SWIFT NORMALASE KIT
For Enzymatic NGS Library
Normalization
Compatible with libraries constructed using
full-length, indexed adapters and PCR amplication
Validated with:
• Swift Hot Start High Fidelity Polymerase
• KAPA HiFi HotStart ReadyMix
• NEBNext Ultra II Q5
®
Master Mix
Protocol for Cat. No. 66096 to be used with indexing kits:
• 2S Set A (Cat. No. 26148)
• 2S Set B (Cat. No. 26248)
• 2S Set A+B (Cat. No. 26396)
• 2S Set A MID (Cat. No. 27148)
• 2S Set B MID (Cat. No. 27248)
• 2S Set A+B MID (Cat. No. 27396)
• 2S Set S1 (Cat. No. 26596)
• 2S Set S2 (Cat. No. 26696)
• 2S Set S3 (Cat. No. 26796)
• 2S Set S4 (Cat. No. 26896)
• 2S Set S1-S4 (Cat. No. 269384)
• 2S Set S1 MID (Cat. No. 27596)
• 2S Set S2 MID (Cat. No. 27696)
• 2S Set S3 MID (Cat. No. 27796)
• 2S Set S4 MID (Cat. No. 27896)
• 2S Set S1-S4 MID (Cat. No. 279384)
Visit swiftbiosci.com/protocols for updates.
PROTOCOL
swiftbiosci.com
Version 1.0
Table of Contents
About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Product Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Swift Normalase Workow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Kit Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Material and Equipment Not Included . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Storage and Usage Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Tips and Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Avoiding Cross-Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Post-PCR Clean-Up Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Library Input Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Meeting the Minimum Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Expected Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Prepare the Reagent Master Mixes and Ethanol. . . . . . . . . . . . . . . . . . . . . . .7
BEGIN YOUR SWIFT NORMALASE PROTOCOL
Perform Library Amplication with Normalase Primers
. . . . . . . . . . . . . . . . . .8
Normalase PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Library Purication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Normalase I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Library Pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Normalase II. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Normalase Inactivation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Recommended Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Section A: Low-plex Pooling Recommendations . . . . . . . . . . . . . . . . . . . . .15
Section B: High sample volume pooling recommendations. . . . . . . . . . . . . . .16
Section C: Helpful Information and Troubleshooting . . . . . . . . . . . . . . . . . . 19
General Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Limitation of Liability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Notice to Purchaser: Limited License . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1
SWIFT NORMALASE KIT
About This Guide
This guide provides instructions for enzymatic normalization of multiplexed next generation sequencing (NGS)
libraries for equimolar pooling and balanced sample representation in sequencing. The protocol is designed
for workflows that produce consistent amplified library yields of 12 nM or more following Normalase PCR and
generates a normalized library pool of 4 nM. This workflow does not introduce more amplification but
replaces the primers in conventional library amplification with Normalase primers, allowing the user to
maintain their conventional library amplification workflow. The Normalase workflow does not require
quantification and is a bead-free normalization method where equal volumes of each library are pooled to
simplify high throughput processing.
Even if library yields have met the 12 nM minimum prior to library amplification using Normalase primers,
perform-ing a minimum of 3 cycles is required to condition the library for the downstream Normalase
enzymology.
IMPORTANT!
Do not begin library construction for Normalase until you read this Protocol. Normalase
PCR primers replace your standard primers and are combined with your amplification
reagents. Conditions for Normalase library amplification are modified from standard
library amplification procedures. Read the entire protocol thoroughly to ensure that you
understand all the important information and modifications needed for successful inte-
gration of Normalase with your library preparation workflow.
Be aware of your downstream sequencing instrument loading concentration and
volume requirements as well as the number of samples to be pooled for multiplexed
sequenc-ing. These factors will affect how you pool and complete the Normalase
workflow. For example, NovaSeq requires 100-310 μl loading volume at 1.5-3 nM
concentration and if multiplexing a lower number of samples, you may need to pool
more than 5 μl of each sample to achieve a sufficient loading volume at the required
concentration. Refer to Appendix A and B for alternate pooling guides if needed.
The Normalase product specification is defined by cluster density of the final 4 nM
Normalase pool when loaded on a MiSeq v2 flow cell at 12 pM to achieve a 1000-1200
K/mm
2
cluster density and CV ≤ 10% within a pool (Swift 2S Turbo Flexible libraries with
an average 200 bp insert size). Across other Illumina platforms, library types, and insert
sizes, optimization of loading concentration may be required to achieve the desired
cluster density.
Library Preparation Library Normalzation
Library Preparation
(Indexed Adapters)
Library Amplification
(Normalase Primers)
Library Amplification
(Conventional Primers)
Normalase I & II
Library Dilution
and Pooling
Sequencing
Library Quantification
(qPCR/Qubit)
2
SWIFT NORMALASE KIT
Product Information
Swift Normalase offers a streamlined solution for equimolar NGS library pooling and uniform multiplexed se-
quencing without the need for concentration adjustment of individual samples.
