TITLE: Protocol for use with NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, E7103)
AUTHORS: New England   Biolabs

[DESCRIPTION]
The NEBNext Ultra II DNA Library Prep Kit for Illumina contains enzymes and buffers required to convert a broad range of input amounts of DNA into high quality libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow also has minimal hands-on time. 
Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated together by construction and sequencing of an indexed library on the Illumina sequencing platform. 
For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/ Bulks department at NEB. Please contact OEM@neb.com for further information.

Figure 1. Workflow demonstrating the use of NEBNext Ultra II DNA Library Prep Kit for Illumina

 
Adaptor trimming sequences: 
The NEBNext libraries for Illumina resemble TruSeq libraries and can be trimmed similar to TruSeq:
Adaptor Read 1 AGATCGGAAGAGCACACGTCTGAACTCCAGTCA
Adaptor Read 2 AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

[BEFORE_START]
Starting Material: 500 pg–1 µg fragmented DNA. NEB recommends that DNA be sheared in 1X TE. If the DNA volume post shearing is less than 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.

[GUIDELINES]
Safe Stop Point: This is a point where you can safely stop the protocol. 

Caution: This signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.

Color: A color listed before or after the reagent name indicates the cap color of the reagent to be added to the reaction. 

[STEPS]
SECTION: NEBNext End Prep
1. Add the following components to a sterile nuclease-free tube:

   Component Volume   NEBNext Ultra II End Prep Enzyme Mix  3 µl   NEBNext Ultra II End Prep Reaction Buffer 7 µl   Fragmented DNA 50 µl   Total volume 60 µl   
  
  
SECTION: NEBNext End Prep
2. Set a 100 μl or 200 μl pipette 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.
 
SECTION: NEBNext End Prep
3. Place in a thermal cycler, with the heated lid set to ≥ 75 °C, and run the following program:
 
30 min @ 20 °C
30 min @ 65 °C
Hold at 4 °C

Safe Stop Point: If necessary, samples can be stored at -20 °C; however, a slight loss in yield (~20%) may be observed. NEB recommends continuing with adaptor ligation before stopping. 
SECTION: Adaptor Ligation
4. Determine whether adaptor dilution is necessary.
 
Caution: If DNA input is ≤ 100 ng, dilute the NEBNext Adaptor for Illumina in 10 mM Tris-HCl, pH 7.5-8.0 with 10 mM NaCl as indicated in the table. 
 
 
 
SECTION: Adaptor Ligation
5. Add the following components directly to the End Prep Reaction Mixture:
   Component Amount   End Prep Reaction Mixture (Step 3) 60 µl   NEBNext Ultra II Ligation Master Mix * 30 µl   NEBNext Ligation Enhancer 1 µl   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 NEBNext Oligo kits. NEB has several oligo options which are supplied separately from the library prep kit. Please see www.neb.com/oligos for additional information. 

 
  
  
  

SECTION: Adaptor Ligation
6. 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.
SECTION: Adaptor Ligation
7. Incubate at 20 °C for 15 min in a thermal cycler with the heated lid off.
SECTION: Adaptor Ligation
8. Add 3 µL of (red or blue) USER Enzyme to the ligation mixture from Step 6.
 

SECTION: Adaptor Ligation
9. Mix well and incubate at 37 °C for 15 min with the heated lid set to ≥ 47 °C.

Safe Stop Point: Samples can be stored overnight at -20 °C

SECTION: Size Selection or Cleanup of Adaptor-ligated DNA
10. Caution: If the starting material is > 50 ng, follow the protocol for size selection in Step Case: Input > 50 ng. For input ≤ 50 ng, size selection is not recommended to maintain library complexity. Follow the protocol for cleanup without size selection in Step Case: Input ≤ 50 ng. 


SECTION: Size Selection of Adaptor-ligated DNA
11. 
Caution: The following section is for cleanup of the ligation reaction. 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 min 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.

 
Caution: The following size selection protocol is for libraries with 200 bp inserts only. For libraries with different size fragment inserts, refer to the table below for the appropriate volumes of beads to be added. The size selection protocol is based on starting volume of 96.5 µL.

To select a different insert size than 200 bp, please use the volumes in this table:
Recommended Conditions for Bead Based Size Selection
 
 
SECTION: Size Selection of Adaptor-ligated DNA
12. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
SECTION: Size Selection of Adaptor-ligated DNA
13. Add 40 µL (~0.4X) of resuspended beads to the 96.5 µL 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 s–5 s on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

SECTION: Size Selection of Adaptor-ligated DNA
14. Incubate samples on bench top for at least 5 min at room temperature.
SECTION: Size Selection of Adaptor-ligated DNA
15. 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.
SECTION: Size Selection of Adaptor-ligated DNA
16. After 5 min (or when the solution is clear), carefully transfer the supernatant containing your DNA to a new tube. 

