bs RAD Library Prep

Edit this page

The protocol for doing a library prep for bs ddRAD.

Note: this protocol is outdated. Not sure if it works

Background

Materials and Equipment

1. DNA Extractions

2. RAD digestion, Adapter ligation, Size Selection and ddRAD library prep

3. BiSulfite Conversion

(based on 96 well plate conversion)

Cells-to-CpG BiSulfite Conversion Kit (2x96 = 550$)

Item Amount
Binding Buffer 1 x 192-rxn bottle
Binding Plates 2 plates
Conversion Buffer 10 mL
Conversion Reagent 2 x 96-rxn bottles
Denaturation Reagent 1.mL
Desulfonation Reagent 1 x 192-rxn bottle
Elution Buffer 20 mL
Elution Plates 2 plates
Lysis Enhancer 2 x 0.5 mL
Wash Buffer 2 x 96-rxn bottles

Other Materials

Item # Needed Cost
MicroAmp Clear Adhesive Film
MicroAmp Optical Film Compression Pad
Nuclease-free Water (not DEPC-treated) (1x500mL)
Reaction Plates
Ethanol, molecular grade, >99.5% or 200 proof
Isopropyl alcohol, ACS, reagent grade, >99.5%
Microcentrifuge tubes (Size)
Pipette tips, nuclease-free (Sizes)

Equipment

Items
ThermoMixer
Microcentrifuge
Pipettors (sizes)
Vortex
Water bath 60oC

4. BiSulfite PCR

KAPA HiFi HotStart Uracil+ ReadyMix

Item Amount Total Amount per Kit
2X KAPA HiFi HotStart Uracil+ ReadyMix 1.25mL (50x50uL) or 6.25mL (250x50uL) 12.5 uL per single reaction

Other Materials

Item # Needed Cost
MicroAmp Clear Adhesive Film
MicroAmp Optical Film Compression Pad
Nuclease-free Water (not DEPC-treated) (1x500mL)
Ethanol, molecular grade, >99.5% or 200 proof
Microcentrifuge tubes (Size)
Pipette tips, nuclease-free (Sizes)

Equipment

Items
Thermocycler
Microcentrifuge
Pipettors (sizes)
Vortex

6. Quality Control Steps

Overview

1. DNA Extractions

2. RAD digestion, Adapter ligation, and Size Selection

3. BiSulfite Conversion

BiSulfite Conversion

4. Bisulfite PCR

KAPA HiFi HotStart is a novel B-family DNA polymerase, engineered to have increased affinity for DNA, without the need for accessory proteins or DNA-binding domains. The intrinsic high processivity of the enzyme results in significant improvement in yield, speed and sensitivity when compared with wild-type B-family DNA polymerases and polymerase blends. When used for the amplification of next-generation sequencing (NGS) libraries, KAPA HiFi HotStart DNA Polymerase exhibits high yields with minimal amplification bias and provides extremely uniform sequence coverage.

The read-ahead function of proofreading DNA polymerases detects pro-mutagenic uracil residues in the template strand and prevents further strand extension, thereby reducing or completely inhibiting PCR amplification. In KAPA HiFi HotStart Uracil+ DNA Polymerase, this uracilbinding pocket is inactivated to enable the amplification of uracil-containing DNA. The enzyme shows the same high yield, low GC-bias and coverage uniformity as the unmodified KAPA HiFi HotStart DNA Polymerase, making it particularly advantageous for applications employing bisulfite DNA conversion, which typically produces low concentrations of AT-rich DNA.

KAPA HiFi HotStart Uracil+ DNA Polymerase has 5’g3’ polymerase and 3’g5’ exonuclease (proofreading) activity, but no 5’g3’ exonuclease activity. The strong 3’g5’ exonuclease activity results in superior accuracy during DNA amplification. A proprietary antibody inactivates the polymerase until the first cycle of denaturation, minimizing spurious amplification products that may result from nonspecific priming events during reaction setup and initiation, and increasing overall reaction efficiency. KAPA HiFi HotStart Uracil+ ReadyMix (2X) is a ready-touse cocktail containing all components required for PCR, except primers and template. The ReadyMix contains 0.2 mM of each dNTP (dATP, dCTP, dGTP, dTTP), and does not contain dUTP.

5. Pooling

6. Quality Control Steps

Protocol

1. DNA Extractions

for oyster extractions see 3a DNA Extraction C. virginica - Protocol

2. RAD digestion

see ‘5 ddRAD seq’ for details on ddRAD digest.

3. Adapter Ligation

SPECIAL NOTES: Using specific methylated adapters!!!

4. BiSulfite Conversion

Link to bisulfite conversion kit.

Prepare the reagents (for new kits)

Before you use a new kit, prepare the Binding Buffer, Wash Buffer, and Desulfonation Reagent.

