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GenTrack Library
Preparation Kit User Guide
For Illumina Sequencing Platforms
- 96 Reactions (Cat. No. NGS09601)
- 384 Reactions (Cat. No. NGS38401)
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Version records
Version No. Release Date Changes
1 Aug 2019 Original version
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Content
Overview ... 5
Introduction ... 5
DNA Input Recommendations ... 5
DNA Quantification ... 6
DNA Quality ... 6
Kit Contents ... 7
Protocol ... 8
Introduction ... 8
Tips and Techniques ... 8
Avoid Cross-Contamination ... 8
Sample or Reagent Transfers ... 9
Handling Beads ... 9
User-Supplied Consumables ... 9
Library Preparation Workflow ... 10
Fragment DNA (Optional) ... 11
Ultrasonic DNA Shearing ... 11
Enzymatic DNA Shearing ... 11
Size Selection (Optional) ... 11
Beads-Based Selection ... 12
Gel Electrophoresis-Based Selection ... 12
Check the Fragmented DNA ... 12
End-Repair and A-Tailing ... 13
Consumables ... 13
Procedure ... 13
Adapter Ligation ... 14
Consumables ... 14
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Procedure ... 15
Purification ... 16
Consumables ... 16
Procedure ... 16
Amplification ... 17
Consumables ... 17
Procedure ... 17
Purification ... 18
Consumables ... 18
Procedure ... 18
Check Libraries ... 20
Quantify ... 20
Qualify ... 20
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Overview
Introduction
This manual shows how to prepare up to 96 pooled indexed DNA libraries from genomic DNA (gDNA) for subsequent Next Generation Sequencing (NGS) on Illumina sequencing platform with the reagents provided in the GenScript GenTrack Library Preparation Kit. The protocol starts with DNA fragmentation followed by end-repair, A-tailing, adapter ligation and libraries amplification, to generate multiplexed paired-end sequencing libraries with dual index.
The GenTrack Library Preparation Kit manual offers:
• Gel-free size selection for fragmented DNA with optimized beads conditions.
• Optimized and complete protocol for DNA library preparation.
• Master-mixed reagents to reduce reagent containers and pipetting.
• High throughput 96 indexes available and supported on all Illumina sequencing platform.
DNA Input Recommendations
The GenTrack Library Preparation Kit protocol is optimized for 1~1000 ng of fragmented genomic DNA with an expected size distribution from about 200 bp to 600 bp. Alternatively, we recommend starting with 1000 ng fragmented genomic DNA input and performing size selection prior to DNA library preparation.
We do not recommend starting from PCR products with this kit.
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DNA Quantification
The success of DNA library preparation will strongly depend on the use of an accurately quantified amount of input DNA.
To obtain an accurate quantification of the DNA input, we recommend quantifying the input DNA with a fluorometric based method specific for double- stranded DNA such as the Qubit dsDNA HS Assay System. Use 1 µL of each DNA sample with 199 µL of the Qubit working solution for sample quantification.
Avoid using methods that measure total nucleic acid content (e.g. NanoDrop or other UV absorbance methods), because common contaminants such as ssDNA, RNA and oligos are not substrates for the GenTrack Library Preparation assays and may decrease the efficiency of reagents for optimized DNA library preparation.
DNA Quality
Start from high-quality DNA sample. Heavily nicked or damaged DNA will lower the yield of DNA libraries output.
Absorbance measurements at 260 nm are commonly used to quantify DNA.
The ratio of absorbance at 260 nm to 280 nm is used as an indication of sample purity. This protocol is optimized for DNA with absorbance ratio values of 1.8–
2.0.
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Kit Contents
Before proceeding, please make sure that all of the reagents identified in the kit are ready. The PB component must be stored at 4°C, and all the other components should be stored at -20°C.
Components 96-Reaction Storage
End Prep Buffer 1.88 mL -20°C
End Prep Enzyme 235 μL -20°C
Ligation Prep Buffer 2.93 mL -20°C
Ligation Prep Enzyme 106.5 μL -20°C
2× PCR Master Mix 2.5 mL -20°C
Primer Mix 500 μL -20°C
PB (Purification Beads) 9 mL 4°C
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Protocol
Introduction
This section describes the GenTrack Library Preparation protocol.
