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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span> The NEBNext</span><span style = "vertical-align:super;">®</span><span>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. </span></div><div class = "text-block">Some of the operational advantages that this kit offers are:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Perform fragmentation, end repair and dA-tailing with a single enzyme mix </li><li style = "counter-reset:ol0;list-style-type:disc;">Experience reliable fragmentation with a single protocol, regardless of DNA input amount or GC content </li><li style = "counter-reset:ol0;list-style-type:disc;">Prepare high quality libraries from a wide range of input amounts: 100 pg–500 ng </li><li style = "counter-reset:ol0;list-style-type:disc;">Generate high yields with increased reaction efficiencies and minimised sample loss </li><li style = "counter-reset:ol0;list-style-type:disc;">Use with DNA in standard buffers (TE, Tris-HCl) and water </li><li style = "counter-reset:ol0;list-style-type:disc;">Vary incubation time to generate a wide range of insert sizes</li></ul></div><div class = "text-block"><span>This protocol has been adapted from the NEBNext</span><span style = "vertical-align:super;">®</span><span>Ultra</span><span style = "vertical-align:super;">™</span><span>II FS DNA Library Prep Kit for Illumina developed by New England BioLabs (NEB).</span></div><div class = "text-block"><span>This is an open-access protocol distributed under the terms of the </span><a href="https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Creative Commons Attribution-NonCommercial-ShareAlike</span></a><span style = ":;">.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://protocols-files.s3.amazonaws.com/files/cuzrbf8zp.png?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWSNCI5SNCPTWTQQ%2F20210112%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20210112T173149Z&X-Amz-Expires=604800&X-Amz-SignedHeaders=host&X-Amz-Signature=1b2f1a454c966fddb1af93dd8213eb2cd4cbeb804c6ceee4a5e1185f39d2f243" /></div></div>

[STEPS]
?. [Before Starting]
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.
?. [Before Starting]
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.
?. [Before Starting]
Starting Material:  purified, genomic DNA.
200 ng
i. It is recommended 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 nuclease-free H2O are also acceptable. ii. If the input DNA (of 200 ng) is less than 26 µl, add 1X TE (provided) to a final volume of 26 µl.
?. [Fragmentation/End Prep]
Fragmentation occurs during a 37°C incubation step. Ensure that the Ultra II FS Reaction Buffer is completely thawed before placing it on ice.
If a precipitate is seen in the buffer, pipette up and down several times to break it up.
?. [Fragmentation/End Prep]
Vortex the Ultra II FS Enzyme Mix  prior to use and place on ice.
It is important to vortex the enzyme mix prior to use for optimal performance.
?. [Fragmentation/End Prep]
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:ComponentVolume per library (yellow) NEBNext Ultra II FS Reaction Buffer 7 µl(yellow) NEBNext Ultra II FS Enzyme Mix 2 µl Total Volume9 µl
ComponentVolume per library (yellow) NEBNext Ultra II FS Reaction Buffer 7 µl(yellow) NEBNext Ultra II FS Enzyme Mix 2 µl Total Volume9 µl
?. [Fragmentation/End Prep]
Gently mix the reaction by pipetting up/down 15 times and then briefly spin in a microcentrifuge. Avoid frothing the solution.
?. [Fragmentation/End Prep]
Using a P10, pipette:  of mastermix into each required well of the 96-plate PCR microplate. 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.
9 µl
26 µl
i. Using a permanent marker, demarcate these wells as 'FRAG' (short for fragmentation). Make sure that the marker ink does not get close to any of the sample wells.ii. The total volume in each sample well should be the same ( )
?. [Fragmentation/End Prep]
Place the sealed PCR microplate in the thermocycler. With a heated lid set to , run the following program:1.              @ 2.              @ 3. Infinite Hold                 @ Using these conditions, we will obtain a mean fragment size of approx. 500 bp for each library sample.
75 °C
37 °C
65 °C
4 °C
Use Table 2 to determine the incubation time required to generate any custom-desired fragment size. Incubation time may need to be optimised for individual samples. See Fig. 1 for a typical fragmentation pattern.
?. [Adaptor Ligation]
Remove the microplate from the thermocycler and immediately place on ice or a cold block.
At this stage, it is recommended that a volume check is performed on a few random wells to ensure that the reaction volume for each sample is still    (+/-  ).
?. [Adaptor Ligation]
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:ComponentVolume per library (red) NEBNext Ultra II Ligation Master Mix* 30 µl(red) NEBNext Ligation Enhancer 1 µl(red) NEBNext Adaptor for Illumina** 2.5 µlTotal Volume 33.5 µl
ComponentVolume per library (red) NEBNext Ultra II Ligation Master Mix* 30 µl(red) NEBNext Ligation Enhancer 1 µl(red) NEBNext Adaptor for Illumina** 2.5 µlTotal Volume 33.5 µl
?. [Adaptor Ligation]
Gently mix the reaction by pipetting up/down 15 times and then briefly spin in a microcentrifuge. Avoid frothing of the solution.
?. [Adaptor Ligation]
Using a P100 or P200, pipette:  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.
