TITLE: C-SOP-301: DNA Library Preparation using the NEBNext Ultra II FS DNA Kit (≥100 ng DNA input)
AUTHORS: Mihir Kekre, Ben Pascoe

[DESCRIPTION]
 The NEBNext®Ultra™ II FS DNA Library Prep Kit meets the dual challenges of constructing high quality libraries from ever-decreasing input material, and scalability of library construction compatible with Illumina sequencing platforms. The fragmentation protocol can tolerate any input amount from 100 picograms–500 nanograms of DNA and varying amounts (%) of GC content. 

Some of the operational advantages that this kit offers are:
Perform fragmentation, end repair and dA-tailing with a single enzyme mix 
Experience reliable fragmentation with a single protocol, regardless of DNA input amount or GC content 
Prepare high quality libraries from a wide range of input amounts: 100 pg–500 ng 
Generate high yields with increased reaction efficiencies and minimised sample loss 
Use with DNA in standard buffers (TE, Tris-HCl) and water 
Vary incubation time to generate a wide range of insert sizes

This protocol has been adapted from the NEBNext®Ultra™II FS DNA Library Prep Kit for Illumina developed by New England BioLabs (NEB).


This is an open-access protocol distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike.
 

[GUIDELINES]
E6177: Product Specifications


[STEPS]
SECTION: Before Starting
1. 
Prior to initiating the protocol, ensure that all active workbenches are cleaned with 80% ethanol, all relevant personal protective clothing is worn and the work area is prepared according to local GLP guidelines for molecular methods.

SECTION: Before Starting
3. Create an organised bench space by clearing away all clutter in order to maximize work efficiency and to avoid unnecessary movements that will minimise exposure of sterile materials to airborne and liquid contaminants. 

Prepare a large bucket of ice to store reagents and samples temporarily during use.
SECTION: Before Starting
2. Starting Material: 200 ng purified, genomic DNA. 
 



SECTION: Fragmentation/End Prep
4. Fragmentation occurs during a 37°C incubation step. 
Ensure that the Ultra II FS Reaction Buffer is completely thawed before placing it on ice. 
 

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


SECTION: Fragmentation/End Prep
6. In a 15 mL Falcon tube, prepare a fresh mastermix of the following components (for each DNA sample + 1 sample overage) as indicated below in Table 1:

 

SECTION: Fragmentation/End Prep
7. Gently mix the reaction by pipetting up/down 15 times and then briefly spin in a microcentrifuge. 

Avoid frothing the solution.

SECTION: Fragmentation/End Prep
8. Using a P10, pipette: 
9 µL of mastermix into each required well of the 96-plate PCR microplate.
26 µL of DNA from each sample (normalised to 200ng from step 2)
 

Gently mix the reaction by pipetting up/down 15 times and then briefly spin in a microcentrifuge to collect liquid from the sides of the well.. 

Avoid frothing of the solution.
SECTION: Fragmentation/End Prep
9. Place the sealed PCR microplate in the thermocycler. With a heated lid set to 75 °C, run the following program:
1. 10 min             @ 37 °C
2. 30 min             @ 65 °C
3. Infinite Hold                 @ 4 °C 

Using these conditions, we will obtain a mean fragment size of approx. 500 bp for each library sample.
 

  
  

SECTION: Adaptor Ligation
10. Remove the microplate from the thermocycler and immediately place on ice or a cold block.
 

SECTION: Adaptor Ligation
11. In a 15 mL Falcon tube, prepare a fresh mastermix of the following components (for each Frag sample + 1 sample overage) as indicated below in Table 3:

 

SECTION: Adaptor Ligation
12. Gently mix the reaction by pipetting up/down 15 times and then briefly spin in a microcentrifuge. 

Avoid frothing of the solution.
SECTION: Adaptor Ligation
13. Using a P100 or P200, pipette: 
33.5 µL of mastermix into each Frag mix-occupied well of the microplate.
 

Gently mix the reaction by pipetting up/down 10 times and then briefly spin in a microcentrifuge to collect liquid from the sides of the well. 

Avoid frothing of the solution.
SECTION: Adaptor Ligation
14. Place the sealed PCR microplate in the thermocycler. With a heated lid OFF, run the following program: 
15 min             @ 20 °C

Remove the plate from the thermocycler immediately upon program completion.

SECTION: Adaptor Ligation
15. To each well, now add 3 µL of (red) USER®Enzyme.