• Follow this protocol in the order described and use the specified volume and parameters for each reagent.
• Before proceeding, confirm the kit contents and the required equipment and consumables.
• When adding adapters, keep records of the adapter index for each sample.
Tips and Techniques
We recommend proceeding each step immediately to the next step, unless a SAFE STOPPING POINT is marked.
Avoid Cross-Contamination
• When adding or transferring samples or mixture containing the samples, always use clean and new tips for each sample.
• When adding adapters, always use clean and new tips for every adapter.
• Remove unused index adapter plate from the working area as soon as possible.
• When using a plate for this process, we recommend to always seal the plate with a new Microseal B Seal after an old one is removed.
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Sample or Reagent Transfers
• Transfer the accurate volume mentioned in this protocol from each well or tube to the corresponding well or tube.
Handling Beads
• Pipet the beads suspension up and down slowly.
• When mixing, mix thoroughly.
• Make sure that the beads remain fully resuspended before use.
• If the beads are aspirated into the pipette tips, dispense them back to the plate or tube(s) on the magnetic stand and wait until the liquid is clear again (~2 min).
• When washing beads:
• Use the appropriate magnet for the plate or tube(s).
• Dispense liquid so that the beads on the side of the wells are wet.
• Keep the plate or tube(s) on the magnet until instructed to remove it.
• Do not agitate the plate or tube(s) while on the magnetic stand.
User-Supplied Consumables
• Prepare absolute ethanol and Nuclease-Free Water.
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Library Preparation Workflow
The following diagram illustrates the workflow using a GenTrack Library Preparation Kit.
SAFE STOPPING POINTS are marked as follows. At this point, samples can be stored at 4°C overnight or -20°C for up to 1 week.
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Fragment DNA (Optional)
This step will fragment genomic DNA to an expected size range for the DNA library preparation and subsequently sequencing on Illumina Sequencing Platforms. We recommend using the following methods to fragment at least 1 μg of genomic DNA.
Note: This step is not supplied in the GenTrack DNA Library Preparation Kit.
Ultrasonic DNA Shearing
Use ultrasonicator instruments (and compatible tubes if necessary) to fragment DNA, such as Covaris® E220 Focused-ultrasonicator, Covaris® S220 Focused- ultrasonicator etc. Refer to the protocol or instruction of the ultrasonicator and set the exact parameter for fragmenting DNA to a size range around 350 bp.
Enzymatic DNA Shearing
Use fragmentase to fragment DNA, such as DNase I, NEB NEBNext® dsDNA Fragmentase etc. Refer to the protocol of the fragmentase used and fragment DNA to a size range around 350 bp.
Size Selection (Optional)
This optional step will select a size-range of input fragmented DNA for an optimized DNA library preparation and subsequent sequencing on Illumina Sequencing Platforms. We recommend to use the following methods to select fragmented DNA.
Note: The reagents required in this size selection step is not supplied in the GenTrack Library Preparation Kit. We do not recommend perform size selection for the input of fragmented DNA less than 50 ng.
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Beads-Based Selection
Use size-selection beads to select the fragmented DNA, such as Beckman Agencourt® AMPure XP Beads, Yeasen Hieff NGS® DNA Selection Beads etc.
Refer to the protocol of the size-selection beads and select the 200~600 bp fragmented DNA with an exact ratio of beads.
Gel Electrophoresis-Based Selection
Use the gel electrophoresis and gel extraction to select the fragmented DNA of an expected size range.
You can perform the normal agarose gel electrophoresis of DNA and cut the gel slice of 200~600 bp DNA bands to do gel extraction using gel extraction kit such as Genscript QuickClean II Gel Extraction Kits, Qiagen QIAquick® Gel Extraction Kit, NEB Monarch® DNA Gel Extraction Kit etc. Refer to the protocol of the gel extraction kit for more details.
Moreover, you can use the gel size selection system to perform size selection, such as ThermoFisher E-Gel SizeSelect II, ThermoFisher E-Gel NGS etc. Refer to the instruction of the system and perform size selection of 200~600 bp fragmented DNA.
