Oct 21, 2024

Public workspaceProtocol for a Workshop on Whole Exome Sequencing (WES) for Students V.1

This protocol is a draft, published without a DOI.
  • 1Carol Davila University of Medicine and Pharmacy, Bucharest
Relu Cocos
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Protocol Citation: Relu Cocos 2024. Protocol for a Workshop on Whole Exome Sequencing (WES) for Students. protocols.io https://protocols.io/view/protocol-for-a-workshop-on-whole-exome-sequencing-dptu5nnw
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 17, 2024
Last Modified: October 21, 2024
Protocol Integer ID: 110164
Disclaimer
This protocol is based on the guidelines provided in the Illumina DNA Prep with Exome 2.5 Enrichment manual. For detailed instructions, reagent specifications, and troubleshooting, please refer to the official Illumina manual. Adjustments and optimizations may be necessary depending on specific laboratory conditions and experimental needs.
Abstract
This protocol outlines the key wet-lab steps for preparing genomic DNA for Whole Exome Sequencing (WES) using the Illumina NextSeq 550Dx. The protocol is specifically designed for medical students participating in a workshop, focusing on mastering the practical techniques of library preparation, exome capture, and DNA sequencing.
Protocol Overview
Protocol Overview
This protocol outlines the key wet-lab steps for preparing genomic DNA for Whole Exome Sequencing (WES) using the Illumina NextSeq 550Dx. The protocol is specifically designed for medical students participating in a workshop, focusing on mastering the practical techniques of library preparation, exome capture, and DNA sequencing.
Learning Objectives
Learning Objectives
Understand the core steps in Whole Exome Sequencing (WES) library preparation.
Learn how to perform targeted enrichment of exonic regions using capture probes.
Prepare and load sequencing-ready libraries on the NextSeq 550Dx.
Gain hands-on experience with DNA quantification, fragmentation, and clean-up techniques.
Materials Needed
Materials Needed
Completing the Illumina DNA Prep with Exome 2.5 Enrichment protocol requires library prep and enrichment reagents, the exome panel, clean up/size selection beads, and index adapters. In order to carry out the WES workflow effectively, the following materials are required:

Reagents
  • Lysozyme (VWR, Australia, Cat.# 0663-10G)
  • Ethanol, Pure (Sigma-Aldrich, Australia, Cat. # E7023)
  • 2-Propanol (Sigma-Aldrich, Australia, Cat. # I8912)
  • Phosphate Buffered Saline (GibcoTM- Thermo Fisher Scientific, UK, Cat. # 10010023)
  • Ultrapure‱ DNase/RNase Free Distilled Water (Invitrogen, Australia Cat. # 10977-015)
  • DNeasy‱ Blood and Tissue Kit (Qiagen, Germany, Cat. # 69504)
  • High Pure PCR Template Preparation Kit (Roche, Germany, Cat. # 11796828001)
  • Qubit‱ dsDNA HS (High Sensitivity) Assay Kit (Invitrogen, Australia, Cat. # Q32851)
  • Nextera‱ DNA Library Preparation Kit (Illumina, USA, Cat. # FC-121-1030)
  • Nextera‱ XT Library Preparation Kit (Illumina, USA, Cat. # FC-131-1024)
  • Nextera‱ XT Index Kit (Illumina, USA, Cat. # FC-131-1001)
  • Miseq Reagent Kit v2 (300 cycles) (Illumina, USA, Cat. # MS-102-2002)
  • KAPA Library Quantification Kit (Illumina, USA, Cat. # 07960140001)
  • Agencourt‱ AMPure XP beads (Beckman Coulter, USA, Cat. # A63880)
  • Illumina DNA Prep with Exome 2.5 Enrichment Kit, Tagmentation Set B (96 Samples, 12-plex, Cat. # 20077595)
Equipment
  • Qubit‱ assay tubes (Life-technologies, USA, Cat. # Q32856)
  • PCR tubes (Molecular Bioproducts, USA, Cat. # MBP3412)
  • Qubit‱ 2.0 Fluorometer (Invitrogen, Australia, Cat. # Q32866)
  • Agencourt Magnetic stand (Beckman Coulter, USA, Cat. # A32782)
  • Applied Biosystems‱ Veriti 96-Well thermal cycler (Thermo Fisher Scientific, USA)
Recipes
  • Qubit working solution: dilute Qubit dsDNA HS Reagent 1:200 in Qubit dsDNA HS buffer. For n samples, prepare n × 200 µl working solution.
  • 80% ethanol: add 2 ml absolute ethanol into 8 ml distilled water.
  • 0.2 M NaOH: weigh 0.04 g of NaOH pellet and dissolve it into 5 ml distilled water.


