Protocol for FS DNA Library Prep Kit (E7805, E6177) with Inputs ≥100 ng

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, as size selection is recommended for this input range. Follow the protocol in Chapter 1 for inputs < 100 ng, as size selection is not recommended for this input range. Follow the protocol in Chapter 3 for inputs ≥ 100 ng and fragment sizes > 550 bp. 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.

2.1. Fragmentation/End Prep
Fragmentation occurs during the 37°C incubation step. Use the chart below to determine the incubation time required to generate the desired fragment sizes. Incubation time may need to be optimized for individual samples. See Figure 2.1 for a typical fragmentation pattern.

 FRAGMENTATION SIZE
 INCUBATION @ 37°C
 OPTIMIZATION
 100 bp-250 bp
 30 min
 30-40 min
 150 bp-350 bp
 20 min
 20-30 min
 200 bp-450 bp
 15 min
 15-20 min
 300 bp-700 bp
 10 min
 5-15 min
 500 bp-1 kb  5 min  5-10 min



Figure 2.1: Example of size distribution on a Bioanalyzer®. Human DNA (NA19240) was fragmented for 5-40 min.
2.1.1. 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.

2.1.2. 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.


2.1.3. 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
 
2.1.4. Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.
 
2.1.5. In a Thermocycler, with the heated lid set to 75°C, run the following program:
5–30 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.

2.2. Adaptor Ligation

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

COMPONENT   VOLUME
FS Reaction Mixture (Step 2.1.5.)  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.


2.2.2. 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).

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

2.2.4. Add 3 µl of (red) USER® Enzyme to the ligation mixture from Step 2.2.3.

Note: Steps 2.2.4. and 2.2.5. 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.

2.2.5. 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.

2.3. Size Selection of Adaptor-ligated DNA for DNA Input > 100 ng.

If the starting material is > 100 ng, follow the protocol for size selection below. For Inputs < 100 ng, size selection is not recommended. Follow the protocol for cleanup without size selection in Chapter 1 Section 1.3. If you want fragment sizes > 550 bp and your input is > 100 follow the entire protocol in Chapter 3


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 2.2.5.). These volumes may not work properly for a size selection at a different step in the workflow, or if this is a second size selection. For size selection of samples contained in different buffer conditions bead volumes may need to be experimentally determined.


The following size selection protocol is for libraries with 150-200 bp inserts only. For libraries with different size fragment inserts, refer to Table 2.3.1. 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 SPRIselect or NEBNext Sample Purification Beads; however, AMPure XP beads can be used following the same conditions. If using AMPure XP beads, please allow the beads to warm to room temperature for at least 30 minutes before use.


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

Table 2.3.1: Recommended conditions for bead based size selection.

   APPROXIMATE
INSERT SIZE DISTRIBUTION
150-250 bp  200-350 bp  275-475 bp  350-600 bp
LIBRARY
PARAMETERS
Approx. Final Library Size Distribution (Insert+Adaptor+primers)
270-370 bp 320-470 bp
400-600 bp
470-800 bp
VOLUME TO
BE ADDED (µl)
 1st Bead Selection
40 30 25 20
 2nd Bead Selection
20 15 10 10

2.3.1. 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).

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

2.3.3. Add 40 μl (0.4X) resuspended beads to the 100 µl sample from Step 2.3.1. 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.

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

2.3.5. 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.

2.3.6. After 5 minutes (or when the solution is clear), carefully transfer the supernatant (~ 140 µl) containing your DNA to a new tube. (Caution: do not discard the supernatant). Discard the beads that contain the unwanted large fragments.

2.3.7. Add 20 µl (~0.2X) resuspended SPRIselect or 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. Incubate samples on the bench top for at least 5 minutes at room temperature.

2.3.8. 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.

2.3.9. 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).

2.3.10. 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.

2.3.11. Repeat Step 2.3.10. 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.

2.3.12. 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.

2.3.13. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 μl 0.1X TE (dilute 1X TE Buffer 1:10 in water).

2.3.14. 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.

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

2.3.16. Proceed to PCR Enrichment of Adaptor-ligated DNA in Section 2.4.
 
Samples can be stored at -20°C.


2.4. PCR Enrichment of Adaptor-ligated DNA

Follow Section 2.4.1A. 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 2.4.1B. if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609)



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

2.4.1A. Forward and Reverse Primers not already combined
 
 Adaptor Ligated DNA Fragments (Step 2.3.16.)
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


2.4.1B. Forward and Reverse Primers already combined
 
Adaptor Ligated DNA Fragments (Step 2.3.16.)
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.

** For use with NEBNext Multiplex Oligos (NEB #E7335, #E7500, #E7710 or #E7730) use only one index primer per PCR reaction. For use with Dual Index Primers (NEB #E7600) use only one i7 primer per reaction.

*** 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.

2.4.2. 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.

2.4.3. 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
3-7*
Final Extension
65°C 5 minutes 1
Hold 4°C

* The number of PCR cycles recommended in Table 2.4.1 are to be seen as a starting point to determine the number of PCR cycles best for standard library prep samples. Use Table 2.4.2 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 2.4.1

INPUT DNA
IN THE FS
REACTION
# OF CYCLES REQUIRED
FOR STANDARD LIBRARY
PREP: YIELD ~100 ng (5-35 nM)*
 500 ng
 3**
 200 ng
 3-4
 100 ng
 4-5

 
* 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.

Table 2.4.2.
 

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
 6-7


* Cycle number was determined for size selected libraries.

2.4.4. Proceed to Cleanup of PCR reaction in Section 2.5.

2.5. 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.

2.5.1. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
 
2.5.2. 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 3-5 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.
 
2.5.3. Incubate samples on bench top for at least 5 minutes at room temperature.
 
2.5.4. 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.

2.5.5. 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).

2.5.6. 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.

2.5.7. Repeat Step 2.5.6. 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.

2.5.8. 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.

2.5.9. 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).

2.5.10. 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.

2.5.11. 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.

2.6. Assess Library Quality on a Bioanalyzer


2.6.1. Dilute library (from Step 2.5.11.) 5-fold in 0.1X TE Buffer.

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

2.6.3. Check that the library size shows a narrow distribution with an expected peak size based on fragmentation time and size selection (Figure 2.2).

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 2.5.11.) to 50 µl with 0.1X TE Buffer and repeat the Cleanup of PCR Reaction in Section 2.5.

Figure 2.2: Example of final library size distributions with size selection. Human DNA (NA19240) was fragmented for 5 or 15 minutes.