Check the Fragmented DNA
This step will check whether the size distribution of fragmented DNA (or size- selected fragmented DNA) is expected. Run the DNA on an Agilent Technologies 2100 Bioanalyzer using a High-Sensitivity DNA chip to check the size distribution.
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Figure 1 shows the size distribution of an expected fragmented DNA for optimized library preparation with a GenTrack DNA Library Preparation Kit.
Figure 1. Size distribution of the expected fragmented DNA
End-Repair and A-Tailing
This step converts the overhangs resulting from fragmentation into blunt ends, repair the nick and then adding a single “A” nucleotide to the 3’ end of the blunt fragments for the next adapter ligation.
Consumables
• End Prep Buffer
• End Prep Enzyme
• Nuclease-Free Water (User-Supplied) Procedure
1. Thaw the End Prep Buffer at room temperature. When thawed, keep it on ice. Gently vortex and centrifuge briefly before use.
2. Thaw and keep the End Prep Enzyme on ice, then mix well by flicking.
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3. In the order listed below, prepare the End Prep Mix in each well of a plate or tube on ice.
Components Volume (μL) per Reaction
End Prep Buffer 17.8
End Prep Enzyme 2.2
Fragmented or size-selected DNA x (1-1000 ng) Nuclease-Free Water 30.0 – x
Total 50.0
4. Gently pipet the mixture up and down for 15~20 times to mix thoroughly.
5. Place the plate or tube(s) on a thermal cycler and run the End Prep Program.
End Prep Program (Lid Set at 100°C)
Temperature Duration
37°C 30 min
75°C 30 min
4°C Hold
Adapter Ligation
This step adds the adapters with index to the ends of the DNA fragment after the end preparation.
Consumables
• Ligation Prep Buffer
• Ligation Prep Enzyme
• GenTrack Adapter Set (Cat. No.: NGS01202/ NGS09602)
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Procedure
1. Thaw the Ligation Prep Buffer and the Adapters at room temperature, then keep them on ice. Gently vortex and centrifuge briefly before use.
2. Thaw and keep the Ligation Prep Enzyme on ice, then mix well by flicking.
3. After the End Prep Program ends, prepare the Adapter Ligation Mix in each well of a plate or tube containing End Prep Mix in the order listed below on ice.
Component Volume (μL) per Reaction
End Prep Mix after Reaction 50.0
GenTrack Adapter* 2.5
Ligation Prep Buffer 26.5
After the buffer is added, mix well before adding the Ligation Prep Enzyme
Ligation Prep Enzyme 1.0
Total 80.0
*Note: Use different GenTrack Adapter from GenTrack Adapter Set for each sample that will be pooled together for sequencing. The Set provides at most 96 different index adapters.
4. Gently pipet the mixture up and down for 15~20 times to mix thoroughly.
5. Place the plate or tube(s) on a thermal cycler and run the Adapter Ligation Program.
Adapter Ligation Program (Without a Heated Lid)
Temperature Duration
22°C 15 min
4°C Hold
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Purification
This step purifies the products of the Adapter Ligation Reaction and removes the unligated adapters.
Consumables
• PB
• Freshly prepared 80% (v/v) ethanol and Nuclease-Free Water (User- Supplied)
Procedure
Warning! Only perform purification with the PB that is equilibrated to room temperature.
1. Thaw the PB at room temperature for at least 30 min before performing the purification, and prepare 450 μL of fresh 80% (v/v) ethanol per sample.
2. Mix the PB thoroughly by vortexing for 15 sec.
3. After the Adapter Ligation Program ends, remove the samples, and then add 40 μL of PB into each sample.
4. Thoroughly pipet the mixture up and down for 15~20 times.
5. Incubate at room temperature for 5 min.
6. Place the plate or tube(s) on the magnet for at least 2 min until the liquid is clear.
7. Carefully discard the supernatant. Do not discard the beads.
8. While keeping the plate or tube(s) on the magnet, add 200 μL of fresh 80%
(v/v) ethanol, incubate for 30 seconds, and then remove the ethanol.
9. Repeat one ethanol wash by performing Step 8 (above).
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10. While keeping the plate or tube(s) on the magnet, allow the beads to air dry for 1~3 min. Do not over dry the beads.