The protocol is readily automatable. A 10% overage volume of reagents is supplied in a 96-reaction kit to
accommodate automation. Swift Biosciences does not supply automated liquid handling instrumentation
or consumables but collaborates with automation solution providers and customers to develop and qualify
optimized automated scripts for use of our kits, in combination with liquid handling platforms routinely used in
NGS library preparation. Please contact your instrument vendor or TechSuppor[email protected] if you plan to
use the Swift Normalase Kit with your particular automated liquid handling system.
Applications
Swift Normalase is suitable for libraries with the following features:
• Libraries with full-length indexed adapters
• Libraries that have an amplied yield of 12 nM or higher (20 µL volume)
• Libraries prepared for direct sequencing (i.e., whole genome, whole transcriptome)
• Target enriched, post-hybridization libraries with indexed adapters
Best results are obtained using libraries with standard size distribution generated by Covaris shearing or
enzy-matic fragmentation (i.e., 150-550 bp inserts) and bead-based size selection.
Libraries with broad or variable size distributions (i.e., transposase-based workows) that demonstrate size
dependent clustering effects that are independent of molarity may have more variable results.
The Swift Normalase Kit is not compatible with these library features:
• PCR-free libraries
• Workows that require indexing PCR primers to complete library construction
• Libraries prepared for pre-hybridization capture which require greater than 4 nM nal library concentrations
3
SWIFT NORMALASE KIT
Swift Normalase Workflow
This workflow provides an easy solution to generate equimolar NGS library pools for balanced sample
representation in multiplexed sequencing. The workflow consists of replacing conventional library
amplification with Normalase primers and amplifying to a minimum yield threshold of 12 nM or more (20 µL
volume), followed by Normalase I, library pooling, and Normalase II to generate 4 nM library pools.
The bead-based clean-up, post-Normalase PCR is used to remove oligonucleotides and small fragments.
NGS Library
Normalase I
(15 Minutes)
Pool
Libraries
Normalase II
(15 Minutes)
Normalase PCR
Purification
Normalized 4nM Pool
Normalase PCR
nM Library
Normalase I: enzymatic selection
of a 4 nM library fraction
Normalase II: enzymatic
normalization to 4 nM
Pooling
Normalized Library Pool
4
SWIFT NORMALASE KIT
Kit Contents
The Swift Normalase Kit is available with reagents (10% excess volume) for the processing of 96 libraries.
Reagents
Quantity (μl)
Storage (ºC)
96 rxn
Reagent R5 528 -20
Buffer Y1 454 -20
Reagent Y2 21 -20
Enzyme Y3 52 -20
Buffer G1* 506 -20
Enzyme G2 21 -20
Reagent B1 105 -20
*These reagents are provided at a 5-fold excess plus overage so that library pooling
and subsequent steps can be repeated up to 5 times for exibility in re-pooling and
re-sequencing.
Reagents
Quantity
(μL)
Storage (ºC)
Low EDTA TE 2x1200 RT
Material and Equipment Not Included
• Library preparation kit and DNA polymerase for amplication (Swift Hot Start High Fidelity Polymerase reagents
provided with the library kit, Kapa HiFi HotStart ReadyMix (KK2602), NEBNext Ultra II Q5 Master Mix (M0544))
• qPCR-based library quantication kit for conrming nal library pool concentration
• Optional Qubit
®
or other uorometric-based assays for determining individual library concentration prior to
Normalase
• Magnetic beads for the clean-up step, e.g., SPRIselect™ beads (Beckman Coulter, Cat. No. B23317/ B23318/ B23319)
• Magnetic rack for the clean-up step, e.g., Invitrogen DynaMag™ or Agencourt
®
SPRIPlate™
• Microfuge
• Programmable thermocycler
• Heat block that adjusts from 37-95 ºC
• 0.2 mL PCR tubes
• 1.5 mL screw cap microfuge tubes
• Aerosol-resistant, low retention pipettes and tips, 2 to 1000 µL (calibrated P10 pipette preferred for accuracy
of 5 µL pipetting during Normalase I and library pooling steps)
• 200-proof/absolute ethanol (molecular biology-grade)
• Nuclease-free water (molecular biology-grade)
Storage and Usage Warning
Upon receipt, store the Swift Normalase Kit at -20 ˚C with the exception of Low EDTA TE solution, which can be
stored at room temperature.
The enzymes provided in this kit are temperature sensitive, and appropriate care should be taken during stor-
age and handling. To maximize use of enzyme reagents when ready to use, remove enzyme tubes from -20 ˚C
storage and place on ice, NOT in a cryocooler, for at least 10 minutes to allow enzymes to reach 4 ˚C prior to
pipetting. Attempting to pipette enzymes at -20 ˚C may result in a shortage of enzyme reagents.
IMPORTANT!
Place the enzymes
on ice, NOT in a cryo-
cooler, for at least
10 minutes to allow
enzymes to reach 4 ˚C
prior to pipetting.
5
SWIFT NORMALASE KIT
Tips and Techniques
Avoiding Cross-Contamination
To reduce the risk of DNA and library contamination, physically separate the laboratory space, equipment, and
supplies where pre-PCR and post-PCR processes are performed. Follow the instructions below to avoid cross-
contamination:
• Clean lab areas using 0.5% sodium hypochlorite (10% bleach).