33.5 µl
i. Using a permanent marker, strikethrough FRAG and replace with 'LIG'. Make sure that the marker ink does not get close to any of the sample wells.ii. The total volume in each sample well should now be the same ( )
?. [Adaptor Ligation]
Place the sealed PCR microplate in the thermocycler. With a heated lid OFF, run the following program:              @ Remove the plate from the thermocycler immediately upon program completion.
20 °C
?. [Adaptor Ligation]
To each well, now add  of (red) USER®Enzyme.Mix by gentle repeated pipetting. Place the sealed PCR microplate in the thermocycler. With a heated lid set at , run the following program:              @
3 µl
47 °C
37 °C
i. Use a fresh tip for subsequent samples if you deposit the enzyme by touching the liquid surface.ii. Step 15 is 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,#E7535and#E6609) Oligos for Illumina.
?. [Adaptor Ligation]
Remove the microplate from the thermocycler, place on ice and proceed to the next section (Size Selection of Adaptor-ligated DNA)
The protocol can be paused here provided the samples are stored at    .
?. [Size Selection of Adaptor-ligated DNA]
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.
At this stage, it is recommended that a volume check is performed on the wells to ensure that the reaction volume for each sample is  .
?. [Size Selection of Adaptor-ligated DNA]
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  0.1X TE (1X TE Buffer diluted 1:10 with water).
28.5 µl
?. [Size Selection of Adaptor-ligated DNA]
Vortex SPRIselect or NEBNext Sample Purification Beads for  to resuspend.
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   for at least   before use.
?. [Size Selection of Adaptor-ligated DNA]
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.
?. [Size Selection of Adaptor-ligated DNA]
Add  (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.
20 µl
i. The SPRI beads tends to be viscous and slow-aspirating. Take extra care to to expel all of the liquid out of the tip during the last mix. ii. Pipette gently to avoid droplet sprays on the inner sides of the well.
?. [Size Selection of Adaptor-ligated DNA]
Incubate the mix on the bench for  at .
4 Room temperature
?. [Size Selection of Adaptor-ligated DNA]
Place the plate on the magnetic stand/rack to separate the beads from the supernatant.
If necessary, quickly spin (3 seconds) the samples to collect the liquid from the sides of the plate wells before placing it on the magnetic stand.
?. [Size Selection of Adaptor-ligated DNA]
After 7 minutes (or when the solution is clear), carefully transfer the supernatant (~ ) containing your DNA to a new tube.
120 µl
Do NOT discard the supernatant. Discard the beads that will now contain the unwanted fragment clusters.
?. [Size Selection of Adaptor-ligated DNA]
Add  (~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.
10 µl
If the SPRIselect have separated out at this stage, vortex for 30 seconds once again.
?. [Size Selection of Adaptor-ligated DNA]
Incubate samples on the bench for  at .
4 Room temperature
?. [Size Selection of Adaptor-ligated DNA]
Place the plate on the magnetic stand to separate the beads from the supernatant.
If necessary, quickly spin (3 seconds) the samples to collect the liquid from the sides of the plate wells before placing on the magnetic stand.
?. [Size Selection of Adaptor-ligated DNA]
After  (or when the solution is clear), carefully remove and discard the supernatant that contains unwanted fragments.
Do NOT discard the beads. Be careful not to disturb the beads that contain the desired DNA when draining the supernatant.
?. [Size Selection of Adaptor-ligated DNA]
Add  of freshly prepared 80% ethanol to the plate wells while they are still on the magnetic stand.
200 µl
?. [Size Selection of Adaptor-ligated DNA]
Incubate at  for , and then carefully remove and discard the liquid.
4 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Size Selection of Adaptor-ligated DNA]
Repeat the ethanol wash steps 29 and 30 two additional times.
?. [Size Selection of Adaptor-ligated DNA]
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  and remove traces of ethanol at the bottom of each well with a p10 pipette tip.
?. [Size Selection of Adaptor-ligated DNA]
Air dry the beads for up to 5 minutes while the plate is on the magnetic stand exposed to the air.
CAUTION: Do not over-dry the beads as this will 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]
Remove the plate from the magnetic stand. Elute the DNA target from the beads by adding  0.1X TE directly onto the clumped beads.
17 µl
?. [Size Selection of Adaptor-ligated DNA]
Mix well by gently pipetting up and down 10 times.Incubate for  at .
4 Room temperature
?. [Size Selection of Adaptor-ligated DNA]
Place the plate on the magnetic stand. After  or when the solution is clear, transfer  of each eluate to a fresh well on the plate.
15 µl
?. [Size Selection of Adaptor-ligated DNA]
Place the microplate on ice and proceed to the next section (PCR Enrichment of Adaptor-ligated DNA).
The protocol can be paused here provided the samples are stored at    .
?. [PCR Enrichment of Adaptor-ligated DNA]
On a fresh 96-PCR microplate, pipette  of the (blue) NEBNext Ultra II Q5 Master Mix into each corresponding sample well from the previous reaction.
25 µl
i. Using a permanent marker, demarcate the fresh wells as 'LIB PCR'. Make sure that the marker ink does not get close to any of the sample wells.