Mix by gentle repeated pipetting. 

Place the sealed PCR microplate in the thermocycler. 
With a heated lid set at 47 °C, run the following program: 
15 min             @ 37 °C
 

SECTION: Adaptor Ligation
16. Remove the microplate from the thermocycler, place on ice and proceed to the next section (Size Selection of Adaptor-ligated DNA)
 

SECTION: Size Selection of Adaptor-ligated DNA
17. In order to select in the right length of library fragments, the volumes of SPRIselect or NEBNext Sample Purification Beads provided are to be used in exact volume ratios to sample in each well. A shift in the volume ratios (of sample : beads) may lead to selection of a different fragment size range.
 

SECTION: Size Selection of Adaptor-ligated DNA
18. The size selection protocol is based on a total starting volume of 100 µl. 

Bring the volume of each well up to 100 µl by adding 28.5 µL 0.1X TE (1X TE Buffer diluted 1:10 with water).

SECTION: Size Selection of Adaptor-ligated DNA
19. Vortex SPRIselect or NEBNext Sample Purification Beads for 30 s to resuspend.
 


SECTION: Size Selection of Adaptor-ligated DNA
20. Table 4 shows the recommended conditions (and volumes) for bead-based size selection:
  

For this protocol, we will aim for insert sizes between 350-600 bp.
SECTION: Size Selection of Adaptor-ligated DNA
21. Add 20 µL (0.2X) resuspended beads to the 100 µl samples from step 18. 

Mix well by pipetting up and down very gently at least 10 times. 
 

SECTION: Size Selection of Adaptor-ligated DNA
22. Incubate the mix on the bench for 7 min at 4 Room temperature.

SECTION: Size Selection of Adaptor-ligated DNA
23. Place the plate on the magnetic stand/rack to separate the beads from the supernatant. 
 

SECTION: Size Selection of Adaptor-ligated DNA
24. After 7 minutes (or when the solution is clear), carefully transfer the supernatant (~ 120 µL) containing your DNA to a new tube.
 


SECTION: Size Selection of Adaptor-ligated DNA
25. Add 10 µL (~0.1X) resuspended SPRIselect or Sample Purification Beads to the supernatant.
 


Mix well by pipetting up and down very gently at least 10 times. Be careful to expel all of the liquid from the tip during the last mix.
SECTION: Size Selection of Adaptor-ligated DNA
26. Incubate samples on the bench for 7 min at 4 Room temperature.

SECTION: Size Selection of Adaptor-ligated DNA
27. Place the plate on the magnetic stand to separate the beads from the supernatant. 
 

SECTION: Size Selection of Adaptor-ligated DNA
28. After 7 min (or when the solution is clear), carefully remove and discard the supernatant that contains unwanted fragments. 
 


SECTION: Size Selection of Adaptor-ligated DNA
29. Add 200 µL of freshly prepared 80% ethanol to the plate wells while they are still on the magnetic stand. 
SECTION: Size Selection of Adaptor-ligated DNA
30. Incubate at 4 Room temperature for 30 s, and then carefully remove and discard the liquid. 
 


SECTION: Size Selection of Adaptor-ligated DNA
31. Repeat the ethanol wash steps 29 and 30 two additional times.
SECTION: Size Selection of Adaptor-ligated DNA
32. After the third wash, be sure to remove all visible traces/droplets of ethanol. 

If necessary, briefly spin the tube/plate, place back on the magnet for 1 min and remove traces of ethanol at the bottom of each well with a p10 pipette tip.

SECTION: Size Selection of Adaptor-ligated DNA
33. Air dry the beads for up to 5 minutes while the plate is on the magnetic stand exposed to the air.
 

SECTION: Size Selection of Adaptor-ligated DNA
34. Remove the plate from the magnetic stand. 

Elute the DNA target from the beads by adding 17 µL 0.1X TE directly onto the clumped beads.
SECTION: Size Selection of Adaptor-ligated DNA
35. Mix well by gently pipetting up and down 10 times.

Incubate for 5 min at 4 Room temperature.

SECTION: Size Selection of Adaptor-ligated DNA
36. Place the plate on the magnetic stand. 

After 7 min or when the solution is clear, transfer 15 µL of each eluate to a fresh well on the plate.


SECTION: Size Selection of Adaptor-ligated DNA
37. Place the microplate on ice and proceed to the next section (PCR Enrichment of Adaptor-ligated DNA).
 