• Use barrier pipette tips to avoid exposure to potential contaminants.
• Always change tips between each sample.
Post-PCR Clean-Up Step
This protocol has been validated with SPRIselect beads (Beckman Coulter); however, it can be used with
Agencourt AMPure
®
XP (Beckman Coulter). If other beads are used, solutions and conditions for DNA
binding may differ.
Consider the information below for performing efcient size selection:
• For the post-Normalase PCR clean-up step, use the same bead ratio as recommended by your library prep
supplier post-PCR in order to achieve consistent results and avoid adapter dimers and short library mol-
ecules that may otherwise result in your nal library.
• However, if the bead ratio recommended by the library prep supplier is greater than a 1.0X ratio (e.g. 1.2X or
higher), we recommend performing a 1.0X ratio to ensure efcient removal of unused Normalase primers.
6
SWIFT NORMALASE KIT
Library Input Considerations
Meeting the Minimum Threshold
In order to ensure that library yields meet 12 nM or higher in the 20 µL eluate follow-ing amplification with
Normalase primers, careful quantification of input material for library prep is important to determine the
required number of PCR cycles to reach the 12 nM yield.
For high quality samples, it is recommended to determine dsDNA concentration using Qubit, or a similar fluo-
rometric method, as it will accurately represent the double-stranded, adaptable DNA content of your sample.
For low quality human DNA samples, we recommend quantification by qPCR using Alu primer pairs provided in
Swift library kits to accurately assess the usable amount of human DNA in the samples and their integrity.
Alternatively, if careful quantification of input material is not possible, determine the lowest usable input
quanti-ty that will be used across the sample set and apply the appropriate number of cycles for that input
quantity to all samples in order to consistently reach the minimum threshold of 12 nM across the sample set,
regardless of varied input quantity or quality.
Use of Normalase primers is required for successful normalization of libraries. Library amplification using Nor-
malase primers (Reagent R5) is required even if your libraries have already met the minimum yield threshold
prior to amplification. A minimum of 3 cycles is required to condition the library for the downstream
Normalase enzymology.
IMPORTANT!
Library input quantities referenced in this protocol refer to total library yield after PCR
amplification with Normalase primers. Amplified library yields must meet the minimum
threshold of 12 nM (in 20 µL) in order to obtain a 4 nM normalized yield. Libraries below
the minimum threshold will result in normalized yields < 4 nM and will be under-repre-
sented in multiplexed sequencing data.
7
SWIFT NORMALASE KIT
Expected Results
As shown in the plot below, 16 Swift 2S Turbo libraries were prepared using differing inputs of Coriell NA12878
DNA from 1-250 ng that were enzymatically fragmented to 350 bp, ligated to Illumina
®
unique dual indexed
adapters, and followed by the recommended number of PCR cycles for the various inputs indicated in this
guide for this library prep. Libraries were prepared by two different individuals and Normalase was performed
using two separate pools of 8 libraries each. The pools were then combined into a single pool using an equal
volume of 5 µL each, then denatured and loaded at 10 pM onto a MiSeq
®
v2 ow cell. The overall cluster den-
sity was 856 K/mm
2
which is within the specications for v2 chemistry, 93% clusters passing lter, and Q30 of
97.5%. Demultiplexing data indicate that individual representation of each library had 7.3% and 8.7% variation
within the pools and a 9.5% variation across pools.
Prepare the Reagent Master Mixes and Ethanol
• To create the master mixes, scale reagent volumes as appropriate, using 10% excess volume to compen-
sate for pipetting loss.
• To assemble reagent master mixes for the Normalase PCR, Normalase I, and Normalase II steps, ensure the
reagent vials and enzymes are at 4 °C. After thawing reagents, briey vortex (except the enzymes) to mix
them well. Spin tubes in a microfuge to collect contents prior to opening.
• Prepare the master mixes at room temperature and always add reagents in the specied order. If preparing
in advance, store master mixes on ice until use, then add to samples at room temperature. Master mixes
can be prepared and used at room temperature if prepared just before use.
• Prepare a fresh 80% ethanol solution using 200-proof/absolute ethanol and nuclease-free water. Approxi-
mately 0.5 mL of 80% ethanol solution will be used per sample.
1.
DNA Input into Turbo (ng)
% Reads Identified
Pool 1
CV=7.3%
20
15
10
5
0
1 1 5 5 10 10 25 25 50 50 75 75 100 100 250 250
Pool 2
CV=8.7%
8
SWIFT NORMALASE KIT
BEGIN YOUR SWIFT NORMALASE PROTOCOL
Perform Library Amplication with Normalase Primers
If your conventional library preparation and amplication step yields ≥ 12 nM in 20 µL, simply replace conventional
amplication primers with Normalase primers and run amplication with minimum adjustments to the nal
extension step. If without amplication your library preparation yields ≥ 12 nM, a minimum of 3 cycles using
Normalase primers (Reagent R5) is required to condition the library for the downstream Normalase enzymology.