Step-by-Step Protocol with Objectives
Step-by-Step Protocol with Objectives
10s
10s

By following this protocol, students will gain a comprehensive understanding of the experimental workflow involved in WES, which is a crucial tool for identifying genetic variants in disease-related genes.


Note
Unless a safe stopping point is specified in the protocol, proceed immediately to the next step.
Avoiding Cross-Contamination: When adding or transferring samples or reagent master mixes, change tips between each sample. When adding index adapters with a multichannel pipette, change tips between each row or each column. If using a single channel pipette, change tips between each sample. Remove unused index adapter tubes or plates from the working area




A. Tagment Genomic DNA
Reagents:
eBLT (Enrichment Bead-Linked Transposomes) (yellow cap)
TB1 (Tagmentation Buffer 1)
Nuclease-free water
Note
eBLT: Must be stored vertically at temperatures above 2°C. Do not use eBLT that has been stored below 2°C.

Prepare the following consumables:


Add Amount20-30 µL DNA to each well of a 96-well PCR plate so that the total input amount is Amount50-1000 ng

Vortex eBLT (yellow cap) vigorously for Duration00:00:10 to resuspend. Repeat as necessary.

10s
Combine Amount11.5 µL eBLT and Amount11.5 µL TB1 to prepare the Tagmentation Master Mix, vortex the Tagmentation Master Mix thoroughly to resuspend and transfer Amount20 µL Tagmentation Master Mix to each well of the plate or tube.
Note
Multiply each volume by the number of samples being processed. These volumes produce 23 µl Tagmentation Master Mix per sample, which includes extra volume for accurate pipetting.



Place on the preprogrammed thermal cycler and run the TAG program. Choose the preheat lid option and set to Temperature100 °C . Set the reaction volume to Amount50 µL .Temperature55 °C for Duration00:05:00 and hold at Temperature10 °C .
Note
Wait until the TAG program has reached the 10°C hold temperature before removing the plate and proceeding.


5m
B. Post Tagmentation Clean Up
This step washes the adapter-tagged DNA on the eBLT before PCR amplification.
Reagents:
ST2 (Stop Tagment Buffer 2) (red cap)
TWB (Tagmentation Wash Buffer)
Prepare the following consumables:


Add Amount10 µL ST2 (red cap) to each well of the plate and slowly pipette each well 10 times to resuspend the beads, then seal, using a 200 µl pipette set to Amount50 µL . and then incubate at room temperature for Duration00:05:00 .

5m
Wash as follows:
Place the plate on the magnetic stand and wait until the liquid is clear (Duration00:03:00 aprox ), then remove and discard supernatant with a pipette set to Amount60 µL .

3m
Remove from the magnetic stand and use a deliberately slow pipetting technique to add Amount100 µL TWB directly onto the beads, and pipette until the beads are fully resuspended
Note
A deliberately slow pipetting technique minimizes the potential of TWB foaming to avoid incorrect volume aspiration and incomplete mixing.


Place the plate on the magnetic stand and wait until the liquid is clear (Duration00:03:00 aprox. ).

3m
Remove and discard supernatant with a pipette set to Amount100 µL .

Repeat steps two times for a total of three washes.
Pipette each well slowly to resuspend the beads. Seal the plate and place on the magnetic stand until the liquid is clear (~3 minutes). Keep on the magnetic stand until step 4 of the Procedure section in Amplify Tagmented DNA.
Note
TWB remains in the wells to prevent overdrying of the beads.