SECTION: PCR Enrichment of Adaptor-ligated DNA
38. On a fresh 96-PCR microplate, pipette 25 µL of the (blue) NEBNext Ultra II Q5 Master Mix into each corresponding sample well from the previous reaction.
 


SECTION: PCR Enrichment of Adaptor-ligated DNA
39. To the Q5 Master Mix in each well, add 10 µL of (blue) Index/Universal Primer combination mix.

Ensure that a fresh tip is used for every well.
 


SECTION: PCR Enrichment of Adaptor-ligated DNA
40. To each well, now add 15 µL of the corresponding size-selected eluates generated in step 36. The PCR reactions in each well will now contain the following:
  
The total volume in each sample well should be the same (50 µL)

SECTION: PCR Enrichment of Adaptor-ligated DNA
41. Using a P100/200 pipette set to 40ul, gently pipette the entire PCR mix 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
42. Place the sealed PCR microplate in the thermocycler. With a heated lid set to 98 °C, run the following program:

 

For standard library prep samples, Table 7 could be used as a starting point to determine the number of PCR cycles.

 

 


SECTION: Cleanup of Amplified Libraries
43. Remove the PCR amplicons from the thermocycler and immediately place on ice.
 

SECTION: Cleanup of Amplified Libraries
44. Vortex SPRIselect or NEBNext Sample Purification Beads for 30 s to resuspend.

SECTION: Cleanup of Amplified Libraries
45. Add 45 µL (0.9X) resuspended beads to the PCR reaction. 

Mix well by gently pipetting up and down at least 10 times.
 

SECTION: Cleanup of Amplified Libraries
46. Incubate the samples on the bench for 7 min at Room temperature.
SECTION: Cleanup of Amplified Libraries
47. Place the plate on the magnetic stand to separate the beads from the supernatant.
 

SECTION: Cleanup of Amplified Libraries
48. After 7 min (or when the solution is clear), carefully remove and discard the supernatant.
 


SECTION: Cleanup of Amplified Libraries
49. Add 200 µL of freshly prepared 80% ethanol to the plate wells while they are still on the magnetic stand. 
SECTION: Cleanup of Amplified Libraries
52. After the third wash, be sure to remove all visible traces/droplets of ethanol. 

If necessary, briefly spin the tube/plate, place back on the magnet for 1 min and remove traces of ethanol at the bottom of each well with a p10 pipette tip.
SECTION: Cleanup of Amplified Libraries
53. Air dry the beads for up to 5 minutes while the plate is on the magnetic stand exposed to the air.
 

SECTION: Cleanup of Amplified Libraries
54. 
Remove the plate from the magnetic stand. 

Elute the DNA target from the beads by swiftly adding 33 µL 0.1X TE directly onto the clumped beads.
SECTION: Cleanup of Amplified Libraries
55. Mix well by gently pipetting up and down 10 times.

Incubate for 7 min at 4 Room temperature.
SECTION: Cleanup of Amplified Libraries
56. Place the plate on the magnetic stand. 

After 7 min (or when the solution is clear), transfer 30 µL of each eluate to a fresh well.
SECTION: Cleanup of Amplified Libraries
50. Incubate at 4 Room temperature for 30 s, and then carefully remove and discard the liquid. 
 

SECTION: Cleanup of Amplified Libraries
51. Repeat the ethanol wash steps 49 and 50 two additional times.
SECTION: Cleanup of Amplified Libraries
57. WGS Library preparation is now complete.
Store the prepared libraries at -20 °C until they are ready for QC.

SECTION: Assessing Library Quality
58. Library QC can be performed using a combination of fragment analysis and real-time PCR to estimate qualitative measurements of 1. median fragment size, 2. distributions of fragments sizes alongside quantitative estimates like mean library concentration. 

Recommended assays:
Fragment analysis - 
Agilent High Sensitivity (HS) Assay on the Bioanalyzer 2100
or
Agilent D1000 ScreenTape Assay on the Tapestation 4150/4200

qPCR (using SYBR fluorescent dyes) - 
NEBNext Library Quant Kit for Illumina (NEB)
or
KAPA Library Quantification Kit Illumina® Platforms (Roche)
 

SECTION: Additional Information &amp; Troubleshooting
59. Troubleshooting Guide for NEBNext®Ultra™ II FS DNA Library Prep Kit

Technical Note: SPRI select: DNA Ratios Affect the Size Range of Library Fragments (10x Genomics)