As a general guide, refer to the table below for the recommended number of cycles when using the Swift 2S Turbo
Flexible Kit and Accel-NGS
®
2S DNA Library Kits. Data in the table below are based on high quality DNA inputs quan-
tied by Qubit. If using lower quality samples or inputs < 1 ng, assess sample quality and quantity by an ALU or other
qPCR-based DNA integrity assay to determine the necessary number of PCR cycles to meet ≥ 12 nM minimum yield.
Swift 2S Turbo Flexible DNA Library Kit and Accel-NGS 2S DNA Library Kits
Input Material (ng)
Minimum Normalase
PCR Cycles
Average Yield (nM)
≥ 100 3
≥ 12
25 5
10 7
1 10
If using a library preparation kit other than Swift 2S Turbo and Accel-NGS 2S kits, you must be familiar with the
number of PCR cycles required to reach at least 12 nM prior to using this kit and these same number of cycles
(minimum of 3 cycles) should be used during the Normalase PCR step in this protocol. If your
standard conditions produce libraries <12 nM library yield, you must increase the number of PCR cycles to
produce at least 12 nM yield in 20 µL volume.
IMPORTANT!
Achieving the minimum threshold of 12 nM is required for optimal performance. Libraries
that do not meet this threshold will be less than 4 nM post-Normalase and will be under-
represented during cluster generation.
9
SWIFT NORMALASE KIT
Normalase PCR
1. Set up your library amplication reactions using standard PCR reagents as shown below, except substi-
tute the standard primers with 5 µL of Reagent R5 (the Normalase PCR primers that have a 600 nM nal
concentration in the reaction).
Swift Hot Start High
Fidelity Polymerase
PCR rxn
Master Mix
KAPA HiFi HotStart ReadyMix (KK2602)
NEBNext Ultra II Q5 Master Mix (M0544)
PCR rxn
Sample: 20 µL Low EDTA TE: 10 µL Reagent R5: 5 µL
Master Mix: 30 µL Reagent R2: 4 µL Sample: 20 µL
Final Volume: 50 μl Buffer R3: 10 µL Master Mix: 25 µL
Enzyme R4: 1 µL Final Volume: 50 μl
Reagent R5: 5 µL
Final Volume: 30 μl
2. Modify the thermocycler PCR program recommended by the library preparation kit or polymerase of
choice with a 5-minute nal extension step. Pre-set the thermocycler in accordance with the cycles
required for your DNA input quantities based on the library preparation guide with adjusted cycle number
if needed to achieve the minimum threshold. Utilize your polymerase of choice with the thermocycler
conditions below:
Swift Hot Start High
Fidelity Polymerase
KAPA HiFi HotStart
ReadyMix
NEBNext Ultra II
Q5 Master Mix
Thermocycler Program
Heated Lid at 105 °C
98 ºC for 30 seconds PCR Cycles:
• 98 ºC for 10 seconds
• 60 ºC for 30 seconds
• 68 ºC for 60 seconds
Final extension 68 ºC for 5 minutes
Hold at 4 ºC — proceed to clean-up
Thermocycler Program
Heated Lid at 105 °C
98 ºC for 45 seconds PCR Cycles:
• 98 ºC for 15 seconds
• 60 ºC for 30 seconds
• 72 ºC for 30 seconds
Final extension 72ºC for 5 minutes
Hold at 4 ºC — proceed to clean-up
Thermocycler Program
Heated Lid at 105 °C
98 ºC for 30 seconds PCR Cycles:
• 98 ºC for 10 seconds
• 60 ºC for 30 seconds
• 65 ºC for 60 seconds
Final extension 65 ºC for 5 minutes
Hold at 4 ºC — proceed to clean-up
Library Purication
3. Ensure the magnetic beads are at room temperature and vortex beads to homogenize the suspension
before use.
4. Utilize the same post-PCR bead ratio as recommended by your library prep supplier in order to achieve
consistent results and avoid adapter dimers and short library molecules that may otherwise result in
your nal library. However, if the bead ratio recommended by the library prep supplier is greater than a
1.0X ratio (e.g., 1.2X or higher), we recommend performing a 1.0X ratio (50 µL beads) to ensure efcient
removal of unused Normalase primers.
5. Mix the sample with beads by moderate vortexing for 5 seconds.
6. Pulse spin the samples in a tabletop microcentrifuge. Do not centrifuge to excess, as marked by the
beads pelleting at the bottom. If this occurs, re-mix your samples and spin again with less force/shorter
duration.
7. Incubate the samples for 5 minutes at room temperature.
10
SWIFT NORMALASE KIT
8. Place the sample on a magnetic rack until the solution clears and a pellet is formed (~ 2 minutes).
9. Remove and discard the supernatant without disturbing the pellet (less than 5 µl may be left behind).
10. Add 200 µl of freshly prepared 80% ethanol solution to the sample while it is still on the magnetic rack.
Use care not to disturb the pellet. Incubate for 30 seconds and then carefully remove the ethanol solution.
11. Repeat step 10 once more for a second wash with the 80% ethanol solution.
12. Gently spin the samples in a tabletop microfuge and place back on the magnetic rack. Remove any residu-
al ethanol solution from the bottom of the tube using a smaller pipette tip.