  1. Add 50 mL isopropyl alcohol (>99.5%) to the bottle of Binding Buffer, then swirl the bottle to mix.
  2. Add 120 mL ethanol (>99.5% or 200 proof) to each bottle of Wash Buffer, then swirl the bottles to mix.
  3. Add 45 mL ethanol (99.5% or 200 proof) to each bottle of Desulfonation Reagent, then swirl the bottles to mix.

After you prepare the reagents, you will have sufficient reagents for 192 reactions (two 96-well plates).

Prepare the conversion reagent

For optimal results, prepare the Conversion Reagent immediately before performing the bisulfite conversion.

  1. Add Denaturation Reagent and water to the powder in the Conversion Reagent bottle, then mix well:
Component Volumne
Denaturation Reagent 260 uL
Water 8 mL
  1. Add 500 uL Conversion Buffer to the Conversion Reagent bottle, then mix again.
  2. To improve solubility of the powder, place the Conversion Reagent bottle in a 60oC water bath for 10 minutes.
  3. Mix by vortexing 2 or 3 times during the 10-minute incubation.

Note: It is normal to see trace amounts of undissolved powder in the Conversion Reagent.

After you prepare the bisulfite conversion reactions, you can store and remaining Conversion reagent at -20oC for up to one month. Before use, thaw the Conversion Reagent at 50oC for 10 minutes, then vortex.

Denature the DNA

  1. Set up the PCR reaction plate. For each sample:
  • Pipet 45 uL into a PCR reaction plate.
  1. Denature the DNA in the reaction plate:
  • Add 5 uL Denaturation Reagent to 45 uL gDNA, then pipet up and down.
  • Incubate at 50oC for 10 minutes.

Perform bisulfite conversion of the denatured DNA

Convert unmethylated cytosines to uracil in the denatured DNA samples.

  1. Add 100 uL prepared Conversion Reagent to each denatured sample for a total reaction volume of 150 uL, then mix the reaction.

    Note: The reaction should not contain any undissolved Conversion Reagent powder after mixing.

  2. Incubate the sample in a thermal cycler, selecting the appropriate thermal cycling conditions for your samples:

  • For gDNA input between 100ng and 2ug, use the general thermal cycling conditions with acceptable recovery and conversion:
Temp Time
65oC 30 minutes
95oC 1.5 minutes
65oC 30 minutes
95oC 1.5 minutes
65oC 30 minutes
4oC up to 4 hours
  • For gDNA input between 50pg and 100ng, use the thermal cycling conditions optimal for recovery but with decreased conversion rates for high sample input:
Temp Time
65oC 30 minutes
95oC 0.5 minutes
65oC 30 minutes
95oC 0.5 minutes
65oC 30 minutes
4oC up to 4 hours
  • For crude gDNA input >2 ug, use thermal cycling conditions optimal for conversion but with slightly decreased recovery for long amplicons:
Temp Time
95oC 3 minutes
65oC 60 minutes
95oC 3 minutes
65oC 30 minutes
4oC up to 4 hours

STOPPING POINT: If you cannot proceed with desalting and desulfonation immediately, you can store the converted sample at -20oC for up to 2 days. Thaw the samples at room temp, then proceed with desalting and desulfonation.

Desalt and desulfonate the samples

Remove salts from the DNA samples, then desulfonate the DNA to remove the sulfonic groups.

  1. Combine the DNA samples with Binding Buffer:
  • Add 600 uL of Binding Buffer to wells of a 2-mL 96-well deep block.
  • Transfer each converted sample (150uL) to the corresponding well in the block.
  • Mix by pipetting up and down with a multichannel pipettor.
  1. Load the samples onto the Binding Plate:
  • Place a Binding Plate on top of a PureLink 96 Receiver Plate.
  • Load each mixed sample onto the corresponding column in the Binding Plate.
  • Spin the stacked plates at 3100 x g for 3 minutes.
  • Discard the flowthrough.
  1. Wash the samples to remove salts:
  • Restack the Binding Plate on top of the 96-well Reciever Plate.
  • Add 600 uL of Wash Buffer to each appropriate column in the Binding Plate.
  • Spin the stacked plates at 3100 x g for 3 minutes, or until all the Wash Buffer passes through the columns.
  • Discard the flowthrough.
  1. Desulfonate the DNA:
  • Restack the Binding Plate on top of the 96-well Receiver Plate.
  • Add 200 uL of Desulfonation Reagent to each appropriate column in the Binding Plate.
  • Cover the Binding Plate with adhesive foil, then let plate stand at room temperature (20-30oC) for 20 minutes.
  • Spin the stacked plates at 3100 x g for 3 minutes.
  • Discard the flowthrough.
  1. Wash the DNA two times. For each wash:
  • Restack the Binding Plate on top of the 96-well Receiver Plate.
  • Add 400 uL of Wash Buffer to each appropriate column in the Binding Plate.
  • Spin the stacked plates at 3100 x g for 3 minutes.
  • Discard the flowthrough.
  1. Remove any residual Wash Buffer:
  • Restack the Binding Plate on top of the 96-well Receiver Plate.
  • Spin the stacked plates at 3100 x g for 20 minutes.

