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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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 DNA into high quality libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow also has minimal hands-on time.</div><div class = "text-block">Note: The Ultra II FS Kit is not compatible with bisulfite conversion workflows.</div></div>

[STEPS]
?. [Fragmentation/End Prep]
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.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 followed by a quick vortex to mix. Place on ice until use.
?. [Fragmentation/End Prep]
Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice.
It is important to vortex the enzyme mix prior to use for optimal performance.There are short videos on NEB.com about handling the FS reagents: Preparing FS Reagents: https://www.neb.com/tools-and-resources/video-library/quick-tips---preparing-the-nebnext-ultra-ii-fs-dna-reaction-buffer-and-enzyme-mixFS master mix: https://www.neb.com/tools-and-resources/video-library/quick-tips---preparing-nebnext-ultra-ii-fs-dna-reaction-buffer-and-enzyme-mix-master-mix
?. [Fragmentation/End Prep]
Add the following components to a 0.2 ml thin wall PCR tube on ice:  AB1ComponentVolume per One Library2DNA26 µl3(yellow) NEBNext Ultra II FS Reaction Buffer7 µl4(yellow) NEBNext Ultra II FS Enzyme Mix2 µl5Total Volume35 µl
AB1ComponentVolume per One Library2DNA26 µl3(yellow) NEBNext Ultra II FS Reaction Buffer7 µl4(yellow) NEBNext Ultra II FS Enzyme Mix2 µl5Total Volume35 µl
?. [Fragmentation/End Prep]
Vortex the reaction for 5 seconds and briefly spin in a microcentifuge.
?. [Fragmentation/End Prep]
In a thermocycler, with the heated lid set to 75°C, run the following program:  AB15 min37°C230 min65°C3Hold4°C
AB15 min37°C230 min65°C3Hold4°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.
?. [Adaptor Ligation]
Add the following components directly to the FS Reaction Mixture:  AB1ComponentVolume2FS Reaction Mixture (Step 5)35 µl3(red) NEBNext Ultra II Ligation Master Mix*30 µl4(red) NEBNext Ligation Enhancer1 µl5(red) NEBNext Adaptor for Illumina**2.5 µl6Total Volume68.5 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 NEBNext or Multiplex Oligos for Illumina. NEB has several Oligo kit options, which are supplied separately from the library prep kit.
AB1ComponentVolume2FS Reaction Mixture (Step 5)35 µl3(red) NEBNext Ultra II Ligation Master Mix*30 µl4(red) NEBNext Ligation Enhancer1 µl5(red) NEBNext Adaptor for Illumina**2.5 µl6Total Volume68.5 µl
The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. Do not premix the adaptor in the Adaptor Ligation Step.
?. [Adaptor Ligation]
Set a 100 μl or 200μl 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.
?. [Adaptor Ligation]
Incubate at 20°C for 15 minutes in a thermocycler with the heated lid off.
?. [Adaptor Ligation]
Add 3 μl of USER Enzyme to the ligation mixture from the previous step.
Steps 9 and 10 are only required for use with standard NEBNext Adaptors. USER Enzyme can be found in the NEBNext Oligos for Illumina.
?. [Adaptor Ligation]
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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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 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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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).
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
Vortex SPRIselecct or NEBNext Sample Purification Beads to resuspend
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
Incubate samples for at least 5 minutes at room temperature.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
Place the tube/plate on an appropriate magnetic stand to separate the bdads 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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
Add 200 μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperautre for 30 seconds, and then carefuly remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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).
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
Incubate samples at room temperautre for at least 5 minutes.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
Place the tube/plate on an appropriate magnetic stand to serparate the beads from the supernatant. If necessary, quickly spin the samples to collect the liquid form the sides of the tube or plate wells before placing on the magnetic stand.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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 the beads.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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 thatcontain DNA targets.
[Incubate]
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
Repeat the previous step 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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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).
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
Mix well on a vortex mixer or by pipetting up and down at least 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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
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.
