Protocol for use with NEBNext FFPE DNA Repair Mix (M6630) and NEBNext Ultra II DNA library Prep Kit for Illumina (E7645)

Symbols
This caution sign signifies a step in the protocol that has multiple paths
leading to the same end point but is dependent on a user variable, like the
amount of input DNA.
Colored bullets indicate the cap color of the reagent to be added to a reaction.

Starting Material: 5 ng–1 µg fragmented FFPE DNA. We recommend that DNA be sheared in 1X TE. If the DNA volume post shearing is less that 50 µl, add 1X TE to a final volume of 50 µl. Alternatively, samples can be diluted with 10 mM Tris-HCl, pH 8.0 or 0.1X TE.

1.1. NEBNext FFPE DNA Repair

1.1.1. Mix the following components in a sterile nuclease-free tube (57 µl final volume):

FFPE DNA 48 µl
 (green) FFPE DNA Repair Buffer
3.5 µl
 (green) NEBNext Ultra II End Prep Buffer 3.5 µl
 (green) NEBNext FFPE DNA Repair Mix 2 µl
Total Volume 57 µl

1.1.2. Mix by pipetting followed by a quick spin to collect all liquid from the sides of the tube.

1.1.3. Incubate at 20°C for 30 minutes (with the heated lid off).

 

1.2. NEBNext Ultra II End Prep

1.2.1. Add 3 µl  (green) NEBNext Ultra II End Prep enzyme mix directly to the FFPE repair reaction mixture from Step 1.1.3.

1.2.2. Set a 100 µl or 200 µl to 50 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.

1.2.3. Place in a thermocycler, with the heated lid set to 75°C, and run the following program:
30 minutes @ 20°C
30 minutes @ 65°C
Hold at 4°C
Proceed to Adaptor Ligation

 

1.3. NEBNext Ultra II Adaptor Ligation

1.3.1. Determine whether adaptor dilution is necessary.

 If DNA input is ≤ 100 ng, dilute the NEBNext Adaptor for Illumina in Tris/NaCl, pH 8.0 as indicated in Table 1.3.

Table 1.3:  Adaptor Dilution

Input

Adaptor Dilution
(Volume Adaptor:
Total Volume)

Working Adaptor
Concentration
1 µg–101 ng No dilution 15 µM
100 ng–5 ng 10-fold (1:10) 1.5 µM
Less that 5 ng 25-fold (1:25) 0.6 µM

Note: Due to the varying degree of quality of FFPE DNA, adaptor dilution may need to be further optimized. The dilutions provided here are a general starting point. Excess adaptor should be removed prior to PCR enrichment.

1.3.2. Add the following components directly to the repaired/End-prepped DNA:

End Prep Reaction Mixture (Step 1.2.3) 60 µl
(red) NEBNext Ultra II Ligation Master Mix*  30 µl
 (red) NEBNext Ligation Enhancer
1 µl
(red) NEBNext Adaptor for Illumina** 2.5 µl
Total Volume 93.5 µl

* Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction

** The NEBNext adaptor is provided in the NEBNext oligos kit. NEB has several oligo kit options, which are supplied separately from the library prep kit.

 

Note: The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. We do not recommend adding adaptor to a premix in the Adaptor Ligation Step.

1.3.3. Set a 100 μl or 200 μl pipette to 80 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. (Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance).  

 

1.3.4. Incubate at 20°C for 15 minutes in a thermocycler with the heated lid off.

 

1.3.5. Add 3 µl of  (red) USER® Enzyme to the ligation mixture from Step 1.3.4

 

Note: Steps 1.3.5 and 1.3.6 are only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Singleplex or Multiplex Oligos for Illumina.

 

1.3.6. Mix well and incubate at 37°C for 15 minutes with the heated lid set to ≥ 47°C.

 

Samples can be stored overnight at –20°C.

 

 

1.4. Cleanup of Adaptor-ligated DNA

 

Note: Size selection is not recommended for FFPE DNA since it contains a large amount of small DNA fragments that would be lost in the size selection process. A cleanup will preserve more of the library.

 

Note: The volumes of SPRIselect or NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step. AMPure XP Beads can be used as well. If using AMPure XP Beads, allow the beads to warm to room temperature for at least 30 minutes before use. These bead volumes may not work properly for a cleanup at a different step in the workflow, or if this is a second cleanup at this step. For cleanups of samples contained in different buffer conditions, the volumes may need to be experimentally determined.

 

1.4.1. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.

 

1.4.2. Add 87 μl (0.9X) resuspended beads to the Adaptor Ligation reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

 

1.4.3. Incubate samples on bench top for 5 minutes at room temperature.

 

1.4.4. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.

 

1.4.5. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard beads).

 

1.4.6. Add 200 μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

 

1.4.7. Repeat Step 1.4.6 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/ plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

 

1.4.8. Air the dry beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of the DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.

