TITLE: NEBNext Ultra II Library Prep for Illumina Low Volume Version (Naturalis)
AUTHORS: Stacey Dubbeldam

[DESCRIPTION]
Low volume version of NEBNext Ultra II DNA library protocol (Naturalis). 

[STEPS]
SECTION: NEBNext Ultra II Library Prep for Illumina Low Volume Version (Naturalis)
1. Make sure that the DNA is eluted in  . If this is not the case, samples can be diluted using 10nM Tris-HCl, pH 8.0 or 0.1X TE.

SECTION: NEBNext Ultra II Library Prep for Illumina Low Volume Version (Naturalis)
2. Check the DNA concentration of your samples using the Fragment Analyzer Genomic DNA 50Kb kit (DNF-467-0500).
SECTION: NEBNext Ultra II Library Prep for Illumina Low Volume Version (Naturalis)
3. Normalise all samples based on the Fragment Analyzer results. The DNA input (ng) should be close to equal for all samples before proceeding with the library prep. 
SECTION: NEBNext Ultra II Library Prep for Illumina Low Volume Version (Naturalis)
3.1. Concentrate plates using the Eppendorf Concentrator (SpeedVac) if starting concentrations are low. 
SECTION: NEBNext End Prep
4. Thaw the   and  , place the Enzyme Mix on ice.

Gently mix and spin down the components before use.

Prepare a master mix of the  and   based on the volume option you choose to work with.
SECTION: NEBNext End Prep
4.1. 
    Volume option 1 (1:8)Volume option 2 (1:12)Volume option 3(1:16)O NEBNext Ultra II End Prep Enzyme Mix0,3750,25 μl0,15O NEBNext Ultra II End Prep Reaction Buffer 0,8750,58 μl0,35Fragmented DNA  (can be adjust with 0,1 xTE)6,264,17 μl2,5Total volume7,553

4.2. Let the I.DOT Mini distribute the appropriate volume of the master mix:
Option 1: 1.25 µL, 0ption 2: 0.83 µL, and option 3: 0.5 µL
I.DOT Liquid setting: H20.
SECTION: NEBNext Adaptor Ligation
5. Thaw the  ,   &   and place on ice. 
4.3. Manually add the appropriate amount of fragmented DNA (see Table 1) using a P10 multichannel.
Gently mix by slowly pipetting the entire volume up and down at least 10 times. 
4.4. Place in a thermocycler, with the heated lid set to ≥ 75 °C, and run the following program:
30 min at 20 °C
30 min at 65 °C
Hold at4 °C
SECTION: NEBNext Adaptor Ligation
5.1. Important note: Before starting the Adapter Ligation, determine whether adaptor dilution* is necessary:
   InputADAPTOR DILUTION (VOLUME OF ADAPTOR: TOTAL VOLUME)WORKING ADAPTOR CONCENTRATION1 µg–101 ngNo Dilution15 µM100 ng–5 ng10-Fold (1:10)1.5 µMless than 5 ng25-Fold (1:25)0.6 µM

Prepare an Adaptor dilution in Tris/NaCl, pH 8.0 (10 mM Tris, 10mM NaCl) if necessary. 
 
 *The NEB adaptor sequence: /5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC/ideoxyU/ACACTCTTTCCCTACACGACGCTCTTCCGATC*T
SECTION: NEBNext Adaptor Ligation
5.2. Prepare a Master Mix of the Ligation Master Mix & Ligation Enhancer based on the volume option chosen (see Table 3). 
Let the I.DOT Mini pipet the appropriate volume of your master mix to the End Prep Reaction Mixture.
Option 1: 3.875 µL, option 2: 2.583 µL, and option 3: 1.55 µL.
     - Liquid class:
Let the I.DOT Mini pipet the appropriate volume of your NEBNext Adaptor for Illumina.
- Liquid class: H20.
    Volume option 1 (1:8)Volume option 2 (1:12)Volume option 3(1:16)End Prep Reaction Mixture from step 17,5 μl5 μl3 μlO NEBNext Ultra II Ligation Master Mix* 3,75 μl2,5 μl1,5 μlO NEBNext Ligation Enhancer0,125 μl0,083 μl0,05 μlO NEBNext Adaptor for Illumina0,31 μl0,208 μl0,0125 μlTotal Volume11,41 μl7,79 μl4,56 μl

Mix by pipetting the entire volume up and down at least 10 times.
Spin down briefly.

SECTION: NEBNext Adaptor Ligation
5.3. Incubate at 20 °C for 15 min in a thermocycler with the heated lid of
SECTION: NEBNext Adaptor Ligation
5.4. Let the I.DOT pipet the appropriate volume (see Table 4) of the   to the ligation mixture:
 
 
    O USER‱ Enzyme0,5 μl0,35 μl0,2 μl
Liquid class: Glycerol 10%
SECTION: NEBNext Adaptor Ligation
5.5. Mix well and incubate at 37 °C for15 min with the heated lid set to ≥47 °C
SECTION: NEBNext Adaptor Ligation
5.6. Bring the volume of the total reaction to 15 µL before proceeding to the following step.

