefly centrifuge, then place the PCR tube in a Thermal Cycler, and initiate the reaction as per the following conditions:

    | Temperature | Time |  |
    |-------------|------|--|
    | Heated lid  | on  |  |
    | 37°C        | 30 min |  |
    | 4°C         | Hold |  |

3. Add 7.5µL 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.

### Purify Digestion Product
**Step 11.**

1. Place AMPure XP magnetic beads and place at room temperature for 30 min in advance. Mix well by vortexing before use.
2. Pipette 168µL AMPure XP magnetic beads to the 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 500µL 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 the tube bottom.
6. Open the cap of a 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 32µL 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°C, ready for preparing DNB.

## Reagents and Their Functions:
- **ERAT Buffer**: Used for end repair and A-tailing of fragmented DNA.
- **ERAT Enzyme**: Enzyme used along with ERAT buffer for end repair and A-tailing.
- **Adapter Mix**: Mix of different adapters used for ligation.
- **Ligation Buffer/Enzyme**: Reagents used for ligating the adapters to the DNA fragments.
- **TE Buffer**: Tris-EDTA buffer used for elution and storage of DNA.
- **PCR Enzyme Mix**: Enzymes for PCR amplification of the ligated DNA.
- **Splint Buffer and Digestion Buffer/Enzyme**: Reagents used for the circularization and digestion steps to create DNA nanoballs.
- **AMPure XP Beads**: Magnetic beads used for purification steps.

---

**endofoutput**
``'