Protocol for FS DNA Library Prep Kit (E7805, E6177) for Large Fragment Sizes (> 550 bp)

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.
Stopping points in the protocol.


Note: Follow the protocol in this chapter for inputs > 100 ng, and fragment sizes > 550 bp. Follow the protocol in Chapter 2 for inputs ≥ 100 ng and fragment sizes < 550 bp. Follow the protocol in Chapter 1 for inputs < 100 ng. 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.

3.1. Fragmentation/End Prep
 
3.1.1. 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.

3.1.2. Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice.

Note: It is important to vortex the enzyme mix prior to use for optimal performance.


3.1.3. Add the following components to a 0.2 ml thin wall PCR tube on ice:

 COMPONENT
VOLUME PER ONE
LIBRARY
DNA  26 µl
(yellow) NEBNext Ultra II FS Reaction Buffer
 7 µl
(yellow) NEBNext Ultra II FS Enzyme Mix
 2 µl
Total Volume
 35 µl
 
3.1.4. Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.
 
3.1.5. In a Thermocycler, with the heated lid set to 75°C, run the following program:
5 min @ 37°C
30 min @ 65°C
Hold @ 4°C
 

If necessary, samples can be stored at –20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.

3.2. Adaptor Ligation

3.2.1. Add the following components directly to the FS Reaction Mixture:

 COMPONENT  VOLUME
 FS Reaction Mixture (Step 3.1.5)
 35 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
 68.5 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 Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600, #E7535 and #E6609) Oligos for Illumina.

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.

3.2.2. 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. (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).

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

3.2.4. Add 3 µl of  (red) USER® Enzyme to the ligation mixture from Step 3.2.3.

Note: Steps 3.2.4. and 3.2.5. are only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600, #E7535 and #E6609) Oligos for Illumina.

3.2.5. 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.

3.3. Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp

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 (71.5 µl; Step 3.2.5.). 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.


3.3.1. 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).

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

3.3.3. 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 3-5 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.

3.3.4. Incubate samples for at least 5 minutes at room temperature.

3.3.5. 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.

3.3.6. 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 the beads).

3.3.7. 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.

3.3.8. Repeat Step 3.3.7. 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.

3.3.9. 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. 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.

3.3.10. 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).

3.3.11. 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.

3.3.12. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 100 μl to a new PCR tube.

3.3.13. 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 3-5 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.

3.3.14. Incubate samples for at least 5 minutes at room temperature.

3.3.15. 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.

3.3.16. After 5 minutes (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 beads).

3.3.17. 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.

3.3.18. Repeat Step 3.3.17. once. 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.

3.3.19. 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 overdry 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.

3.3.20. 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).

3.3.21. Mix well on a vortex mixer or by pipetting up and down 10 times. 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.

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

3.3.23. Proceed to PCR Enrichment of Adaptor-ligated DNA in Section 3.4.

 Samples can be stored overnight at -20°C.


3.4. PCR Enrichment of Adaptor-ligated DNA

Follow Section 3.4.1A. if you are using the following oligos (10 µM primer):
NEBNext Singleplex Oligos for Illumina (NEB #E7350)
NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335)
NEBNext Multiplex Oligos for Illumina (Set 2, NEB #E7500)
NEBNext Multiplex Oligos for Illumina (Set 3, NEB #E7710)
NEBNext Multiplex Oligos for Illumina (Set 4, NEB #E7730)
NEBNext Multiplex Oligos for Illumina (Dual Index Primers, NEB #E7600)

Follow Section 3.4.1B. if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609)


3.4.1. Add the following components to a sterile strip tube:

3.4.1A. Forward and Reverse Primers not already combined
 
 Adaptor Ligated DNA Fragments (Step 3.3.23.)
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


3.4.1B. Forward and Reverse Primers already combined
 
Adaptor Ligated DNA Fragments (Step 3.3.23.)
15 µl
  (blue) NEBNext Ultra II Q5 Master Mix
 25 µl
  (blue) Index/Universal Primer****
 10 µl
 Total Volume
 50 µl

* The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600) Oligos for Illumina. For use with Dual Index Primers (NEB #E7600), look at the NEB #E7600 manual for valid barcode combinations and tips for setting up PCR reactions.

** The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600) Oligos for Illumina. For use with Dual Index Primers (NEB #E7600), look at the NEB #E7600 manual for valid barcode combinations and tips for setting up PCR reactions.

*** For use with Dual Index Primers (NEB #E7600) use only one i5 Primer per reaction.

**** The primers are provided in NEBNext Multiplex Oligos for Illumina (NEB #E6609). Please refer to the NEB #E6609 manual for valid barcode combinations and tips for setting up PCR reactions.

3.4.2. 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.

3.4.3. 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
Annealing/Extension
98°C
65°C
10 seconds
75 seconds
3-8*
Final Extension
65°C 5 minutes 1
Hold 4°C

* The number of PCR cycles recommended in Table 3.4.1 are to be seen as a starting point to determine the number of PCR cycles best for standard library prep samples. Use Table 3.4.2 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 3.4.1
 
INPUT DNA
IN THE FS
REACTION
# OF CYCLES REQUIRED
FOR STANDARD LIBRARY
PREP: YIELD ~100 ng (5-35 nM)*
 500 ng
 3-4
 200 ng
 4-5
 100 ng
 5-7
 
* Cycle number was determined for size selected libraries.

Table 3.4.2.
 
INPUT DNA
IN THE FS
REACTION
# OF CYCLES REQUIRED
FOR TARGET ENRICHMENT
LIBRARY PREP (YIELD ~750 ng-1 µg)*
 500 ng
 4-5
 200 ng
 5-6
 100 ng
 7-8

* Cycle number was determined for size selected libraries.

3.4.4. Proceed to Cleanup of PCR reaction in Section 3.5.

3.5. Cleanup of PCR Reaction

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.


3.5.1. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
 
3.5.2. 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 3-5 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.
 
3.5.3. Incubate samples on bench top for at least 5 minutes at room temperature.
 
3.5.4. 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.

3.5.5. 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 the beads).

3.5.6. 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.

3.5.7. Repeat Step 3.5.6. 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.

3.5.8. 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. 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.

3.5.9. 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).

3.5.10. 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.

3.5.11. 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 and store at –20°C.

3.6. Assess Library Quality on a Bioanalyzer

3.6.1. Dilute library (from Step 3.5.11.) 5-fold in 0.1X TE Buffer.

3.6.2. Run 1 µl on a DNA High Sensitivity Chip.

3.6.3. Check that the library size shows a narrow distribution with an expected peak size > 700 bp (Figure 3.1).

Note: If a peak ~80 bp (primers) or 128 bp (adaptor-dimer) is visible in the Bioanalyzer trace, bring up the sample volume (from Step 3.5.11.) to 50 µl with 0.1X TE Buffer and repeat the Cleanup of PCR Reaction in Section 3.5.

Figure 3.1: Example of final library size distribution with size selection. 100 ng Human DNA (NA19240) was fragmented for 5 minutes.
Note: Due to the preference of the Illumina sequences to preferentially cluster smaller fragments, the average insert size from the sequence data may be smaller than expected (see Figure 3.2).

We recommend gel size selection if you need an average sequence insert size > 700 bp.
Figure 3.2: Example of insert size distribution with size selection