13. Remove samples from magnetic rack. Add 22 µl of Low EDTA TE buffer and re-suspend the pellet. Mix well
by pipetting up and down until homogenous.
14. Place the sample tubes on a magnetic rack and wait 2 minutes.
15. Carefully transfer 20 µl of the sample to a new 0.2 mL PCR tube without carry over of any beads.
Optional Quantication
If you wish to confirm that library yields have achieved the minimum threshold of 12 nM, perform a library
quantification (i.e., Qubit, BioAnalyzer or qPCR) before proceeding with Normalase I reaction.
Safe Stopping Point
For long term storage (i.e., > 2 weeks), libraries can be stored at -20 ˚C post-Normalase PCR purication. If
planning to sequence within the next two weeks, proceed to Normalase I.
11
SWIFT NORMALASE KIT
Normalase I
16. Pre-set a thermocycler program for 15 minutes at 30 °C with open lid or lid heating OFF.
17. Prepare the Normalase I Master Mix (listed in the table below). The mix can be prepared at room
temperature and stored on ice until use if prepared in advance. Ensure that it is thoroughly mixed by mod-
erate vortexing followed by a pulse spin to collect contents prior to use.
Per Library 24 Libraries 96 Libraries
Buffer Y1
4.3 µl 103.2 µl 412.8 µl
Reagent Y2
0.2 µl 4.8 µl 19.2 µl
Enzyme Y3
0.5 µl 12.0 µl 48.0 µl
Total Volume 5.0 μl 120.0 μl 480.0 μl
IMPORTANT!
The Normalase I Master Mix should be built for a minimum 10 reactions to ensure
pipetting accuracy.
18. Using a calibrated P10 pipette, carefully add 5 µl of Normalase I Master Mix into each 20 µl library eluate
at room temperature and thoroughly mix by moderate vortexing for 5 seconds.
19. Spin down the sample tube in a microfuge and place in the thermocycler and run the program.
Thermocycler Program
15 min at 30 ˚C with open lid or lid heating OFF
Safe Stopping Point
For long term storage (i.e., > 2 weeks), libraries can be stored at -20 ˚C post-Normalase I. If planning to se-
quence within the next two weeks, proceed to Library Pooling step.
12
SWIFT NORMALASE KIT
Library Pooling
This step can be repeated up to 5 times to enable various re-pooling combinations as only 5 µl of post-Norma-
lase I library (25 µl volume) is utilized for pooling, and sufcient reagents are provided. Also note that stability
of normalized pools is limited with a storage time of two weeks since the resulting normalized pools are single
stranded DNA libraries. Therefore, if re-sequencing is required after two weeks, for best results re-pool the
librar-ies and repeat Normalase II and inactivation.
Note: If you are pooling less than 5 libraries or post-hybridization capture library pools, see Appendix Section A
for low-plex pooling recommendations.
Note: If pooling 5 μl per sample does not generate a nal 4 nM pool of sufcient volume for instrument loading,
see Appendix Section B for high sample volume pooling recommendations.
IMPORTANT!
There is no minimum or maximum limit to the number of samples that can be placed
into a single pool. However, note the following recommendations:
• Consider your desired number of reads for each sample and only pool those samples
together that have the same required depth. For example, samples each requiring 10
million reads can be pooled together whereas samples requiring 100 million reads
should be combined in a separate pool. Thus, you can adjust your ratio of pools when
loading the instrument to achieve the desired sequence depth for each pool.
• Additionally, consider index compatibility as well as insert size. Pool libraries of
comparable insert sizes that can be co-sequenced together to avoid size dependent
clustering effects that are independent of molarity that can lead to higher variation in
sample representation in sequencing data. Also, do not pool libraries with index
combinations that have not been validated by the supplier or demultiplexing errors
and loss of data may result.
20. Following the Normalase I incubation, generate a library pool (or pools) by carefully placing 5 µl of each
individual library into a 0.2 mL PCR tube if pooling 30 libraries or less (achieves up to a final volume of 186
µl). Alternatively, use a 1.5 mL screw cap microfuge tube, particularly when pooling greater than 30 librar-
ies as the volume will exceed the PCR tube maximum volume.
As mentioned previously to ensure even pooling, use of a calibrated P10 pipette will produce the best
results.
21. Thoroughly mix, and spin the library pools in a microfuge, and proceed to Normalase II reaction with the
library pools created.
13
SWIFT NORMALASE KIT
Normalase II
22. Pre-set a thermocycler program for 15 minutes at 37 °C with open lid or lid heating OFF. Alternatively, if us-
ing a 1.5 mL screw cap microfuge tube, set a heat block at 37 °C.
23. Pre-mix Normalase II Master Mix (listed in the table below). The master mix can be stored on ice until use,
and then added to pools at room temperature.
Reagents* Per Library 48 Libraries 96 Libraries
Buffer G1
0.96 µl 48 µl 96 µl
Enzyme G2
0.04 µl 2 µl 4 µl
Total Volume 1.00 µl 50 µl 100 µl
*We recommend preparing Normalase II master mix for 48 samples even if you are processing less than 48 samples in order to avoid
pipetting extremely low volumes; recommended volumes have been rounded up for convenient pipetting. Although sufcient reagents are
supplied for up to 5 repeated Normalase II reactions per sample, repeatedly processing a lower number of samples will result in signicant
loss of Normalase II reagents.
