TITLE: NEBNext® Ultra™ DNA Library Prep Protocol for Illumina® (E7370)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
The NEBNext®Ultra™ DNA Library Prep Kit for Illumina®contains reagents for preparation of libraries for next-generation sequencing on the Illumina platform from 5 ng – 1 µg input DNA, in a streamlined workflow. Please note that adaptors and primers are not included in the kit and are available separately.

Each kit component must pass rigorous quality control standards, and each set of reagents is functionally validated together by construction and sequencing of a 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 custom@neb.com for further information.

[GUIDELINES]
The NEBNext®Ultra™ DNA Library Prep Kit for Illumina®contains reagents for preparation of libraries for next-generation sequencing on the Illumina platform from 5 ng – 1 µg input DNA, in a streamlined workflow. Please note that adaptors and primers are not included in the kit and are available separately.

Each kit component must pass rigorous quality control standards, and each set of reagents is functionally validated together by construction and sequencing of a 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 custom@neb.com for further information.


Ultra DNA Library Preparation Workflow for Illumina
 

NEBNext Ultra™ DNA provides high library yields even with low inputs and difficult samples
 
All libraries were prepared with 5 ng of input DNA and run on an Agilent Bioanalyzer®.
A.E. coligenomic DNA libraries. 1: Ladder; 2: Library prepared using NEBNext DNA Library Prep Master Mix Set (E6040); 3: Library prepared using NEBNext Ultra DNA Library Prep Kit (E7370). With 5 ng inputs, library yields are significantly higher using the NEBNext Ultra™ DNA Kit.
B. FFPE sample. Human lung tumor genomic DNA from a 66-year old male, extracted from a 10-year old FFPE block (Biochain, Inc.). 1: Library prepared using NEBNext Ultra DNA Library Prep Kit (E7370). 2: Input DNA (highly degraded FFPE genomic DNA).


Read depth correlation shows consistent high coverage for 5 ng - 1 µg input amounts
 
Libraries were prepared with various amounts of human IMR90 gDNA (5, 50, and 1000 ng) and PCR amplified. A PCR-free library was also prepared with 1000 ng human IMR90 gDNA. Sequence coverage of 10 kb windows of the human genome was analyzed on Galaxy, and Pearson correlation coefficient (R2 values) were calculated using Microsoft® Excel®. All libraries are highly correlated with each other, independent of DNA input as well as PCR amplification. High coverage was achieved for all DNA input amounts.


NEBNext Ultra DNA Kits display strong performance with input amounts as low as 5 ng
 
Input DNA: IMR90 genomic DNA (human fibroblast).

PF Mismatch Rate: The rate of bases mismatching the reference for all bases aligned to the reference sequence.

Percent Duplication: The percentage of mapped sequence that is marked as duplicate.

Percent Chimeras: The percentage of reads that map outside of a maximum insert size or that have the two ends mapping to different chromosomes.

This data illustrates the strong performance of the NEBNext Ultra DNA Library Prep Kit for Illumina, even with 5 ng of human genomic DNA input. Mismatch rate, % duplication and % chimeras are all low, and similar, regardless of input amount.


Lot Control
The lots provided are managed separately and qualified by additional functional validation. Individual reagents undergo standard enzyme activity and quality control assays, and also meet stringent criteria in the additional quality controls listed on each individual component page

[STEPS]
SECTION: NEBNext End Prep
1. Add the following components in a sterile nuclease-free tube:
   Reagent Cap Color Volume   Fragmented DNA    55.5 μl   End Prep Enzyme Mix Green 3.0 μl   End Repair Reaction Buffer (10x) Green 6.5 μl   

SECTION: NEBNext End Prep
3. Place in a thermocycler, with the heated lid set to ≥ 75°C, and run the following program:
   Time Temperature   30 minutes 20°C   30 minutes 65°C   Hold 4°C   
SECTION: Adaptor Ligation
4. Add the following components directly to the End Prep reaction mixture:
 

   Reagent Cap Color Volume   Blunt/TA Ligase Master Mix Red 15 μl   Ligation Enhancer Red 1 μl   NEBNext Adaptor For Illumina Red 2.5 μl   
 
 

SECTION: Adaptor Ligation
6. Incubate at 20 °C for 15 min in a thermal cycler. 

SECTION: Adaptor Ligation
7. Add 3 µL to the ligation mixture. 
 

SECTION: Adaptor Ligation
8. Mix well and incubate at 37 °C for 15 min with heated lid set to ≥ 47 °C. 
SECTION: Size Selection
11. 
 

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 a starting volume of 100 µl. Size selection conditions were optimized with AMPure XP beads; however, SPRIselect beads can be used following the same conditions.

To select a different insert size than 200 bp, please use the volumes in this table: 
 

SECTION: Size Selection
13. Add 13.5 µL to the ligation reaction for a 100 μl total volume.
SECTION: Size Selection
14. Add 55 µL to the 100 µL (for 200bp selection -- for other size selections, refer to the table above for the amount of 1st bead selection to add). 