?. [Size Selection of Adaptor-ligated DNA for Fragment Sizes > 550 bp]
Proceed to PCR Enrichment of Adaptor-ligated DNA in next Section: PCR Enrichment of Adaptor-ligated DNA.
?. [PCR Enrichment of Adaptor-ligated DNA]
• Use Option A for any kit where NEBNext index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes.• Use Option B for any kit where NEBNext index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined.
?. [PCR Enrichment of Adaptor-ligated DNA]
Add the following components to a sterile strip tube:Option A: Forward and Reverse Primers NOT Already Combined:  AB1ComponentVolume per One Library2Adaptor Ligated DNA Fragments (Step 33)15 µl3(blue) NEBNext Ultra II Q5 Master Mix25 µl4Index Primer/i7 Primer *,**5 µl5Universal PCR Primer/i5 Primer *,**5 µl6Total Volume50 µlOption B: Forward and Reverse Primers Already Combined  AB1ComponentVolume per One Library2Adaptor Ligated DNA Fragments (Step 33)15 µl3(blue) NEBNext Ultra II Q5 Master Mix25 µl4Index/Universal Primer *10 µl5Total Volume50 µl*NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual 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.
AB1ComponentVolume per One Library2Adaptor Ligated DNA Fragments (Step 33)15 µl3(blue) NEBNext Ultra II Q5 Master Mix25 µl4Index Primer/i7 Primer *,**5 µl5Universal PCR Primer/i5 Primer *,**5 µl6Total Volume50 µl
AB1ComponentVolume per One Library2Adaptor Ligated DNA Fragments (Step 33)15 µl3(blue) NEBNext Ultra II Q5 Master Mix25 µl4Index/Universal Primer *10 µl5Total Volume50 µl
?. [PCR Enrichment of Adaptor-ligated DNA]
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.
?. [PCR Enrichment of Adaptor-ligated DNA]
Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:* 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.* Cycle number was determine for size selected libraries.Table 3.4.2.* Cycle number was determined for size delected libraries.
?. [PCR Enrichment of Adaptor-ligated DNA]
Proceed to Cleanup of PCR reaction is next Section: Cleanup of PCR Reaction.
?. [Cleanup of PCR Reaction]
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.
?. [Cleanup of PCR Reaction]
Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
?. [Cleanup of PCR Reaction]
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.
?. [Cleanup of PCR Reaction]
Incubate samples on bench top for at least 5 minutes at room temperature.
?. [Cleanup of PCR Reaction]
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.
?. [Cleanup of PCR Reaction]
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).
?. [Cleanup of PCR Reaction]
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.
?. [Cleanup of PCR Reaction]
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.
?. [Cleanup of PCR Reaction]
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.
?. [Cleanup of PCR Reaction]
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).
?. [Cleanup of PCR Reaction]
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.
?. [Cleanup of PCR Reaction]
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.
?. [Assess Library Quality on a Bioanalyzer]
Dilute library  (from previous step) 5-fold in 0.1X TE Buffer.
?. [Assess Library Quality on a Bioanalyzer]
Run 1 μl on a DNA High Sensitivity Chip.
?. [Assess Library Quality on a Bioanalyzer]
Check that the library size shows a narrow distribution with an expected peak size > 700 bp (See Figure 3.1)Figure 3.1: Example of final library size distribution after size selection. 100 ng Human DNA (NA19240) was fragmented for 5 minutes.(Note: Due to the preperence of the Illumina sequences to preperentially cluster smaller fragments, the average insert sizes from the sequence data may be smaller than expected (See Figure 3.2. below).We recommend gel size selection if you need an average sequence insert size > 700 bp.Figure 3.2: Example of insert size distribution after bead-based size selection.
If a peak ~80 bp (primers) or 128 bp (adaptor-dimer) is visible in the Bioanalyzer trace, bring up the sample volume (from Step 51) to 50 μl with 0.1X TE Buffer and repeat the Cleanup of PCR Reaction in Section Cleanup of PCR Reaction.