24. Add 1 µl of Normalase II Master Mix multiplied by the total number of libraries within each prepared pool,
see examples below:
Reagents Per Library 24-Plex Pool 96-Plex Pool
Normalase II Master Mix 1 µl 24 µl 96 µl
25. Mix well by vortexing for 5 seconds, and spin down the library pools in a microfuge.
26. Place the library pools in the thermocycler and advance the program or place the 1.5 mL screw cap mi-
crofuge tubes into the 37˚ C heat block.
Thermocycler Program Heat Block (1.5 mL screw cap microfuge tube)
15 min at 37 ˚C with open lid or lid heating OFF 15 min at 37 ˚C
14
SWIFT NORMALASE KIT
Normalase Inactivation
27. Following the Normalase II reaction, pre-set a thermocycler program as listed below.
28. Add 0.2 μl of Reagent B1 multiplied by the total number of libraries within each prepared pool, see exam-
ples below:
Reagent Per Library 24-Plex Pool 96-Plex Pool
Reagent B1
0.2 µl 4.8 µl 19.2 µl
29. Place the library pools in the thermocycler and advance the program or place the 1.5 mL screw cap mi-
crofuge tubes into the heat block. If using a 1.5 mL screw cap microfuge tube, set a heat block at 95 °C to
incubate your library pools, being careful to not incubate the samples longer than 2 minutes.
Thermocycler Program Heat Block (1.5 mL screw cap microfuge tube)
Hold at 95 ºC
2 min at 95 ºC with lid kept at 95 ºC
Hold at 4 ºC
2 min at 95 ºC
30. Your nal library pools are at 4 nM and are ready for further pooling, dilution, and loading the instrument. It
is not necessary to perform an additional purication step.
Final pools contain single stranded DNA and can be stored at - 20 ºC for up to two weeks before sequencing.
For longer term storage, refer back to the safe stopping point following post-Normalase I, and perform the
pooling and subsequent steps as indicated above prior to sequencing.
Recommended Quality Control
To ensure optimal sequencing results we recommend that you perform a qPCR quantification on your final
Normalase pool(s). Final library pools are ssDNA and cannot be quantied by uorometric methods or Bio-
analyzer. If you have a validated qPCR assay that reproducibly predicts an optimal number of reads on your
sequencing instrument, concentration adjustment of your nal pool can be performed based on your qPCR
results. If you do not have a validated qPCR assay, we recommend a commercially available kit such as KAPA
Library Quantication Kit (Cat. No. KK4828). Follow the manufacturer’s protocol assuming a 4 nM concentra-
tion and conrm the accuracy of the assay and results before proceeding with concentration adjustment of the
nal pool(s) based on the qPCR results.
• If qPCR results indicate gross concentration deviation from 4 nM expected normalized pool concentration,
indicative of library failure or Normalase processing error, consult the Troubleshooting section of this protocol
(Appendix, Section C) or contact Swift technical support at TechSuppor[email protected] for assistance.
• If qPCR quantication indicated pools were below 4 nM, this may be due to a failure of a subset of samples
within a pool. If this is observed, quantify your Normalase I-treated libraries to identify which libraries have
failed or are < 12 nM.
• The Normalase product specication is dened by cluster density of the nal 4 nM Normalase pool when
loaded on a MiSeq v2 ow cell at 12 pM to achieve a 1000-1200 K/mm
2
cluster density and CV ≤ 10% within
a pool (Swift 2S Turbo Flexible libraries with an average 200 bp insert size). Across other Illumina platforms,
library types, and insert sizes, optimization of loading concentration may be required to achieve the optimal
number of reads supported by the ow cell of choice.
If you experience problems with your Swift Normalase Kit, please contact us at TechSuppor[email protected],
or by phone at 734.330.2568 (9:00 am-5:00 pm ET, Monday-Friday).
15
SWIFT NORMALASE KIT
Appendix
Section A: Low-plex Pooling Recommendations
Please use the following recommendations for pooling, Normalase II, and Normalase Inactivation if pooling
less than 5 libraries or post-hybridization capture library pools. These pooling recommendations are to avoid
pipetting errors from dispensing extremely small volumes.
Number of
Libraries or Pools
Volume Per
Library or Pool
Total Volume
Normalase II
MasterMix Volume
Reagent B1 Inactivation
Volume
1* 25.00 µl 25 µl
5 µl 1 µl
2 12.50 µl 25 µl
3 8.30 µl 25 µl
4 6.25 µl 25 µl
5 5.00 µl 25 µl
* Do not proceed with the above recommendations unless sequencing within the next two weeks as remaining library will not be
available to repeat the pooling and subsequent steps. Final libraries are single stranded and can be stored at –20 ºC for up to two
weeks before sequencing.
20. Following the Normalase I incubation, generate a library pool (or pools) by carefully placing the specied
amount of each individual library as shown in the table above into a 0.2 mL PCR tube. As mentioned previ-
ously, use of a calibrated pipette will produce best results with this pipetting step to ensure even pooling.