11. Remove the sample plate or tube(s) from the magnet and elute in 22 μL of Nuclease-Free Water or equivalent buffer.
12. Thoroughly pipet the mixture up and down for 15~20 times.
13. Incubate for 5 min at room temperature.
14. Place the plate tube(s) on a magnet for at least 2 min until the liquid is clear.
15. Transfer 20 μL of elution to a new plate or tube(s). Make sure that no beads are carried over.
Amplification
This PCR step selectively enriches the DNA fragments with adapter on both ends, and amplifies the amount of DNA libraries output for sequencing.
Consumables
• 2× PCR Master Mix
• Primer Mix
• Nuclease-Free Water (User-Supplied) Procedure
1. Thaw the Primer Mix at room temperature, and then gently vortex and centrifuge briefly.
2. Thaw and keep the 2× PCR Master Mix on ice, and then gently vortex and centrifuge briefly.
3. In the order listed below, prepare the PCR Amplification Mix in each well of a plate or tube(s) containing the samples on ice.
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Components Volume (μL) per Reaction Adapter Ligated DNA 20.0
2× PCR Master Mix 25.0
Primer Mix 5.0
Total 50.0
4. Gently pipet the mixture up and down for 15~20 times to mix thoroughly, and then centrifuge briefly.
5. Place the plate or tube(s) on a thermal cycler and run the PCR Amplification Program.
PCR Amplification Program (Lid Set at 105°C)
Temperature Duration Cycles
98°C 2 min 1
98°C 30 sec
2~17 cycles
62°C 30 sec
72°C 30 sec
72°C 3 min 1
4°C Hold 1
Purification
This step purifies the products of the PCR Amplification reaction.
Consumables
• PB
• Freshly prepared 80% (v/v) ethanol and Nuclease-Free Water (User- Supplied)
Procedure
Warning! Only perform purification with the PB that is equilibrated to room temperature.
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1. Thaw the PB at room temperature for at least 30 min before performing the purification, and prepare 450 μL of fresh 80% (v/v) ethanol per sample.
2. Mix the PB thoroughly by vortexing for 15 sec.
3. After the PCR Amplification Program ends, remove the samples, and then add 50 μL of PB into each sample.
4. Thoroughly pipet the mixture up and down for 15~20 times.
5. Incubate at room temperature for 5 min.
6. Place the plate or tube(s) on the magnet for at least 2 min until the liquid is clear.
7. Carefully discard the supernatant. Do not discard the beads.
8. While keeping the plate or tube(s) on the magnet, add 200 μL of fresh 80%
(v/v) ethanol, incubate for 30 seconds, and then remove the ethanol.
9. Repeat one ethanol wash by performing Step 8 (above).
10. While keeping the plate or tube(s) on the magnet, allow the beads to air dry for 1~3 min. Do not over dry the beads.
11. Remove the sample plate or tube(s) from the magnet and elute in 22 μL of Nuclease-Free Water or equivalent buffer.
12. Thoroughly pipet the mixture up and down for 15~20 times.
13. Incubate for 5 min at room temperature.
14. Place the plate tube(s) on a magnet for at least 2 min until the liquid is clear.
15. Transfer 20 μL of elution (DNA library) to a new plate or tube(s). Make sure that no beads are carried over.
16. Store the DNA library at -20°C for the subsequent quantification, qualification and sequencing.
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Check Libraries
This is a quality control step to quantify and qualify the DNA library that you prepared.
Quantify
To achieve the highest-quality data, quantification of DNA library is needed to load an appropriate input on Illumina sequencing platforms and create optimum cluster densities. Quantify your DNA library prepared using a double stranded DNA-specific fluorescent dye method, such as Qubit dsDNA HS Assay System.
For a more accurate quantification, a real-time qPCR quantification is needed.
Qualify
Run the DNA library on an Agilent Technologies 2100 Bioanalyzer using a High- Sensitivity DNA Chip to check the size distribution.
Figure 2 shows the size distribution of a successful DNA library preparation with this GenTrack DNA Library Preparation Kit.
Figure 2. Example of the size distribution of DNA library