C. Amplify Tagmented DNA
This step amplifies the tagmented DNA using a limited-cycle PCR program. The PCR step adds prepaired 10 base pair Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for library clustering on the flow cells.
Reagents:
1. EPM (Enhanced PCR Mix)
2. Index adapter plate
3. Nuclease-free water
4. Low DNA binding PCR plate
5. 1.7 ml microcentrifuge tubes
6. Microseal 'B' adhesive seal
Prepare the following consumables:


eBLT PCR program:
Choose the preheat lid option and set to Temperature100 °C . Set the reaction volume to Amount50 µL . Temperature72 °C for Duration00:03:00 . Temperature98 °C for Duration00:03:00 . (X) cycles of: (98°C for 20 seconds, 60°C for 30 seconds, 72°C for 1 minute) and 72°C for 3 minutes. Hold at 10°C.
Note
The total running time is ~38 minutes for 9 cycles and ~46 minutes for 12 cycles.




38m
For each sample, combine EPM (Amount23 µL ) with Nuclease-free water (Amount23 µL ) to prepare the PCR Master Mix, vortex, and then centrifuge the PCR Master Mix at 280 × g for Duration00:00:10 .

10s
With the plate on the magnetic stand remove and discard supernatant with a pipette set to Amount100 µL , then remove from the magnetic stand, immediately add Amount40 µL PCR Master Mix directly onto the beads in each sample well and pipette 10 times to mix until the beads are fully resuspended.

Seal the sample plate and centrifuge at 280 × g for Duration00:00:10 . Centrifuge the index adapter plate at 1000 × g for 1 minute.

10s
Prepare the index adapter plate: [ < 96 samples] Pierce the foil seal on the index adapter plate with a new pipette tip for each well. Pierce only the number of samples being processed. [96 samples] Align a new Low DNA binding PCR Plate above the index adapter plate and press down to puncture the foil seal. Discard the Eppendorf PCR plate used to puncture the foil seal.
Using a new pipette tip, add Amount10 µL prepaired Index 1 (i7) and Index 2 (i5) index adapters to each well, and then using a pipette set to Amount40 µL , pipette 10 times to mix, and then seal the plate with Microseal 'B'.

Centrifuge at 280 × g for Duration00:00:30 and place on the preprogrammed thermal cycler and run the eBLT PCR program.
Note
SAFE STOPPING POINT
if you are stopping, store at Temperature-25 °C to Temperature-15 °C for up to 30 days.




30s
D. Clean Up Libraries
This step uses a double-sided bead purification procedure to purify the amplified and indexed libraries.
Reagents:
1. IPB (Illumina Purification Beads)
2. RSB (Resuspension Buffer)
3. EtOH (Freshly prepared 80% ethanol)
4. Nuclease-free water

Note
About Reagents: IPB must be at room temperature before use. Vortex before each use. Vortex frequently to make sure the beads are evenly distributed and aspirate and dispense slowly due to the viscosity of the solution.


Prepare the following consumables:


Note
For each sample, prepare 400 µl fresh 80% EtOH from absolute ethanol. Including an overage of 20% is recommended.

Use a plate shaker to shake the 96-well PCR plate at 1800 rpm for Duration00:01:00 and then place the plate on the magnetic stand and wait until the liquid is clear (Duration00:01:00 aprox. ).

2m
Transfer Amount45 µL supernatant from each well of the PCR plate to the corresponding well of a new MIDI plate and resuspend Illumina Purification Beads.

Add Amount77 µL nuclease-free water to each well-containing supernatant and add Amount88 µL IPB to each well-containing supernatant.

Place on the magnetic stand and wait until the liquid is clear (~Duration00:05:00 ).
5m
Pipette each well 10 times to mix. Seal the plate and incubate at room temperature for Duration00:05:00 , and then add 20 µl to each well of a new MIDI plate.
Note
During incubation, vortex the IPB!


5m
Remove seal and transfer Amount200 µL supernatant from each well of the first plate to the corresponding well of the new MIDI plate containingAmount20 µL IPB, and then pipette each well of the MIDI plate 10 times to mix.
Note
Discard the first plate.


Incubate at room temperature for Duration00:05:00 , and place on the magnetic stand and wait until the liquid is clear (~Duration00:05:00 ), and then without disturbing the beads, remove and discard all supernatant.

10m
Wash two times as follows:
a. with the plate on the magnetic stand, add Amount200 µL freshConcentration80 % (v/v) EtOH without mixing.
b. wait for Duration00:00:30 .
c. without disturbing the beads, remove and discard supernatant.