SECTION: Size Selection
16. Incubate samples on bench top for at least 5 min at -20 Room temperature.
SECTION: Size Selection
17. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. 
 
SECTION: Size Selection
18. 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
19. Add 25 µL to the supernatant (for 200bp selection -- for other size selections, refer to the table above for the amount of 2nd bead selection to add). 

SECTION: Size Selection
22. Place the tube/plate on an appropriate magnetic stand for 5 min (or until solution is clear) to separate the beads from the supernatant. 
 

SECTION: Size Selection
23. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant that contains unwanted DNA. (Caution: do not discard beads).
 
SECTION: Size Selection
24. Add 200 µL to the tube/plate while in the magnetic stand. Incubate at -20 Room temperature for 30 s, and then carefully remove and discard the supernatant. 
 

SECTION: Size Selection
25. Repeat the previous step: Add 200 µL to the tube/plate while in the magnetic stand. Incubate at -20 Room temperature for 30 s, and then carefully remove and discard the supernatant. 
 

 

SECTION: Size Selection
26. Air the dry beads for up to 5 min while the tube is on the magnetic stand with the lid open. 
 

SECTION: Size Selection
27. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads into 17 µL of 10 Milimolar (mM) or 0.1 X.
SECTION: Size Selection
28. Mix well on a vortex mixer or by pipetting up and down 10 times. Incubate for at least 2 min at -20 Room temperature. 
 

SECTION: Size Selection
29. 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).

 

SECTION: PCR Enrichment of Adaptor-ligated DNA
30. 
 

Mix the following components in sterile strip tubes:
 
For index kits with primers separate: 
 

   Reagent Cap Color Volume   Adaptor Ligated Fragments n/a 15 µl   NEBNext Q5 Hot Start HiFi PCR Master Mix Blue 25 µl   Index Primer/ I7 primer Blue 5 µl   Universal PCR primer/ I5 Primer Blue 5 µl   

For Index kits where the primers are already combined: 
 

   Reagent Cap Color Volume   Adaptor Ligated Fragments n/a 15 µl   NEBNext Q5 Hot Start HiFi PCR Master Mix Blue 25 µl   Index/ Universal Primer   10 µl   

SECTION: PCR Enrichment of Adaptor-ligated DNA
32. Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:

 
* Please note the number of PCR cycles recommended in the following table are to be seen as s starting point to determine the number of PCR cycles best for your samples. The number of cycles should be chosen based on the input amount and sample type. Thus, samples prepared with a different method prior to library prep may require further 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). 
   DNA Input Number of PCR Cycles   1 µg 4   50 ng 7-8   5 ng 12   
 
 

SECTION: Cleanup of PCR Amplification
33. 
 
SECTION: Cleanup of PCR Amplification
35. Add 45 µL to the PCR reaction. Mix well by pipetting up and down at least 10 times.
 

SECTION: Cleanup of PCR Amplification
36. Incubate for at least 5 min at -20 Room temperature.

SECTION: Cleanup of PCR Amplification
37. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant (5 min or until the solution is clear). 
 

SECTION: Cleanup of PCR Amplification
38. After the solution is clear (about 5 min), 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 PCR Amplification
39. Add 200 µL to the tube/plate while in the magnetic stand. Incubate at -20 Room temperature for 30 s, and then carefully remove and discard the supernatant.
 

SECTION: Cleanup of PCR Amplification
40. Repeat the previous step once for a total of two washes: Add 200 µL to the tube/ plate while in the magnetic stand. Incubate at -20 Room temperature for 30 s, and then carefully remove and discard the supernatant.
 

 

SECTION: Cleanup of PCR Amplification
41. Air dry the beads for up to 5 min while the tube/plate is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of PCR Amplification
42. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 33 µL. 
SECTION: Cleanup of PCR Amplification
43. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at -20 Room temperature.
 

SECTION: Cleanup of PCR Amplification
44. 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 Amplification
45. Check the size distribution on an Agilent Bioanalyzer® High Sensitivity DNA chip. The sample may need to be diluted before loading.
SECTION: NEBNext End Prep
2. Set a 100 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 the liquid from the side of the tube. 
 

SECTION: Adaptor Ligation
5. Set a 100 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 the liquid from the side of the tube. 
 

SECTION: Adaptor Ligation
9. Safe Stopping Point: It is safe to store the library at -20 °C.
 

10. Size selection is optional. If the starting material is > 50 ng, follow the protocol for size selection. For input less than 50 ng, size selection is not recommended. Follow the protocol for cleanup without size selection.
SECTION: Size Selection
12. Vortex SPRIselect beads to resuspend. AMPure XP beads can be used as well. If using AMPure XP beads, please allow the beads to warm to -20 Room temperature for at least 30 min before use.
SECTION: Size Selection
15. Mix well by pipetting up and down at least 10 times. 
 

