TITLE: E7805 NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® Protocol for Large Fragment Sizes (&gt; 550 bp) (Chapter 3)
AUTHORS: New England   Biolabs

[DESCRIPTION]
The NEBNext Ultra II FS DNA Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of intact gDNA, cDNA, plasmids and amplicons* into high quality libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow also has minimal hands-on time.
*Fragmentation times for cDNA, plasmids and amplicons may vary from gDNA times. 

Note: The Ultra II FS Kit is not compatible with bisulfite conversion workflows and FFPE DNA. 
See the following FAQ's for more information:
Is the NEBNext Ultra II FS DNA library prep kit for Illumina compatible with all sample types?
Can enzymatically fragmented DNA be used as input material for EM-seq?

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 indexed libraries on an Illumina sequencing platform. 

For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM & Customized Solutions at NEB. Please contact custom@neb.com for further information. 

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

 
Adaptor trimming sequences
The NEBNext libraries for Illumina resemble TruSeq libraries and can be trimmed similar to TruSeq: 
AdaptorRead1 AGATCGGAAGAGCACACGTCTGAACTCCAGTCA
AdaptorRead2 AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT


[BEFORE_START]
Note: Follow this protocol for inputs ≥ 100 ng and fragment sizes > 550 bp. 

Follow the protocol in Chapter 1 for inputs ≤ 100 ng
https://www.protocols.io/view/e7805-nebnext-ultra-ii-fs-dna-library-prep-kit-for-k8tczwn

Follow the protocol in Chapter 2 for inputs ≥ 100 ng and fragments ≤ 550 bp.
https://www.protocols.io/view/e7805-nebnext-ultra-ii-fs-dna-library-prep-kit-for-nwxdffn
 
For 100 ng inputs, either the no size selection protocol (Chapter 1) or a size selection protocol (Chapter 2 or 3) can be followed. 

Starting Material: 100–500 ng purified, genomic DNA*. We recommend that the DNA be in 1X TE (10 mM Tris pH 8.0, 1 mM EDTA), however, 10 mM Tris pH 7.5–8, low EDTA TE or H2O are also acceptable. If the input DNA is less than 26 μl, add TE (provided) to a final volume of 26 μl.

* This kit can also be used for plasmids, cDNA and some amplicons. It is not recommended for use with FFPE DNA. See FAQ:
https://www.neb.com/faqs/2020/10/16/is-the-nebnext-ultra-ii-fs-dna-library-prep-kit-for-illumina-compatible-with-all-sample-types

For FFPE DNA we recommend NEBNext Ultra II DNA Library Pre Kit for Illumina + Covaris or NEBNext FFPE DNA Library Prep Kit (NEB #E6650) or NEBNext UltraShear FFPE DNA Library Prep Kit (NEB #E6655).

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

Caution: This signifies a step in the protocol that has multiple paths that lead 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 a reagent name indicates the cap color of the reagent to be added to a reaction.

[STEPS]
SECTION: Fragmentation/End Prep
1. Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.

SECTION: Fragmentation/End Prep
2. Vortex the Ultra II FS Enzyme Mix 5 s - 8 s prior to use and place on ice.
 




SECTION: Fragmentation/End Prep
3. Add the following components to a 0.2 ml thin wall PCR tube on ice:
 
*Intact gDNA, cDNA, plasmids and some amplicons.
  
  

SECTION: Fragmentation/End Prep
4. Vortex the reaction for 5 s and briefly spin in a microcentifuge.

SECTION: Fragmentation/End Prep
5. In a thermal cycler, with the heated lid set to 75 °C, run the following program:
   5 min 37°C   30 min 65°C   Hold 4°C   
 

SECTION: Adaptor Ligation
6. Add the following components directly to the FS Reaction Mixture:
   Component Volume   FS Reaction Mixture (Step 5) 35 µl   NEBNext Ultra II Ligation Master Mix* 30 µl   NEBNext Ligation Enhancer 1 µl   NEBNext Adaptor for Illumina** 2.5 µl   Total Volume 68.5 µl   
 
* Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding 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. Please see www.neb.com/oligios for additional information.

 
  
  
  

SECTION: Adaptor Ligation
7. 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.
 
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
8. Incubate at 20 °C for 15 min in a thermal cycler with the heated lid off.

SECTION: Adaptor Ligation
9. Add 3 µL of (red or blue) USER Enzyme to the ligation mixture from the previous step.

 
  




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




SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
11. Caution: 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 (71.5 µL; Step 10). 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.

SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
12. Bring the volume of the reaction up to 100 µL by adding 28.5 µL 0.1X TE (dilute 1X TE Buffer 1:10 with water).
  

SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
13. Vortex SPRIselecct or NEBNext Sample Purification Beads to resuspend
  

SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
14. Add 40 µL (0.4X) 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: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
15. Incubate samples for at least 5 min at room temperature.

SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
16. 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 for Fragment Sizes > 550 bp
17. 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 the beads.
SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
18. 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 for Fragment Sizes > 550 bp
19. Repeat the previous step 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: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
20. 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. 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 for Fragment Sizes > 550 bp
21. Remove the tube/ plate from the magnetic stand. Elute the DNA target from the beads by adding 102 µL 0.1X TE (dilute 1X TE Buffer 1:10 in water).
  


SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
22. 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: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
23. Place the tube/ plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 100 µL to a new PCR tube. 

SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
24. Add 50 µL (~ 0.5X) of resuspended SPRIselect or Sample Purification Beads to the sample. 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 for Fragment Sizes > 550 bp
25. Incubate samples at room temperature for at least 5 min.

SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
26. 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 for Fragment Sizes > 550 bp
27. 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.
 
Caution: Do not discard the beads.
SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
28. 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 for Fragment Sizes > 550 bp
29. Repeat the previous step 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: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
30. 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. 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 for Fragment Sizes > 550 bp
31. Remove the tube/ plate from the magnetic stand. Elute the DNA target from the beads into 17 µL 0.1X TE (dilute 1X TE Buffer 1:10 in water).
  

SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
32. Mix well on a vortex mixer or by pipetting up and down at least 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 for Fragment Sizes > 550 bp
33. 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. 

SECTION: Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp
34. Proceed to PCR Enrichment of Adaptor-ligated DNA in next Section: PCR Enrichment of Adaptor-ligated DNA

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

SECTION: PCR Enrichment of Adaptor-ligated DNA
35. • 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 serparate tubes. Primers are supplied at 10 μM each.
 
• 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
36. Add the following components to a sterile strip tube:
 
Option A: Forward and Reverse Primers NOT Already Combined:
   Component Volume per One Library   Adaptor Ligated DNA Fragments (Step 34) 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 seperately 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 manuals 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
   Component Volume per One Library   Adaptor Ligated DNA Fragments (Step 34) 15 µl   NEBNext Ultra II Q5 Master 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"  in the materials section. 
  

SECTION: PCR Enrichment of Adaptor-ligated DNA
37. 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
38. Place the tube on a thermal cycler with the heated lid set to 105 °C and perform PCR amplification using the following PCR cycling conditions:

 

* The number of PCR cycles recommended in Table A are to be seen as a starting point to determine the number of PCR cycles best for standard library prep samples. Use Table B for applications requiring high library yields, such as target enrichment. 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).
 
Table A
 

* Cycle number was determine for size selected libraries.
 
Table B
 

* Cycle number was determined for non-size selected libraries.
SECTION: PCR Enrichment of Adaptor-ligated DNA
39. Proceed to Cleanup of PCR reaction in next Section: Cleanup of PCR Reaction.
SECTION: Cleanup of PCR Reaction
40. 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.
SECTION: Cleanup of PCR Reaction
41. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
  

SECTION: Cleanup of PCR Reaction
42. Add 30 µL (0.6X) 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
43. Incubate samples on bench top for at least 5 min at room temperature.

SECTION: Cleanup of PCR Reaction
44. 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
45. 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 the beads.

SECTION: Cleanup of PCR Reaction
46. 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
47. Repeat the previous step 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
48. 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. 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
49. Remove the tube/ plate from the magnetic stand. Elute the DNA target from the beads by adding 33 µL of 0.1X TE (dilute 1X TE Buffer 1:10 in water).
  
SECTION: Cleanup of PCR Reaction
50. 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
51. 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: Assess Library Quality on a Bioanalyzer
52. Dilute library (from previous step) 5-fold in 0.1X TE Buffer.
  
SECTION: Assess Library Quality on a Bioanalyzer
53. Run 1 µL on a DNA High Sensitivity Chip.

SECTION: Assess Library Quality on a Bioanalyzer
54. Check that the library size shows a narrow distribution with an expected peak size > 700 bp (See Figure 1)

 

Figure 1: Example of final library size distributions without size selection. 100 ng Human DNA (NA 19240) was fragmented for 5 minutes.

a


 

We recommend gel size selection if you need an average sequence insert size > 700 bp.
 
Figure 2: Example of insert size distribution after bead-based size selection.

a