30s
Use a Amount20 µL pipette to remove and discard residual EtOH, and air-dry on the magnetic stand for Duration00:05:00 , and then remove from the magnetic stand.

5m
Add 17 µl RSB to the beads, and seal the plate, and then use a plate shaker at 1800 rpm for Duration00:02:00 .

2m
Incubate at room temperature for Duration00:02:00 , place the plate on the magnetic stand and wait until the liquid is clear (~Duration00:02:00 ).

4m
Transfer 15 µl supernatant to a new 96-well PCR plate.
Note
SAFE STOPPING POINT
If you are stopping, seal the plate with Microseal 'B' or Microseal 'F', and store at ‑25°C to ‑15°C for up to 30 days.


E. Qualify Pre-Enrichment Libraries
It is recommended to check the quality or to qualify pre-enrichment libraries before proceeding to enrichment.
If you elect not to check pre-enrichment libraries, perform the following procedure instead to
reserve samples for potential troubleshooting later.
Note
Qualify Pre-Enrichment Libraries: Pre-enrichment libraries can be qualified individually, one library at a time, or as a pool before enrichment. Perform the following procedure to qualify pre-enrichment libraries.
1. Assess the quality of 1 µl library or pooled libraries using one of the following methods.
• Add 1 µl RSB to the library, and then analyze the 2 µl volume using the Advanced Analytical Fragment Analyzer with HS-NGS High Sensitivity 474 kit.
• Analyze 1 µl library or pooled libraries using the Agilent Technology 2100 Bioanalyzer using a DNA 1000 kit.
Expect the mean fragment size to be between 300–400 bp and a distribution of DNA fragment size range of 150–1500 bp. The following figures provide example traces.

F. Pool Pre-Enrichment Libraries
This step combines DNA libraries with unique indexes into one pool of up to 12 libraries. Fewer pre-enrichment libraries may be pooled, but you may need to perform additional optimization. If using fewer pre-enrichment libraries, you cannot process the full 96 samples through enrichment, as only eight enrichment reactions are supported with this kit.
You can pool by volume or mass. Use the following table to determine the appropriate method for your input.
* If starting with ≥ 50 ng DNA input, the pre-enrichment library yields were normalized during tagmentation, which uses eBLT. This normalization enables you to pool equal volumes of each pre-enrichment library in a final pool volume ≤ 30 µl (target 250–500 ng per sample).

Quantify Pre-Enrichment Libraries: After pre-enrichment library quantification, all sample input types can be pooled by mass to achieve optimal library balance and a similar number of sequencing reads per library.
Determine library concentration (ng/µl) by proceeding as follows:
1. Quantify 1 μl of each pre-enrichment library using the Qubit dsDNA BR Assay Kit to determine library concentration (ng/μl). Expect the following pre-enrichment library yield based on sample type and input.

Concentration results may differ depending on the quantification method used. The Qubit dsDNA BR Assay is recommended, but validation will be needed when using an alternative method.

Poll by Volume:
when the input is 50–1000 ng gDNA, quantifying and normalizing individual libraries generated in the same experiment is not required.
For a standard 12-plex pool: Combine Amount2.5 µL of each pre-enrichment library in a Amount1.7 µL l microcentrifuge tube to generate a 12-plex pool at a total final pool volume of Amount30 µL .
When preparing a pool of lower plexity (<12 pre-enrichment libraries per pool): Combine Amount2.5 µL of each pre-enrichment library in a Amount1.7 mL microcentrifuge tube, then add RSB to bring the total final pool volume up to Amount30 µL .
Pool pre-enrichment libraries based on the sample volumes in the following table:



Note
SAFE STOPPING POINT
If you are stopping, cap the 1.5 ml microcentrifuge tube and store at ‑25°C to ‑15°C for up to 30 days.


G. Hybridize Probes
This step binds target regions of DNA within the pre-enrichment library with the exome capture probes, and optional mitochondrial genome or custom capture probes.
Reagents:
1. EHB2 (Enrich Hyb Buffer 2)
2. NHB2 (Hyb Buffer 2 + IDT NXT Blockers) (blue cap)
3. Twist BioScience for Illumina Exome 2.5 Panel (green cap)
4. Nuclease-free water

Note
NHB2 precipitates and separates during storage. Follow the NHB2 preparation instructions before first use.