 

1.4.9. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 μl of 10 mM Tris-HCl or 0.1X TE (pH 8.0).

 

1.4.10. Mix well by pipetting 10 times. Incubate for at least 2 minutes at room temperature. If necessary, quickly spin the sample to collect all liquid from the sides of the tube before placing back on the magnetic stand.

 

1.4.11. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 15 µl to a new PCR tube.

 

 Samples can be stored at –20°C.

 

 

1.5. PCR-enrichment of Adaptor-ligated DNA

 

Note: Check and verify that the concentration of your oligos is 10 µM. 

 

Use Option A for any NEBNext oligos kit where index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes.

 

Use Option B for any NEBNext oligos kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined.

 

1.5.1A. Forward and Reverse Primer Not Already Combined

Add the following components to a sterile strip tube:

 

Adaptor Ligated DNA Fragments (from Step 1.4.11) 15 µl
 (blue) NEBNext Ultra II Q5 Master Mix 25 µl
 (blue) Index Primer/i7 Primer*,** 5 µl
 (blue) Universal PCR Primer/i5 Primer*,** 5 µl
Total Volume 50 µl

*NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations.

**Use only one i7 primer/ index primer per sample. Use only one i5 primer (or the universal primer for single index kits) per sample

 

1.5.1B. Forward and Reverse Primer Already Combined

 

Adaptor Ligated DNA Fragments (from 1.4.11) 15 µl
 (blue) NEBNext Ultra II Q5 Master Mix 25 µl
 (blue) Universal PCR Primer/index Primer* 10 µl
Total Volume 50 µl

 

*NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations.

 

1.5.2. Set a 100 µl or 200 µl pipette to 40 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

 

1.5.3. Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:

Cycle Step Temp Time Cycles
Initial Denaturation 98°C 30 seconds 1
Denaturation 98°C 10 seconds 3-12*
Annealing/Extension 65°C 75 seconds
Final Extension 65°C 5 minutes 1
Hold 4°C  

* The number of PCR cycles should be chosen based on input amount and sample type. Thus, samples prepared with a different method prior to library prep may require re-optimization of the number of PCR cycles. The number of cycles should be high enough to provide sufficient library fragments for a successful sequencing run, but low enough to avoid PCR artifacts and over-cycling (high molecular weight fragments on Bioanalyzer). The number of PCR cycles recommended in Table 1.5.4 are to be seen as a starting point to determine the number of PCR cycles best for standard library prep samples.

 

1.5.4.

FFPE DNA Input # PCR Cycles
Recommended*
 1 µg 3–4**
500 ng 5–6
100 ng 6–7
50 ng 7–8
10 ng 10–11
5 ng 11–12

* The higher end of the recommended cycle number for the Ultra II DNA library prep kit was used for FFPE DNA due to the lower fraction of starting DNA that can be converted into a library. The number of cycles will need to be determined experimentally by the user depending upon the quality of the FFPE DNA used.

** NEBNext adaptors contain a unique truncated design. Libraries constructed with NEBNext adaptors require a minimum of 3 amplification cycles to add the complete adaptor sequences for downstream processes.

 

 

1.6. 1X SPRI Bead Cleanup of PCR Amplification

 

Note: The volumes of SPRIselect or NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step. AMPure XP Beads can be used as well. If using AMPure XP Beads, allow the beads to warm to room temperature for at least 30 minutes before use. These volumes may not work properly for a cleanup at a different step in the workflow. For cleanups of samples contained in different buffer conditions, the volumes may need to be experimentally determined.

 

1.6.1. Vortex SPRIselect or Sample Purification Beads to resuspend. AMPure XP beads can be used as well. If using AMPure XP beads, allow the beads to warm to room temperature for at least 30 minutes before use.

 

1.6.2. Add 50 μl (1X) resuspended SPRIselect beads to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

 

1.6.3. Incubate samples on bench top for 5 minutes at room temperature.

 

1.6.4. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.

 

1.6.5. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard beads).

 

1.6.6. Add 200 μl of 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

 

1.6.7. Repeat Step 1.6.6 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/ plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

 

1.6.8. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.

 

1.6.9. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 33 μl of 0.1X TE.

 

1.6.10. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

 

1.6.11. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 30 μl to a new PCR tube for and store at –20°C.

 

1.6.12. Check the size distribution on an Agilent Bioanalyzer High Sensitivity DNA chip. The sample may need to be diluted before loading.

 

1.6.13. A sharp peak at 128 bp corresponds to adaptor-dimer. We recommend repeating Steps 1.6.1 to 1.6.11 if this occurs.

 

 Samples can be stored at –20°C.

 

Figure 1.1: Example of a library prepared with normal liver FFPE DNA.