Caution: Do not discard the supernatant.

Discard the beads that contain the unwanted large fragments.


SECTION: Size Selection of Adaptor-ligated DNA
17. Add 20 µL (0.2X) resuspended SPRIselect or NEBNext Sample Purification Beads to the supernatant and mix at least 10 times. Be careful to expel all of the liquid from the tip during the last mix. Then incubate samples on the bench top for at least 5 min at room temperature.
  

SECTION: Size Selection of Adaptor-ligated DNA
18. 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.
SECTION: Size Selection of Adaptor-ligated DNA
19. After 5 min (or when the solution is clear), carefully remove and discard the supernatant that contains unwanted DNA. Be careful not to disturb the beads that contain the desired DNA targets.

Caution: Do not discard beads.
SECTION: Size Selection of Adaptor-ligated DNA
20. Add 200 µL of 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
SECTION: Size Selection of Adaptor-ligated DNA
21. Repeat step 20 once for a total of two washes. Be sure to remove all visable 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. 


SECTION: Size Selection of Adaptor-ligated DNA
22. Air dry the beads for up to 5 min 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.
SECTION: Size Selection of Adaptor-ligated DNA
23. Remove the tube/ plate from the magnetic stand. Elute the DNA target from the beads into 17 µL of 10 mM Tris-HCl or 0.1X TE.
  

SECTION: Size Selection of Adaptor-ligated DNA
24. Mix well on a vortex mixer or by pipetting up and down 10 times. Incubate for at least 2 min 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.
SECTION: Size Selection of Adaptor-ligated DNA
25. Place the tube/ plate on a magnetic stand. After 5 min (or when the solution is clear), transfer 15 µL to a new PCR tube for amplification. 

Safe Stop Point: Samples can be stored at -20 °C



SECTION: PCR Enrichment of Adaptor-ligated DNA: PCR Amplification
26. Use Option A if you are using the following oligos: 
Use Option A for any NEBNext Oligo Kit where index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes. Primers are supplied at 10 µM each. 
 
Use Option B if you are using the following oligos: 
Use Option B for any kit where NEBNext Oligo Kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined. Primers are supplied at 10 µM (5 µM each). 
SECTION: PCR Enrichment of Adaptor-ligated DNA: PCR Amplification
27. PCR Amplification
Add the following components to a sterile strip tube:
 
Option A: Forward and Reverse Primers NOT Already Combined
   Component Volume per Reaction   Adaptor Ligated DNA Fragments 15 µl   NEBNext Ultra II Q5 Master Mix 25 µl   Index Primer/i7 Primer*,** 5 µl   Universal PCR Primer/i5 Primer*,**  5 µl   Total Volume 50 µl   
 
Option B: Forward and Reverse Primer Already Combined
   Comonent Volume per Reaction   Adaptor Ligated DNA Fragments 15 µl   NEBNext Ultra II Q5 aster Mix 25 µl   Index/Universal Primer* 10 µl   Total Volume 50 µl   
  
SECTION: PCR Enrichment of Adaptor-ligated DNA: PCR Amplification
28. 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.
SECTION: PCR Enrichment of Adaptor-ligated DNA: PCR Amplification
29. Place the tube on a thermal cycler 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 3-15 *   Annealing/Extension 65°C 75 seconds   Final Extension 65°C 5 minutes 1   Hold 4°C ∞     

 

 

SECTION: PCR Enrichment of Adaptor-ligated DNA: PCR Amplification
30. Proceed to Cleanup of PCR Amplification in the next Section.
SECTION: Cleanup of PCR Reaction
31. 
 

SECTION: Cleanup of PCR Reaction
32. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
  

SECTION: Cleanup of PCR Reaction
33. Add 45 µL (0.9X) resuspended 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 s–5 s on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
SECTION: Cleanup of PCR Reaction
34. Incubate samples on bench top for at least 5 min at room temperature.
SECTION: Cleanup of PCR Reaction
35. 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.
SECTION: Cleanup of PCR Reaction
36. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets.

Caution: Do not discard the beads.
SECTION: Cleanup of PCR Reaction
37. Add 200 µL 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

SECTION: Cleanup of PCR Reaction
38. Repeat Step 37 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. 

SECTION: Cleanup of PCR Reaction
39. Air dry the beads for up to 5 min 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.
SECTION: Cleanup of PCR Reaction
40. Remove the tube/ plate from the magnetic stand. Elute the DNA target from the beads by adding 33 µL of 0.1X TE. 
SECTION: Cleanup of PCR Reaction
41. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min 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.