Save the following IEE (Illumina Exome Enrichment)-HYB program.
Note
AICI VERIFICA daca lipsesc pasi!!!

H. Capture Hybridized Probes
This step uses Streptavidin Magnetic Beads (SMB3) to capture the Exome 2.5 and optional mitochondrial probes hybridized to the target regions of interest within the libraries.
Reactivi:
1. EE1 (Enrichment Elution Buffer 1)
2. EEW (Enhanced Enrichment Wash) (amber cap)
3. ET2 (Elute Target Buffer 2)
4. HP3 (2 N NaOH)
5. SMB3 (Streptavidin Magnetic Beads)
Prepare the following consumables:

Preheat a microheating system with a MIDI heat block insert to incubate the sample plate to 62°C. An optional second microheating system can be used to preheat EEW.
1. CAPTURE
Centrifuge the sample plate or tubes of pooled enriched libraries at 280 × g for Duration00:00:30 , and transfer pool from each well of the PCR plate or 8-tube strip to the corresponding well of a new MIDI plate or 1.7 ml microcentrifuge tube.

30s
Add Amount250 µL SMB3 to each well or tube, and then mix thoroughly as follow: [Plate] Seal the plate and shake at 1200 rpm for 4 minutes; [Tube] Cap the tube and vortex at high speed three times for 10 seconds each. (step4) Place the sample plate or tube on the MIDI heat block insert on the microheating system. Close the lid, and incubate for 15 minutes at 62°C.
Note
Proceed to the next step while the pooled libraries incubate.


Preheat EEW (amber tube) by laying the tube on its side on the MIDI heat block insert on the microheating system to 62°C.
Note
Alternatively, lay EEW on top of the MIDI plate or next to the 1.7 ml microcentrifuge tube on the MIDI heat block insert during the incubation in step 4. Keep EEW heated until step 2 of the Wash.

Immediately centrifuge the plate or tube at 280 × g for Duration00:00:30 . Immediately place on a magnetic stand and wait until the liquid is clear (~Duration00:02:00 ), and using a pipette set to Amount350 µL , remove and discard all supernatant from each well or from the tube.

2m 30s
2. WASH
Remove from the magnetic stand. Add 200 µl preheated EEW (amber tube) to each well or microcentrifuge tube, and then mix thoroughly as follows. [Seal and shake at 1800 rpm for Duration00:04:00 If splashing occurs, reduce the speed to 1600 rpm. [tube] Cap the tube and vortex at high speed three times for Duration00:04:00 s each. Return unused EEW to the microheating system and keep heated.

8m
Place the sample plate or tube on the MIDI heat block insert on the microheating system. Close the lid, and incubate for Duration00:05:00 at Temperature62 °C . [tube] Centrifuge briefly for 3 seconds, and immediately place the plate or microcentrifuge tube on a magnetic stand and wait until the liquid is clear (~Duration00:02:00 ).


7m
Using a pipette set to 200 µl, remove and discard all supernatant from each well or from the tube.
Note
Repeat steps two additional times for a total of three washes.

3. TRANSFER WASH
Remove the plate or tube from the magnetic stand. Add Amount200 µL preheated EEW (amber tube) to each well or to the tube, and then mix thoroughly as follows. Cap the tube, and then vortex at high speed three times for Duration00:00:10 each

10s
Transfer Amount200 µL resuspended bead solution to a new MIDI plate or to a new Amount1.7 µL microcentrifuge tube.
Note
Transferring the reagent minimizes carryover of residual reagents that can inhibit downstream PCR.


Place the sample plate or tube on the MIDI heat block insert on the microheating system. Close the lid, and incubate for Duration00:05:00 at Temperature62 °C . Centrifuge briefly for Duration00:00:03 .

5m 3s
Immediately place on a magnetic stand and wait until the liquid is clear (~Duration00:02:00 ). Using a pipette set to Amount200 µL remove and discard all supernatant from each well or from the tube.

2m
Centrifuge the plate or the tube at 280 × g for Duration00:00:30 . Place on a magnetic stand for Duration00:00:10 .

40s
Use a 20 µl pipette to remove and discard residual liquid from each well or from the tube.
Note
Immediately proceed to Elute to prevent excessive drying of the beads and library yield loss.

