LCM Zymo Trio WGBS Library Prep

Product: Zymo-Seq™ Trio WGBS Library Kit

Here’s the Zymo Trio WGBS library prep workflow:

Materials

Kit contents
PCR tubes, 1.5 mL tubes
Two thermocyclers
Heating block
Mini centrifuge
Vortex, shaker
Aluminum beads (to keep things on ice)
Magnetic stand for PCR tubes
Custom index primers for Illumina if you have more than 12 samples (Zymo kit provides 12)

Kit contents

Zymo-Seq™ Trio WGBS Library Kit	D5462 (25 preps)	Storage Temperature
Lightning Conversion Reagent	4.5 ml	Room Temp.
M-Binding Buffer	20 ml	Room Temp.
M-Wash Buffer	6 ml (conc.)	Room Temp.
L-Desulphonation Buffer	10 ml	Room Temp.
DNA Elution Buffer	4 ml	Room Temp.
Zymo-Spin™ IC Columns	25	Room Temp.
Collection Tubes	25	Room Temp.
DNA Wash Buffer	6 ml (conc.)	Room Temp.
Select-a-Size MagBeads	10 mL	4 ºC
E coli Non-Methylated gDNA	5 μg/20 μL	-20 °C
Adapter Ligation Buffer 1	48 μL	-20 °C
Adapter Ligation Buffer 2	48 μL	-20 °C
Adapter Ligation Buffer 3	48 μL	-20 °C
Adapter Ligation Master Mix	625 μL	-20 °C
2X Index PCR Premix	600 μL	-20 °C
Zymo-Seq UDI Primer Set (1-12)	20 μL/Index	-20 °C

Sample Input

Sample Input Material: Purified cell-free DNA (cfDNA), Sonicated FFPE DNA (average size of 300-600 bp), Sheared genomic DNA (average size of 300-600 bp).
- If the formalin-fixed, paraffin-embedded (FFPE) derived DNA input is sufficient (≥20 ng), we recommend performing sonication to shear the FFPE DNA to an average size of 300-600 bp for best results. This should yield enough sheared DNA for the 5 ng minimum input required for the kit (concentration of the DNA may need to be performed for best results).
Minimum Input: 5 ng
Recommended Maximum Input: 10 ng. Higher inputs can be used to create libraries; however, the diversity of the library may decline as input increases.
Input Quality: For optimal results, use minimum input of DNA with no RNA or genomic DNA contamination. cfDNA can be concentrated using the DNA Clean & ConcentratorTM (D4013) prior to processing. cfDNA can be suspended in water, DNA Elution Buffer, or TE buffer.

Protocol

Buffer preperation

Once buffers are prepared for a kit, they do not need to be prepared again.

Add 300 uL of the Select-a-Size MagBead Concentrate to the 10 mL Select-a-Size MagBead Buffer bottle
- Resuspend by vortexing and store at 4 ºC
Add 24 mL of 100% EtOH to the M-Wash Buffer concentrate
Add 24 mL of 100% EtOH to the DNA Wash Buffer concentrate

Best practices

Preset the thermocycler programs
Thaw and keep -20°C components on ice unless instructed otherwise. Flick to mix and centrifuge before use
- Avoid multiple freeze-thaws, make aliquots if needed
Allow beads to equilibrate to room temperature >30 mins before use
Resuspend magnetic particles immediately before each use by vortexing until homogenous

I am following the modifications for FFPE DNA! (Appendix F)

In Section 2, Step 11: After the 1-hour adapter ligation reaction, add 85 μL of DNA Elution Buffer to the sample to bring the volume up to 135 μL and mix well by pipetting.
In Section 2, Step 12: Follow the clean-up protocol in Appendix A on pg. 13 using 50 μL of Select-a-Size MagBeads. Allow beads to equilibrate to room temperature for 30 minutes prior to use. For elution, resuspend the beads in 15 μL of DNA Elution Buffer and aspirate all 15 μL eluate into a new tube after separation from the beads.
In Section 3, Step 3: The recommended number of PCR cycles may be increased to the following depending on the FFPE DNA sample input amount and type used (see table below in Section 3)
In Section 3, Step 4: Follow the clean-up protocol in Appendix A on pg. 13 using 40 μL of Select-a-Size MagBeads. Allow beads to equilibrate to room temperature for 30 minutes prior to use. For elution, resuspend the beads in 20 μL of DNA Elution Buffer and aspirate all 20 μL eluate after separation from the beads.

Section 1: Bisulfite Conversion of DNA (same as Pico Kit)

Thaw samples on ice
Prepare samples with Tris buffer to desired input amount (5-10ng) in 20 uL in strip tubes
Add 130 uL of Lightning Conversion Reagent to each tube and mix well by pipetting
Centrifuge briefly
Run thermocycler program (lid temp 105°C):
Store at 4°C for <20 hours (overnight)

Section 1, continued

This is a safe stopping point. Bisulfite-converted DNA can be safely stored at ≤ −20°C for up to one month.

Section 2: Adapter Ligation

Appendix A: Select-a-Size MagBead Clean-Up Protocol

Before Starting:

Ensure that the indicated volume of ethanol has been added to the DNA Wash Buffer.
Allow the Select-a-Size MagBeads to equilibrate to room temperature for 30 minutes prior to use.
Resuspend the magnetic particles immediately before use by vortexing the Select-a-Size MagBeads until homogenous.