21. Thoroughly mix, and spin the library pools in a microfuge, and proceed to Normalase II reaction with the
library pools created.
Normalase II
22. Pre-set a thermocycler program for 15 minutes at 37 °C with open lid or lid heating OFF.
23. Pre-mix Normalase II Master Mix (listed in the table below). The master mix can be stored on ice until use,
and then added to pools at room temperature.
Reagents* Per Pool 5 Pools
Buffer G1
4.8 µl 24 µl
Enzyme G2
0.2 µl 1 µl
Total Volume 5.0 µl 25 µl
* We recommend constructing Normalase II master mix for 5 pools even if you are
processing less than 5 pools in order to avoid pipetting extremely low volumes.
Normalase Inactivation
24. Following the Normalase II reaction, pre-set a thermocycler program as listed below.
25. Add 1 μl of Reagent B1 to each pool.
Reagent Per Pool
Reagent B1
1 µl
16
SWIFT NORMALASE KIT
26. Place the library pools in the thermocycler and advance the program.
Thermocycler Program
Hold at 95 ºC
2 min at 95 ºC with lid kept at 95 ºC
Hold at 4 ºC
27. Your nal library pools are at 4 nM and are ready for further pooling, dilution, and loading the instrument. It
is not necessary to perform an additional purication step.
Final pools contain single stranded DNA and can be stored at - 20 ºC for up to two weeks before sequencing.
For longer term storage, refer back to the safe stopping point following post-Normalase PCR purication, and
perform the pooling and subsequent steps as indicated above prior to sequencing.
For Quality Control recommendations, please refer to page 14.
Section B: High sample volume pooling recommendations
Please use the following recommendations for Sample Pooling, Normalase II, and Normalase Inactivation
steps if you require a higher nal volume of 4 nM pool than the volume produced by the standard protocol that
uses 5 µl per sample. These pooling recommendations can enable you to sequence on NovaSeq or generate
higher volumes for loading a single pool over multiple lanes or ow cells.
For example, NovaSeq requires 100-310 μl loading volume at 1.5-3 nM concentration and if multiplexing a
lower number of samples, you may need to pool more than 5 μl of each sample to achieve a sufcient loading
volume at the required concentration. The table below outlines the exible options available to support a broad
range of nal 4 nM pool volumes when pooling 24 samples or less.
Per Sample pooling volume (μl) 5 7.5 10 15 20 25
Normalase II
Master Mix per
sample volume (μl)
G1 0.96 1.44 1.92 2.88 3.84 4.80
G2 0.04 0.06 0.08 0.12 0.16 0.20
Total 1.00 1.50 2.00 3.00 4.00 5.00
Inactivation step per
sample volume (μl)
B1 0.20 0.30 0.40 0.60 0.80 1.00
Final 4nM pool
volume (μl)
1 sample 6.20 9.30 12.40 18.60 24.80 31.00
6 samples 37.20 55.80 74.40 111.59 148.80 186.00
12 samples 74.40 111.60 148.80 223.19 297.60 372.00
18 samples 111.60 167.40 223.20 334.78 446.40 558.00
24 samples 148.80 223.20 297.60 446.37 595.20 744.00
Minimum number of reactions
recommended for Normalase II Master Mix
24 18 12 10 6 5
A. Alternate per sample pooling volumes. Be aware that higher pooling volumes will reduce the number of re-pooling iterations avail-
able with your remaining sample.
B. The per sample Normalase II master mix reagent volumes. Use these reagent quantities to prepare your Normalase II master mix.
C. The per sample Normalase Inactivation reagent volumes.
(Table legend continues on next page.)
A
B
C
D
E
17
SWIFT NORMALASE KIT
D. Final 4 nM volumes for pooling 1-24 samples over the range of individual sample volume options of 5-25 µl. These nal volumes
are the sum of the sample pool volume, Normalase II master mix volume and Inactivation volume. Final volumes in white cells can
be prepared in a 0.2 ml PCR tube and thermocycler, whereas nal volumes in gray cells must be prepared in a 1.5 ml screw cap mi-
crofuge tube and heat block to accommodate the larger volumes.
E. Minimum number of reactions recommended for Normalase II Master Mix assembly. We recommend preparing Normalase II mas-
ter mix for the minimum reaction number shown to avoid pipetting extremely low volumes. Keep in mind that repeatedly processing
fewer samples than the recommended master mix volume will result in signicant loss of Normalase II reagents.
20. Following the Normalase I incubation, generate a library pool (or pools) by carefully placing the specied
amount of each individual library as shown in the table above into a 0.2 mL PCR tube or a 1.5 mL screw
cap microfuge tube, depending on the nal desired volume.
As mentioned previously, use of a calibrated pipette will produce best results with this pipetting step to
ensure even pooling.
21. Thoroughly mix, and spin the library pools in a microfuge, and proceed to Normalase II reaction with the
library pools created.
Normalase II
22. Pre-set a thermocycler program for 15 minutes at 37 °C with open lid or lid heating OFF. Alternatively, if us-
ing a 1.5 mL screw cap microfuge tube, set a heat block at 37°C.