SECTION: 1,2X Reaction Clean-up
6. Let the MN beads get to room temperature (remove from fridge 15 min before use).
Prepare fresh 80% EtOH (200 µL per sample, e.g. 41.7 mL EtOH 96% + 8.3 mL MilliQ).
Mix the MN beads well by vortexing.
Add 1,2x MN beads to the ligation reaction.
Mix well by pipetting.
Incubate at room temperature for 5 min.
Place the plate on the magnetic rack.
When the solution is clear (5 min), carefully remove the supernatant (LEAVE THE PLATE ON THE MAGNET).
Wash the beads 2x with 50 µL 80% ethanol (LEAVE THE PLATE ON THE MAGNET).
Let the beads air dry for 1 min(LEAVE THE PLATE ON THE MAGNET).
Take the plate off the magnet and resuspend the beads with e.g.12 µL 0,1x TE, by pipetting up and down. 
Wait 5 min and put the plate back on the magnet.
When the solution is clear (5 min), move 12 µL of the supernatant to a clean plate.

NOTE: The plate can be concentrated again in the Eppendorf Concentrator (SpeedVac) if volume option 2 or volume option 3 is chosen and low concentrations are expected.


SECTION: NEBNext PCR Enrichment
7. Distribute the   with the chosen volume option to all wells of a new PCR plate.
Add the Adaptor Ligated DNA Fragments and IDT_10 primers manually using a P10 multichannel.
    Volume option 1 (1:8)Volume option 2 (1:12)Volume option 3(1:16)Adaptor Ligated DNA Fragments 5,25 μl3,15 μl2,1 μlONEBNext Ultra II Q5 Master Mix 2X6,25 μl3.75 μl2,5 μlO Illumina IDT_10 i7 Primer*0,5 μl0,3 μl0,2 μlO Illumina IDT_10 i5 Primer*0,5 μl0,3 μl0,2 μlTotal volume12,5 μl7,5 μl5 μl

SECTION: NEBNext PCR Enrichment
7.1. Mix by pipetting the entire volume up and down at least 10 times
Place the plate in a thermocycler and perform PCR amplification using the following PCR cycling conditions
    Cycle StepTempTimeCyclesInitial Denaturation98°C30 seconds1Denaturation98°C10 seconds3–15*dilurAnnealing/Extension65°C75 secondsFinal Extension65°C5 minutes1Hold4°C∞

SECTION: NEBNext PCR Enrichment
7.2. The number of PCR cycles should be chosen based on input amount and sample type (see Table 7). 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 TapeStation or Fragment Analyzer). Use the table below for applications requiring high library yields (~1 µg).
  INPUT DNA IN THE END PREP REACTION# OF CYCLES REQUIRED FOR TARGET ENRICHMENT LIBRARY PREP (~1 µg):1 µg*3–4*, ** 500 ng*4–5*100 ng*6–7* 50 ng7–810 ng9–105 ng10–111 ng12–130.5 ng14–15

SECTION: Check PCR succes
8. Check the success of a few amplified samples on the TapeStation using the D5000 High Sensitivity kit:
Dilute1 µLof amplified sample in 9 µL of MilliQ (10x) and measure the sample.
- Optional: add 0.2 µL of amplified sample and 1.8 µL of MilliQ directly to the TapeStation strip.
If the amplification was not sufficient, add additional cycles.
If overcycling has occurred, redo the amplification with the remaining sample from the previous step with fewer cycles.

Bring the volume of the PCR reaction to at least 10 µLbefore proceeding to the next step.
SECTION: Reaction Clean-up
9. Let the MN beads get to room temperature (remove from fridge 15 min before use).
Prepare fresh 80% EtOH (200 µL per sample, e.g. 41.7 mL EtOH 96% + 8.3 mL MilliQ).
Mix the MN beads well by vortexing.
Add 0,9x MN beads* to the ligation reaction.
Mix well by pipetting.
Incubate at room temperature for 5 min.
Place the plate on the magnetic rack.
When the solution is clear (5 min), carefully remove the supernatant (LEAVE THE PLATE ON THE MAGNET).
Wash the beads 2x with 50 µL 80% ethanol (LEAVE THE PLATE ON THE MAGNET).
Let the beads air dry for 1 min(LEAVE THE PLATE ON THE MAGNET).
Take the plate off the magnet and resuspend the beads with e.g.12 µL 0,1x TE, by pipetting up and down.
Place the plate back on the magnet.
When the solution is clear (5 min), move 12 µL of the supernatant to a clean plate.

*Bead to sample ratio is dependent on the library size. Carefully choose the correct ratio. 
SECTION: Measuring libraries and equimolar pooling of samples
10. Measure the concentration of all samples on the Fragment Analyzer. 
Export a smear analysis and pool all samples equimolarly by either using the Qiagility pipetting robot or manually.

SECTION: Measuring pools
11. Measure the pool(s) in triplicate on the TapeStation using the High Sensitivity D5000 kit.
Use the mean concentration per pool for calculations.
If equimolar pooling of subpools is required, use the average molar concentration of the three measured subpools for pooling of these pools (calculate this based on the lowest pool concentration).