SECTION: Cleanup of PCR Reaction
42. Place the tube/ plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 30 µL to a new PCR tube and store at -20 °C.
SECTION: Cleanup of PCR Reaction
43. Check the size distribution on an Aglient Bioanalyzer High Sensitivity DNA chip. The sample may need to be diluted before loading. 
 


Examples of libraries prepared with human DNA (NA19240):
a

SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
11. 
 

 

SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
12. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
13. 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 s–5 s on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
14. Incubate samples on bench top for at least 5 min at room temperature.
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
15. 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.
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
16. After 5 min (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. 

SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
17. Add 200 µL of 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
18. Repeat Step 17 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. 
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
19. Air dry the beads for up to 5 min 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. 
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
20. 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. 
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
21. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min 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.
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection (for input ≤ 50 ng)
22. Place the tube/ plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 15 µL to a new PCR tube.

Safe Stop Point: Samples can be stored at -20 °C.


SECTION: PCR Enrichment of Adaptor-ligated DNA
23. Use Option A if you are using the following oligos: 
Use Option A for any NEBNext Oligo Kit where index primers are supplied in tubes. There kits have the forward and reverse primers supplied in separate tubes. Primers are supplied at 10 µm each. 
 
Use Option B if you are using the following oligos: 
Use Option B for any NEBNext Oligo Kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined. Primers are supplied at 10 µM combined. (5 µm each). 
SECTION: PCR Enrichment of Adaptor-ligated DNA
24. PCR Amplification
Add the following components to a sterile strip tube:
 
Option A: Forward and Reverse Primer NOT Already Combined
   Component Volume per Reaction   Adaptor Ligated DNA Fragments 15 µl   NEBNext Ultra II Q5 Master Mix 25 µl   Index Primer/i7 Primer*,** 5 µl   Universal PCR Primer/i5 Primer*,**  5 µl   Total Volume 50 µl   
 *NEBNext Oligos must be purchased separately from the library prep kit. For oligo purchasing options refer to "Required Materials Not Included" section in the Materials Section. 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. 

Option B: Forward and Reverse Primers Already Combined
   Comonent Volume per Reaction   Adaptor Ligated DNA Fragments 15 µl   NEBNext Ultra II Q5 aster Mix 25 µl   Index/Universal Primer* 10 µl   Total Volume 50 µl   
*NEBNext Oligos must be purchased separately from the library prep kit. For oligo purchasing options refer to "Required Materials Not Included" section in the Materials Section. 
SECTION: PCR Enrichment of Adaptor-ligated DNA
25. 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.
SECTION: PCR Enrichment of Adaptor-ligated DNA
26. Place the tube on a thermal cycler 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 3-15 *   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 4.1 are to be seen as a starting point to determine the number of PCR cycles best for standard library prep samples. Use Table 4.2 for applications requiring high library yields (~1 μg) such as target enrichment.

Table 4.1
 
* These input ranges will work best when size selection is done 
** 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.

Table 4.2
 
* Cycle number was determined for size selected libraries. 
** 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.
SECTION: PCR Enrichment of Adaptor-ligated DNA
27. Proceed to Cleanup of PCR Amplification in next Section.
SECTION: Cleanup of PCR Reaction
28. 
 

SECTION: Cleanup of PCR Reaction
29. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of PCR Reaction
30. Add 45 µL (0.9X) resuspended 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 s–5 s on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
SECTION: Cleanup of PCR Reaction
31. Incubate samples on bench top for at least 5 min at room temperature.
SECTION: Cleanup of PCR Reaction
32. 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.
SECTION: Cleanup of PCR Reaction
33. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets.

Caution: Do not discard the beads.
SECTION: Cleanup of PCR Reaction
34. Add 200 µL of 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

SECTION: Cleanup of PCR Reaction
35. Repeat Step 34 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. 

SECTION: Cleanup of PCR Reaction
36. Air dry the beads for up to 5 min 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.
SECTION: Cleanup of PCR Reaction
37. Remove the tube/ plate from the magnetic stand. Elute the DNA target from the beads by adding 33 µL of 0.1X TE.
SECTION: Cleanup of PCR Reaction
38. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min 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.

SECTION: Cleanup of PCR Reaction
39. Place the tube/ plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 30 µL to a new PCR tube and store at -20 °C.
SECTION: Cleanup of PCR Reaction
40. Check the size distribution on an Aglient Bioanalyzer High Sensitivity DNA chip. The sample may need to be diluted before loading. 

Safe Stop Point: Samples can be stored at -20 °C.


Examples of libraries prepared with human DNA (NA19240):
a