23. Pre-mix Normalase II Master Mix depending on the chosen pooling volume and minimum master mix reac-
tion volume (listed in the table below). The master mix can be stored on ice until use, and then added to
pools at room temperature.
Reagents 5 µl (24 rxn) 7.5 µl (18 rxn) 10 µl (12 rxn) 15 µl (10 rxn) 20 µl (6 rxn) 25 µl (5 rxn)
Buffer G1
23.04 µl 25.92 µl 23.04 µl 28.8 µl 23.04 µl 24.00 µl
Enzyme G1
0.96 µl 1.08 µl 0.96 µl 1.2 µl 0.96 µl 1.00 µl
Total Volume
24.00 µl
27.00 µl
24.00 µl 30.00 µl 24.00 µl 25.00 µl
24. Add the recommended volume of Normalase II Master Mix based on your per sample pooling volume mul-
tiplied by the total number of samples within each prepared pool, see volumes below:
Reagents 5 µl 7.5 µl 10 µl 15 µl 20 µl 25 µl
Normalase II Master
Mix Per Sample
1 µl 1.5 µl 2.00 µl 3.00 µl 4.00 µl 5.00 µl
25. Mix well by vortexing for 5 seconds, and spin down the library pools in a microfuge.
26. Place the library pools in the thermocycler and advance the program or place the 1.5 mL screw cap
microfuge tubes into the 37°C heat block.
Thermocycler Program Heat Block (1.5 mL screw cap microfuge tube)
15 min at 37 ºC with open lid or lid heating OFF 15 min at 37 ºC
18
SWIFT NORMALASE KIT
Normalase Inactivation
27. Following the Normalase II reaction, pre-set a thermocycler program as listed below.
28. Add the recommended volume of Reagent B1 based on your per sample pooling volume multiplied by the
total number of samples within each prepared pool, see volumes below:
Reagents 5 µl 7.5 µl 10 µl 15 µl 20 µl 25 µl
Reagent B1
Per Sample
0.20 µl 0.30 µl 0.40 µl 0.60 µl 0.80 µl 1.00 µl
29. Place the library pools in the thermocycler and advance the program or place the 1.5 mL screw cap mi-
crofuge tubes into the heat block. If using a 1.5 mL screw cap microfuge tube, set a heat block at 95°C to
incubate your library pools, being careful to not incubate the samples longer than 2 minutes.
Thermocycler Program Heat Block (1.5 mL screw cap microfuge tube)
Hold at 95 ºC
2 min at 95 ºC with lid kept at 95 ºC
Hold at 4 ºC
2 min at 95 ºC
30. Your nal library pools are at 4 nM and are ready for further pooling, dilution, and loading the instrument. It
is not necessary to perform an additional purication step.
Final pools contain single stranded DNA and can be stored at -20°C for up to two weeks before sequenc-
ing. For longer term storage, refer back to the safe stopping point following post-Normalase I, and perform
the pooling and subsequent steps as indicated above prior to sequencing.
For Quality Control recommendations, please refer to page 14.
19
SWIFT NORMALASE KIT
Section C: Helpful Information and Troubleshooting
Problem Possible Cause Suggested Remedy
Variation of read counts
(CV > 10%) between
libraries within a
Normalase pool
1. Inconsistent pipetting of
Normalase I Master Mix or
inconsistent pipetting of
individual libraries into a pool.
2. Artifact of flow cell loading
b
eing outside of Illumina’s
recommended range
1. Use a P10 pipettor if available and ensure
pipettes are maintained and calibrated
2. Adjust loading concentration accordingly
Some libraries were
signicantly under-
represented in the
sequence data
These libraries may not have met
the minimum threshold of 12 nM
in 20 µl library yield
1. Quantify affected libraries to determine if library
concentration was less than 12 nM
2. Review whether the number of PCR cycles was
appropriate for your sample quality and quantity
Recommended qPCR
quantication of pools
following Normalase II
indicated unexpected
yields
See page 14 See page 14
Recommended qPCR
quantication of pools
following Normalase
indicated no library
yield present
Library preparation, amplication,
or Normalase I failure
Contact TechSuppor[email protected] for specic
troubleshooting recommendations
Recommended qPCR
quantication of pools
following Normalase
indicated signicant
excess yield of 12 nM
or greater
Normalase II failure, selected 4
nM fraction was not enzymati-
cally normalized
Re-pool the post-Normalase I libraries and repeat
Normalase II and inactivation
Fluorometric methods
such as BioAnalyzer or
Qubit indicated no library
Normalase libraries are single
stranded and cannot be detected
by dsDNA interclating dyes
Perform qPCR quantication
Overall cluster density/
reads passing filter was
lower than expected
1. Normalase pools were stored
for greater than two weeks
2. Libraries were consistenly
less than 12 nM post-library
amplication
1. Re-pool the post-Normalase I libraries and repeat
Normalase II and inactivation
2. Review whether the number of PCR cycles was
appropriate for your sample quality and quantity
If you experience problems with your library prep, please contact us at TechSuppor[email protected], or by
phone at 734.330.2568 (9:00 am-5:00 pm ET, Monday-Friday).
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