Important! Trace amounts of Wash Buffer could inhibit downstream reactions.

  • Discard the flowthrough and the 96-well Receiver Plate.
  1. Elute the DNA:
  • Place the Binding Plate on top of a new Elution Plate.
  • Add 100 uL of Elution Buffer directly to the center of each appropriate column in the Binding Plate. Note: If you started with less than 10ng gDNA, you can reduce the elution volume to 50 uL.
  • Let the plate stand at room temp (20-30oC) for 5 minutes.
  • Spin the stacked plates for 3100 x g for 5 minutes.

STOPPING POINT Store the converted DNA at 4oC for up to 6 months. For long-term storage, store at -20oC or -70oC. Store aliquots to avoid multiple freeze-thaw cycles.

Assess the yield and quality of the bisulfite-converted DNA

5. KAPA HiFi Hotstart Uracil+ PCR

IMPORTANT! The KAPA HiFi HotStart Uracil+ ReadyMix contains an engineered B-family (proofreading) DNA polymerase and uniquely-formulated buffers, and requires specialized reaction conditions. If these conditions are not adhered to, reaction failure is likely. Refer to Important Parameters for more information.

1. Prepare the PCR master mix

1.1 KAPA HiFi HotStart Uracil+ reactions MUST be set up on ice since the high proofreading activity of the enzyme will result in rapid primer degradation at room temperature.

1.2 Ensure that all reagents are properly thawed and mixed.

1.3 Prepare a PCR master mix containing the appropriate volume of all reaction components common to all or a subset of reactions to be performed.

1.4 Calculate the required volumes of each component based on the following table:

Component 25 uL reaction Final conc.
PCR-grade water Up to 25 uL N/A
2X KAPA HiFi Hotstart Uracil+ ReadyMix 12.5 uL 1X
10 uM Forward Primer 0.75 uL 0.3 uM
10 uM Reverse Primer 0.75 uL 0.3 uM
Template DNA As required As required

2. Set up individual reactions

2.1 Transfer the appropriate volumes of PCR master mix, template and primer to individual PCR tubes or wells of a PCR plate. 2.2 Cap or seal individual reactions, mix and centrifuge briefly.

3. Run the PCR

3.1 Perform PCR with the following cycling protocol:

Step Temp. Duration Cycles
Initial denaturation 95oC 3 min 1
Denaturation 98oC 20 sec 15-36
Annealing 60oC-75oC 15 sec 15-35
Extension 72oC 15-60 sec/kb 15-35
Final Extension 72oC 1 min/kb 1
Hold 4oC Infinite 1

4. Notes and troubleshooting

Amplification of bisulfite-converted DNA

Amplification of bisulfite-treated DNA can be problematic due to DNA damage arising from the harsh conditions required for near-complete conversion of unmethylated cytosines. Conversion conditions should be optimized to minimize the extent of DNA damage while ensuring sufficient conversion efficiency. Because bisulfite treatment converts cytosines to uracils, which are subsequently substituted with thymines during PCR, bisulfite-converted DNA is typically much more AT-rich than the original source DNA. This should be considered carefully when designing PCR primers, and cycling parameters such as annealing temperature may require special attention.

MgCl2 concentration

KAPA HiFi HotStart Uracil+ ReadyMix contains a final (1X) MgCl2 concentration of 2.5 mM, which is sufficient for most applications. Applications which are likely to require higher MgCl2 concentrations include long PCR (>10 kb) and AT-rich PCR, as well as amplification using primers with a low GC content (<40%). Note that bisulfiteconverted DNA is typically very AT-rich, as are the primers used for amplification of these templates; this application may therefore require additional MgCl2.

Prevention of amplicon contamination with UDG

For effective removal of carryover contamination, dUTP must be added routinely to PCR reactions with KAPA HiFi HotStart Uracil+ ReadyMix. Typically, dUTP is added to a concentration of 0.2 mM, but short AT-poor amplicons may require up to 0.3 mM dUTP for effective removal using UDG. For amplicons >600 bp, a lower dUTP concentration (≤ 0.1 mM) may be required for efficient amplification. Low amplification efficiencies may result from incomplete UDG inactivation, in which case a longer initial denaturation may be required to inactivate the UDG prior to cycling. Refer to the manufacturer’s recommendations for optimal UDG concentration, and incubation/inactivation conditions.

