License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: November 06, 2023
Last Modified: November 07, 2023
Protocol Integer ID: 90449
Abstract
Low volume version of NEBNext Ultra II DNA library protocol (Naturalis).
Protocol materials
NEBNext Ligation Master MixNew England BiolabsCatalog #E7648AAVIAL
Step 5
NEBNext® Ligation EnhancerNew England BiolabsCatalog #E7374AAVIAL
Step 5
NEBNext Adaptor for IlluminaNew England BiolabsCatalog #E7337 in Kits E7335, E7500, E771
Step 5
NEB USER® EnzymeNew England BiolabsCatalog #M5505S/L
Step 5.4
NEBNext Ultra II Q5 Master Mix - 250 rxnsNew England BiolabsCatalog #M0544L
Step 7
NEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646
In 2 steps
NEBNext dA-Tailing Reaction Buffer - 2.0 mlNew England BiolabsCatalog #B6059S
In 2 steps
TE Buffer
Step 1
NEBNext Ultra II Library Prep for Illumina Low Volume Version (Naturalis)
NEBNext Ultra II Library Prep for Illumina Low Volume Version (Naturalis)
Make sure that the DNA is eluted in TE BufferContributed by users. If this is not the case, samples can be diluted using 10nM Tris-HCl, pH 8.0 or 0.1X TE.
Check the DNA concentration of your samples using the Fragment Analyzer Genomic DNA 50Kb kit (DNF-467-0500).
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.
Concentrate plates using the Eppendorf Concentrator (SpeedVac) if starting concentrations are low.
NEBNext End Prep
NEBNext End Prep
Thaw the NEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646 and NEBNext dA-Tailing Reaction Buffer - 2.0 mlNew England BiolabsCatalog #B6059S, place the Enzyme Mix on ice.
Gently mix and spin down the components before use.
Prepare a master mix of the NEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646and NEBNext dA-Tailing Reaction Buffer - 2.0 mlNew England BiolabsCatalog #B6059S based on the volume option you choose to work with.
A
B
C
D
Volume option 1 (1:8)
Volume option 2 (1:12)
Volume option 3 (1:16)
NEBNext Ultra II End Prep Enzyme Mix
0,375
0,25
0,15
NEBNext Ultra II End Prep Reaction Buffer
0,875
0,58
0,35
Fragmented DNA (can be adjust with 0,1 xTE)
6,26
4,17
2,5
Total volume
7,5
5
3
Table 1: Amount (μl) per volume option.
Let the I.DOT Minidistribute the appropriate volume of the master mix:
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.
Place in a thermocycler, with the heated lid set to ≥ 75 °C, and run the following program:
00:30:00 at 20 °C00:30:00 at 65 °C
Hold at4 °C
1h
NEBNext Adaptor Ligation
NEBNext Adaptor Ligation
30m
Thaw theNEBNext Ligation Master MixNew England BiolabsCatalog #E7648AAVIAL, NEBNext® Ligation EnhancerNew England BiolabsCatalog #E7374AAVIAL & NEBNext Adaptor for IlluminaNew England BiolabsCatalog #E7337 in Kits E7335, E7500, E771 and place on ice.
Important note: Before starting the Adapter Ligation, determine whether adaptor dilution* is necessary:
A
B
C
Input
ADAPTOR DILUTION (VOLUME OF ADAPTOR: TOTAL VOLUME)
WORKING ADAPTOR CONCENTRATION
1 µg–101 ng
No Dilution
15 µM
100 ng–5 ng
10-Fold (1:10)
1.5 µM
less than 5 ng
25-Fold (1:25)
0.6 µM
Table 2: Input Adaptor Dilution.
Prepare an Adaptor dilution in Tris/NaCl, pH 8.0 (10 mM Tris, 10mM NaCl) if necessary.
Let the I.DOT Mini pipet the appropriate volume of your NEBNext Adaptor for Illumina.
- Liquid class: H20.
A
B
C
D
Volume option 1 (1:8)
Volume option 2 (1:12)
Volume option 3 (1:16)
End Prep Reaction Mixture from step 1
7,5 μl
5 μl
3 μl
NEBNext Ultra II Ligation Master Mix*
3,75 μl
2,5 μl
1,5 μl
NEBNext Ligation Enhancer
0,125 μl
0,083 μl
0,05 μl
NEBNext Adaptor for Illumina
0,31 μl
0,208 μl
0,0125 μl
Total Volume
11,41 μl
7,79 μl
4,56 μl
Table 3: Amount (μl) per volume option.
Mix by pipetting the entire volume up and down at least 10 times.
Spin down briefly.
Incubate at 20 °C for 00:15:00 in a thermocycler with the heated lid of
15m
Let the I.DOT pipet the appropriate volume (see Table 4) of the NEB USER® EnzymeNew England BiolabsCatalog #M5505S/L to the ligation mixture:
A
B
C
D
USER Enzyme
0,5 μl
0,35 μl
0,2 μl
Table 4: Amount (μl) per volume option.
Liquid class: Glycerol 10%
Mix well and incubate at 37 °C for00:15:00 with the heated lid set to ≥47 °C
15m
Bring the volume of the total reaction to 15 µL before proceeding to the following step.
1,2X Reaction Clean-up
1,2X Reaction Clean-up
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 00:05:00.
Place the plate on the magnetic rack.
When the solution is clear (00:05:00), 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 00:01:00(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 00:05:00 and put the plate back on the magnet.
When the solution is clear (00:05:00), 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.
21m
NEBNext PCR Enrichment
NEBNext PCR Enrichment
30m
Distribute the NEBNext Ultra II Q5 Master Mix - 250 rxnsNew England BiolabsCatalog #M0544L 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.
A
B
C
D
Volume option 1 (1:8)
Volume option 2 (1:12)
Volume option 3 (1:16)
Adaptor Ligated DNA Fragments
5,25 μl
3,15 μl
2,1 μl
NEBNext Ultra II Q5 Master Mix 2X
6,25 μl
3.75 μl
2,5 μl
Illumina IDT_10 i7 Primer*
0,5 μl
0,3 μl
0,2 μl
Illumina IDT_10 i5 Primer*
0,5 μl
0,3 μl
0,2 μl
Total volume
12,5 μl
7,5 μl
5 μl
Table 5: Amount (μl) per volume option.
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
A
B
C
D
Cycle Step
Temp
Time
Cycles
Initial Denaturation
98°C
30 seconds
1
Denaturation
98°C
10 seconds
3–15*
Annealing/Extension
65°C
75 seconds
Final Extension
65°C
5 minutes
1
Hold
4°C
∞
Table 6: PCR program
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).
A
B
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 ng
7–8
10 ng
9–10
5 ng
10–11
1 ng
12–13
0.5 ng
14–15
Table 7: Number of cycles per input DNA.
Check PCR succes
Check PCR succes
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.
Reaction Clean-up
Reaction Clean-up
16m
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 00:05:00.
Place the plate on the magnetic rack.
When the solution is clear (00:05:00), 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 00:01:00(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 (00:05:00), 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.
16m
Measuring libraries and equimolar pooling of samples
Measuring libraries and equimolar pooling of samples
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.
Measuring pools
Measuring pools
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).