Add 50 (60 for non-FFPE) μL of the Select-a-Size MagBeads to the tube. Mix thoroughly by pipetting until homogenous and incubate for 5−10 minutes at room temperature.
1. This is a 0.37X bead cleanup (0.55X for non-FFPE)
Place the tube on a magnetic stand for 3 minutes, or until the supernatant is clear.
Carefully remove the supernatant without disturbing the magnetized bead pellet.
1. Avoid aspirating any beads when removing the supernatant.
Without removing from the magnetic stand, add 200 μL of DNA Wash Buffer to the tube, incubate for at least 30 seconds, and then remove the supernatant completely without disturbing the magnetized bead pellet. Repeat this wash step for two washes total.
Remove the tube from the magnetic stand and centrifuge very briefly. Then return the tube to the magnetic stand, wait for the beads to pellet, and remove any residual DNA Wash Buffer with a 10 μL pipette tip.
Leave the tube on the magnetic stand and keep the cap open for 2−3 minutes to allow the beads to air dry.
1. Do not over dry the beads as this may negatively impact recovery. Beads should remain a glossy brown color.
2. When performing the clean-up in Section 2: Adapter Ligation, the beads are more likely to disperse around the tube and are more susceptible to drying faster than normal.
Cap and remove the tube from the magnetic stand. Add the indicated volume of DNA Elution Buffer and fully resuspend the beads by pipetting up and down. Incubate for 5 minutes at room temperature.
1. When performing the clean-up in Section 2: Adapter Ligation, the resuspended beads behave differently and will be more challenging to remove from the sides of the tube. Briefly centrifuge tubes if necessary. Take care to ensure the beads are still fully resuspended.
2. If performing clean-up in Section 2: Adapter Ligation, add 15 μL of DNA Elution Buffer to fully resuspend the beads.
Place the tube back on the magnetic stand for 2 minutes or until the supernatant is clear.
Transfer the indicated volume of eluate to a new tube. Discard the beads.
If performing clean-up in Section 2: Adapter Ligation, aspirate all 15 μL eluate and transfer to a 0.2 mL PCR tube.

This is a safe stopping point. The purified adapter-ligated DNA can be safely stored at ≤ −20°C for up to one month.

Section 3: Index PCR Amplification

Combine the following on ice to a 0.2 mL PCR tube containing the purified adapter-ligated DNA:
- Adapter-Ligated DNA: 15 μL
- Zymo-SeqTM UDI Index Primers: 10 μL
- 2X Index PCR Premix: 25 μL
- Total Volume: 50 μL
Mix entire reaction thoroughly by pipetting or gently vortexing then briefly centrifuge.
If performing the clean-up same-day, equilibrate to room temperature for 30 minutes prior to use
Perform the following steps in a thermal cycler (lid temp 105°C):
- The recommended number of PCR cycles may be increased to the following depending on the FFPE DNA sample input amount and type used:

This is a safe stopping point. Amplified DNA samples can be safely stored overnight at 4°C. Otherwise, continue directly to Step 4 on the next page.

Follow the clean-up protocol in Appendix A using 40 μL of Select-a-Size MagBeads (60 μL for non-FFPE)
- For elution, resuspend the beads in 20 μL of DNA Elution Buffer and aspirate all 20 μL eluate after separation from the beads.

Appendix A: Select-a-Size MagBead Clean-Up Protocol

Before Starting:

Ensure that the indicated volume of ethanol has been added to the DNA Wash Buffer.
Allow the Select-a-Size MagBeads to equilibrate to room temperature for 30 minutes prior to use.
Resuspend the magnetic particles immediately before use by vortexing the Select-a-Size MagBeads until homogenous.

Add 40 (60 for non-FFPE) μL of the Select-a-Size MagBeads to the tube. Mix thoroughly by pipetting until homogenous and incubate for 5−10 minutes at room temperature.
1. This is a 0.8X bead cleanup (1.2X for non-FFPE)
Place the tube on a magnetic stand for 3 minutes, or until the supernatant is clear.
Carefully remove the supernatant without disturbing the magnetized bead pellet.
1. Avoid aspirating any beads when removing the supernatant.
Without removing from the magnetic stand, add 200 μL of DNA Wash Buffer to the tube, incubate for at least 30 seconds, and then remove the supernatant completely without disturbing the magnetized bead pellet. Repeat this wash step for two washes total.
Remove the tube from the magnetic stand and centrifuge very briefly. Then return the tube to the magnetic stand, wait for the beads to pellet, and remove any residual DNA Wash Buffer with a 10 μL pipette tip.
Leave the tube on the magnetic stand and keep the cap open for 2−3 minutes to allow the beads to air dry.
1. Do not over dry the beads as this may negatively impact recovery. Beads should remain a glossy brown color.
Cap and remove the tube from the magnetic stand. Add the indicated volume of DNA Elution Buffer and fully resuspend the beads by pipetting up and down. Incubate for 5 minutes at room temperature.
1. If performing clean-up in Section 3: Index PCR Amplification, add 20 μL of DNA Elution Buffer to fully resuspend the beads.
Place the tube back on the magnetic stand for 2 minutes or until the supernatant is clear.
Transfer the indicated volume of eluate to a new tube. Discard the beads.
If performing clean-up in Section 3: Index PCR Amplification, aspirate all 20 μL eluate and transfer to a new 1.5 mL microcentrifuge tube.

THIS IS THE FINAL WGBS LIBRARY. DNA libraries can be safely stored at ≤ −20°C for months.

QC

Run to visualize libraries. Here’s an example of what the library should look like on a Tapestation:

“If adapter dimers are present, they will form an approximately 130-180 bp band. Yields will vary depending on the total quantity and quality of sample input DNA.

Written on December 3, 2024