4. ELUTE
Combine the following volumes to prepare an Elution Master Mix. Multiply each volume by the number of samples being processed EE1 (Amount28.5 µL ) + HP3 (Amount1.5 µL ), and vortex, and then centrifuge the master mix at 280 × g for Duration00:00:10 .

10s
Remove the sample plate or tube from the magnetic stand, and add Amount23 µL Elution Master Mix to each well or to the tube, and then mix thoroughly as follows: cap the tube, and then vortex at high speed three times for Duration00:00:10 each, and ncubate the plate or the tube at room temperature for Duration00:02:00 .

2m 10s
Centrifuge at 280 × g for Duration00:00:30 , and place on a magnetic stand and wait until the liquid is clear (~2 minutes).

30s
Transfer Amount21 µL supernatant from the MIDI plate or Amount1.7 mL microcentrifuge tube to the corresponding well of a new 96-well PCR plate or to a new 8-tube strip, and add 4 μl ET2 to each well or to the tube containing 21 μl supernatant. Set pipette to 20 µl and slowly pipette each well or the tube 10 times to mix.

Centrifuge the sample plate or the tube at 280 × g for Duration00:00:30 .

30s
I. Amplify Exome-Enriched Library Pools
This step uses PCR to amplify the enriched whole exome library pools.
Reagents:
1. EPM (Enhanced PCR Mix)
2. PPC (PCR Primer Cocktail)
Prepare the following consumables:
Save the AMP program on the thermal cycler.
Add Amount5 µL PPC to each well or tube, and add 20 µl EPM to each well or tube and mix thoroughly as follows, pipette 10 times to mix, and then cap the 8‑tube strip.

Centrifuge the plate or 8-tube strip at 280 × g for Duration00:00:30 s.

30s
Place on the preprogrammed thermal cycler and run the AMP program.
Note
SAFE STOPPING POINT
If you are stopping, store at 2°C to 8°C for up to two days. Alternatively, leave on the thermal cycler for up to 24 hours at 10°C.

J. Clean Up Amplified Exome-Enriched Library Pools
Reagents:
1. IPB (Illumina Purification Beads)
2. RSB (Resuspension Buffer)
3. Freshly prepared 80% ethanol (EtOH)
Prepare the following consumables:

For each sample, prepare 400 μl fresh 80% EtOH from absolute ethanol. Including an overage of 20% is recommended.
Centrifuge the PCR samples at 280 × g for Duration00:00:30 , and transfer Amount45 µL from each well of the PCR plate or 8-tube strip to the corresponding well of a new MIDI plate or 1.7 ml microcentrifuge tube.

30s
Add Amount40.5 µL IPB to each well or tube, and then mix thoroughly as follows. Cap the tube, and then vortex at high speed for 10 seconds. Repeat two times and incubate the plate or the tube TemperatureRoom temperature for Duration00:05:00 .

5m
Centrifuge at 280 × g for Duration00:01:00 , and place on a magnetic stand and wait until the liquid is clear (~Duration00:05:00 .

6m
Using a pipette set to Amount85 µL , remove and discard all supernatant from each well or tube.

Wash two times as follows:
1. With the plate on the magnetic stand, add Amount200 µL fresh 80% EtOH without mixing.
2. Wait for Duration00:00:30 .
3. Without disturbing the beads, remove and discard supernatant.




30s
Use a Amount20 µL pipette to remove and discard residual EtOH from each well or from the tube.

Air-dry on the magnetic stand for Duration00:05:00 .

5m
Remove from the magnetic stand and add Amount32 µL RSB each well or to the tube. Cap the tube, and then vortex at high speed three times for Duration00:00:10 . Repeat two times


10s
Incubate the plate or the tube at room temperature for Duration00:05:00 , and then Centrifuge at 280 × g for Duration00:00:30 .

5m 30s
PROTOCOLUL INITIAL


DNA Quantification and Quality ControlDuration00:00:00

DNA FragmentationDuration00:00:00

End Repair, A-Tailing, and Adapter Ligation
PCR AmplificationDuration00:00:00

Exome Capture
Post-Capture PCR Amplification
Sequencing on the NextSeq 550Dx