?. [PCR Enrichment of Adaptor-ligated DNA]
To the Q5 Master Mix in each well, add  of (blue) Index/Universal Primer combination mix.Ensure that a fresh tip is used for every well.
10 µl
The primers are provided in NEBNext Multiplex Oligos for Illumina (NEB Cat no. E6609). Please refer to the NEB #E6609 manual for valid barcode combinations and tips for setting up PCR reactions.
?. [PCR Enrichment of Adaptor-ligated DNA]
To each well, now add  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 ()
15 µl
50 µl
?. [PCR Enrichment of Adaptor-ligated DNA]
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.
?. [PCR Enrichment of Adaptor-ligated DNA]
Place the sealed PCR microplate in the thermocycler. With a heated lid set to , run the following program:CYCLE STEPTEMPERATURETIMECYCLESInitial Denaturation98°C30 seconds1Denaturation98°C10 seconds3-7Annealing/Extension65°C75 secondsFinal Extension65°C5 minutes1Hold4°C∞For standard library prep samples, Table 7 could be used as a starting point to determine the number of PCR cycles.INPUT DNA IN THE FS REACTION# OF CYCLES REQUIRED FOR STANDARD LIBRARY PREP: YIELD ~100 ng (5–35 nM)*500 ng3**200 ng3-4100 ng4-5
98 °C
CYCLE STEPTEMPERATURETIMECYCLESInitial Denaturation98°C30 seconds1Denaturation98°C10 seconds3-7Annealing/Extension65°C75 secondsFinal Extension65°C5 minutes1Hold4°C∞
INPUT DNA IN THE FS REACTION# OF CYCLES REQUIRED FOR STANDARD LIBRARY PREP: YIELD ~100 ng (5–35 nM)*500 ng3**200 ng3-4100 ng4-5
For the aims and outputs of this protocol, our recommendation would be to apply 5 CYCLES.
?. [Cleanup of Amplified Libraries]
Remove the PCR amplicons from the thermocycler and immediately place on ice.
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 Amplified Libraries]
Vortex SPRIselect or NEBNext Sample Purification Beads for  to resuspend.
?. [Cleanup of Amplified Libraries]
Add  (0.9X) resuspended beads to the PCR reaction. Mix well by gently pipetting up and down at least 10 times.
45 µl
i. The SPRI beads tends to be viscous and slow-aspirating. Take extra care to to expel all of the liquid out of the tip during the last mix. ii. Pipette gently to avoid droplet sprays on the inner sides of the well.
?. [Cleanup of Amplified Libraries]
Incubate the samples on the bench for  at .
0 Room temperature
?. [Cleanup of Amplified Libraries]
Place the plate on the magnetic stand to separate the beads from the supernatant.
If necessary, quickly spin (3 seconds) the samples to collect the liquid from the sides of the plate wells before placing on the magnetic stand.
?. [Cleanup of Amplified Libraries]
After  (or when the solution is clear), carefully remove and discard the supernatant.
Do NOT discard the beads. Be careful not to disturb the beads that contain the desired DNA when draining the supernatant.
?. [Cleanup of Amplified Libraries]
Add  of freshly prepared 80% ethanol to the plate wells while they are still on the magnetic stand.
200 µl
?. [Cleanup of Amplified Libraries]
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  and remove traces of ethanol at the bottom of each well with a p10 pipette tip.
?. [Cleanup of Amplified Libraries]
Air dry the beads for up to 5 minutes while the plate is on the magnetic stand exposed to the air.
CAUTION: Do not over-dry the beads as this will 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 Amplified Libraries]
Remove the plate from the magnetic stand. Elute the DNA target from the beads by swiftly adding  0.1X TE directly onto the clumped beads.
33 µl
?. [Cleanup of Amplified Libraries]
Mix well by gently pipetting up and down 10 times.Incubate for  at .
4 Room temperature
?. [Cleanup of Amplified Libraries]
Place the plate on the magnetic stand. After  (or when the solution is clear), transfer  of each eluate to a fresh well.
30 µl
?. [Cleanup of Amplified Libraries]
Incubate at  for , and then carefully remove and discard the liquid.
4 Room temperature
Be careful not to disturb the beads that contain DNA targets.
?. [Cleanup of Amplified Libraries]
Repeat the ethanol wash steps 49 and 50 two additional times.
?. [Cleanup of Amplified Libraries]
WGS Library preparation is now complete.Store the prepared libraries at  until they are ready for QC.
-20 °C
?. [Assessing Library Quality]
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 2100orAgilent D1000 ScreenTape Assay on the Tapestation 4150/4200qPCR (using SYBR fluorescent dyes) - NEBNext Library Quant Kit for Illumina (NEB)orKAPA Library Quantification Kit Illumina® Platforms (Roche)
For a guide to library QC for Illumina sequencing, refer to: "C-SOP-401: Quality Control (QC) of DNA Libraries for Whole Genome Sequencing" on protocols.io
?. [Additional Information & Troubleshooting]
Troubleshooting Guide for NEBNext®Ultra™ II FS DNA Library Prep KitTechnical Note: SPRI select: DNA Ratios Affect the Size Range of Library Fragments (10x Genomics)