SECTION: Size Selection
20. Mix at least 10 times. 
 

SECTION: Size Selection
21. Incubate samples on the bench top for at least 5 min at -20 Room temperature.
SECTION: PCR Enrichment of Adaptor-ligated DNA
31. 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: Cleanup of PCR Amplification
34. Vortex SPRIselect beads to resuspend. AMPure XP beads can be used as well. If using AMPure XP beads, allow the beads to warm to -20 Room temperature for at least 30 min before use.
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection
11. 
 

Vortex SPRIselect 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 min before use.
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection
12. Add 86.5 µL to the Adaptor Ligation reaction. Mix well by pipetting up and down at least 10 times. 
 

SECTION: PCR Enrichment of Adaptor-ligated DNA
22. 
 

Mix the following components in sterile strip tubes (Total volume  50 μl):
 
For index kits with primers separate: 
 

   Reagent Cap Color Volume   Adaptor Ligated Fragments n/a 15 µl   NEBNext Q5 Hot Start HiFi PCR Master Mix Blue 25 µl   Index Primer/ I7 primer Blue 5 µl   Universal PCR primer/ I5 Primer Blue 5 µl   

For Index kits where the primers are already combined: 
 

   Reagent Cap Color Volume   Adaptor Ligated Fragments n/a 15 µl   NEBNext Q5 Hot Start HiFi PCR Master Mix Blue 25 µl   Index/ Universal Primer   10 µl   

SECTION: PCR Enrichment of Adaptor-ligated DNA
23. 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
24. Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:
 
 
 
 * Please note the number of PCR cycles recommended in this table are to be seen as s starting point to determine the number of PCR cycles best for your samples. The number of cycles should be chosen based on the input amount and sample type. Thus, samples prepared with a different method prior to library prep may require further 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). 
   DNA Input Number of PCR Cycles   1 µg 4   50 ng 7-8   5 ng 12   
 
 

SECTION: Cleanup of PCR Reaction
25. 
 
SECTION: Cleanup of PCR Reaction
26. Vortex SPRIselect beads to resuspend. AMPure XP beads can be used as well. If using AMPure XP beads, allow the beads to warm to -20 Room temperature for at least 30 min before use.
SECTION: Cleanup of PCR Reaction
27. Add 45 µL to the PCR reaction. Mix well by pipetting up and down at least 10 times.
 

SECTION: Cleanup of PCR Reaction
28. Incubate for at least 5 min at -20 Room temperature.
SECTION: Cleanup of PCR Reaction
29. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant (5 min or until the solution is clear). 
 

SECTION: Cleanup of PCR Reaction
30. After the solution is clear (about 5 min), 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 PCR Reaction
31. Add 200 µL to the tube/ plate while in the magnetic stand. Incubate at -20 Room temperature for 30 s, and then carefully remove and discard the supernatant.
 

SECTION: Cleanup of PCR Reaction
32. Repeat the previous step once for a total of two washes: Add 200 µL to the tube/ plate while in the magnetic stand. Incubate at -20 Room temperature for 30 s, and then carefully remove and discard the supernatant.
 

 

SECTION: Cleanup of PCR Reaction
33. Air dry the beads for up to 5 min while the tube/plate is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of PCR Reaction
34. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 33 µL. 
SECTION: Cleanup of PCR Reaction
35. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at -20 Room temperature.
 

SECTION: Cleanup of PCR Reaction
36. 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
37. Check the size distribution on an Agilent Bioanalyzer® High Sensitivity DNA chip. The sample may need to be diluted before loading.
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection
13. Incubate samples on bench top for at least 5 min at 20 Room temperature.
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection
14. 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
15. 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 Adaptor-ligated DNA without Size Selection
16. Add 200 µL to the tube/ plate while in the magnetic stand. Incubate at 20 Room temperature for 30 s, and then carefully remove and discard the supernatant. 
 

SECTION: Cleanup of Adaptor-ligated DNA without Size Selection
17. Repeat previous step once for a total of two washes: Add 200 µL to the tube/ plate while in the magnetic stand. Incubate at 20 Room temperature for 30 s, and then carefully remove and discard the supernatant.  
 

 

SECTION: Cleanup of Adaptor-ligated DNA without Size Selection
18. Air dry the beads for up to 5 min while the tube/plate is on the magnetic stand with the lid open.
 

SECTION: Cleanup of Adaptor-ligated DNA without Size Selection
19. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 µL of 10 Milimolar (mM) or 0.1 X.
SECTION: Cleanup of Adaptor-ligated DNA without Size Selection
20. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at 20 Room temperature.
 

SECTION: Cleanup of Adaptor-ligated DNA without Size Selection
21. 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.
 

SECTION: Cleanup of PCR Amplification
46. Safe Stop: Samples can be stored at -20 °C.
SECTION: Cleanup of PCR Reaction
38. Safe Stop: Samples can be stored at -20 °C.