Denaturation

Due to the high salt concentration of the KAPA HiFi HotStart Uracil+ ReadyMix, it is important to use appropriate denaturation parameters. An initial denaturation time of 2–5 min at 95°C is recommended to ensure that complex template DNA is fully denatured before the first primer annealing step. Use 5 min for complex, genomic DNA and/ or GC-rich targets, and at least 45 sec for less complex templates such as purified viral or plasmid DNA.

Annealing temperature

Due to the high salt concentration of the KAPA HiFi HotStart Uracil+ ReadyMix, the optimal annealing temperature for a given primer set is usually different when compared with other PCR buffer systems. When using the kit with a specific primer pair for the first time, determine the optimal annealing temperature with annealing temperature gradient PCR. We recommend a gradient from 60–72°C, although some assays may require even higher annealing temperatures. For assays with optimal annealing temperatures of 68°C or higher, 2-step cycling may be performed at the optimal annealing temperature. Optimal annealing temperatures below 60°C are typically rare, but may be necessary when using primers with a high AT content, as is often the case with amplification of bisulfite-converted DNA. If a gradient PCR is not feasible, use an annealing temperature of 60°C as a first approach, and adjust the annealing temperature based on the results obtained: • If a low yield of only the specific product is obtained, lower the annealing temperature in 1–2°C increments. • If nonspecific products are formed in addition to the specific product, increase the annealing temperature in 1–2°C increments. • If no product is formed (specific or nonspecific), reduce the annealing temperature by 5°C. MgCl2 concentration may have to be increased. If only nonspecific products are formed (in a ladder-like pattern), increase the annealing temperature by 5°C.

Amplicon length

KAPA HiFi HotStart Uracil+ ReadyMix can amplify targets up to 18 kb in length from simple purified template such as plasmid DNA, and up to 15 kb in length from high quality (i.e. not bisulfite-treated), complex genomic DNA. For efficient amplification of fragments ≥10 kb, higher template concentrations, and optimization of the Mg2+ concentration, may be required.

Primer and template DNA quality

Primers should be designed to minimize the possibility of primer-dimer formation, self-priming, or nonspecific priming. Primer pairs should have similar theoretical melting temperatures (Tm), and should have a GC content of 40–60%, although this may not be feasible for bisulfite-converted template DNA. Primers with a GC content 60% may require higher denaturation temperatures or longer denaturation times, while primers with a GC content 40% may require lower annealing temperatures and increased MgCl2 concentrations. Template DNA quality has a significant impact on the success of PCR amplification. Degraded, damaged, or sheared template DNA is usually problematic. While KAPA HiFi HotStart Uracil+ ReadyMix tolerates uracil, deamination of dCMP to dUMP in the DNA template will generate G/C to A/T mutations during amplification. NOTE: Always dilute and store primers and DNA in a buffered solution (e.g. 10 mM Tris-HCl, pH 8.0 – 8.5) instead of PCR-grade water, and minimize freeze-thaw cycles to limit degradation and maintain primer quality.

Cycle number

Excessive library amplification should be avoided to minimize the following adverse effects: • increased duplicate reads • uneven coverage depth and sequence dropout • chimeric library inserts • nucleotide substitutions • heteroduplex formation To minimize over-amplification and associated unwanted artifacts, the number of amplification cycles should be optimized to ensure a sufficient amount of amplified library for the next step in the workflow (capture or sequencing), plus the amount needed for library QC and/or archiving. Depending on the sequencing application and degree of multiplexing, 100 ng – 1.5 μg of amplified library is typically required. The number of cycles typically required will vary, depending on input amount and quality. Size selection of libraries at any part in the library construction process results in significant loss of material and as a result, 2–4 additional cycles are required for workflows which include a sizeselection step prior to library amplification.

Primer depletion and library over-amplification

In library amplification reactions (set up according to the recommended protocol), primers are typically depleted before dNTPs. When DNA synthesis can no longer take place due to primer depletion, subsequent rounds of DNA denaturation and annealing result in the separation of complementary DNA strands, followed by imperfect annealing to non-complementary partners. This presumably results in the formation of so-called “daisychains” or tangled knots, comprising large assemblies of improperly annealed, partially double-stranded, heteroduplex DNA. These species migrate slower and are observed as secondary, higher molecular weight peaks during the electrophoretic analysis of amplified libraries. However, they are typically comprised of library molecules of the desired length, which are separated during denaturation prior to target enrichment (capture) or cluster amplification. Since these heteroduplexes contain significant portions of single-stranded DNA, over-amplification leads to the under-quantification of library molecules with assays employing dsDNA-binding dyes. qPCR-based library quantification methods, such as that employed by the KAPA Library Quantification Kit, quantify DNA by denaturation and amplification, thereby providing a more accurate measurement of the amount of adapter-ligated molecules—even if the library was over-amplified.

6. Pooling

7. Quality Control Steps