MicroPlex Library
Preparation Kit v2
High Performance Library Preparation for Illumina® NGS Platforms

Cat. No. C05010012 (12 single indexes - 12 rxns)
C05010013 (12 single indexes - 48 rxns)
C05010014 (48 single indexes - 48 rxns)

USER GUIDE
Version 1

01_06_2018

Please read this manual carefully
before starting your experimentContents
Introduction

5

Kit method overview

6

Kit materials

8

Required materials not provided

10

Remarks before starting

12

Manual processing

16

Protocol for library preparation (manual version)16

Library processing (manual version)

26

Option A: Library purification using AMPure® XP beads

27

Option B: Library size selection using AMPure XP beads

28

Automated processing

31	Protocol for library preparation using IP-Star® Compact AutomatedSystem32

	Library processing using IP-Star Compact Automated System

42

Option A: Library purification using AMPure® XP beads

43

Option B: Library size selection using AMPure XP beads

45

Appendix: Indexing reagents

49

Single index sequences, multiplexing and index pooling

50

FAQs

55

Related products

57

Technical support

58

3

4

MICROPLEX LIBRARY PREPARATION KIT MANUALIntroduction
The MicroPlex Library Preparation Kit v2 contains all necessary reagentsincluding indexes for multiplexing up to 48 samples using single barcoding  
on llumina NGS platforms. Generated libraries are compatible withsingle-end or paired-end sequencing.  MicroPlex chemistry is specificallydeveloped and optimized to generate DNA libraries with high molecularcomplexity from the lowest input amounts. Only 50 pg to 50 ng offragmented double-stranded DNA is required for library preparation. The
entire three-step workflow takes place in a single tube or well in about
2 hours. No intermediate purification steps and no sample transfers arenecessary to prevent handling errors and loss of valuable samples. The
kit provides excellent results for• DNA-seq (high-coverage, deep sequencing: de novo sequencing,whole genome resequencing, whole exome sequencing, cell-freeplasma DNA sequencing)• Enrichment techniques: ChIP-seq, MethylCap-seq• RNA-seq (cDNA)

The kit MicroPlex Library Preparation Kit v2 can be used manually orwith automation using the IP-Star Compact Automated System – thecorresponding protocols are included in this manual.

5

Kit method overview
The MicroPlex Library Preparation Kit v2 is based on patented MicroPlex
technology (Figure 1). Unlike other NGS library preparation kits, which
are based on ligation of Y-adapters, MicroPlex uses stem-loop adapters
to construct high quality libraries with a fast and efficient workflow:

5 minutes*

STEP 1 Template preparation
Template:
Fragmented
ds DNA/c DNA

DNA
Repair5 minutes*

STEP 2 Library synthesis
Stem-loop
Adaptor
3’

Additionof Adaptors

5’

5’

3’
Blunt-end Ligation

Cleavable
Replication
Stop

Nick

Cleavable
Replication
Stop
Stem-loop
Adaptor5’

5’ Nick

5 minutes*

STEP 3 Library amplification

Re-amplification

Extension and Cleavage

Primer A

High Fidelity Amplification
BarcodePrimer B* hands-on-time

Library processing
Purification or size selection
Quantification and pooling

Ready for sequencing

Figure 1. MicroPlex technology.
6

MICROPLEX LIBRARY PREPARATION KIT MANUAL

Optional
Intermediate Library
Quantification

Step 1. Template Preparation provides efficient repair of the fragmented
double-stranded DNA input.
In this step, the DNA is repaired and yields molecules with blunt ends.
Step 2. Library Synthesis enables ligation of MicroPlex patented stemloop adapters.
In the next step, stem-loop adaptors with blocked 5’ ends are ligated withhigh efficiency to the 5’ end of the genomic DNA, leaving a nick at the 3’
end. The adaptors cannot ligate to each other and do not have singlestrand tails, both of which contribute to non-specific background foundwith many other NGS preparations.
Step 3. Library Amplification enables extension of the template, cleavageof the stem-loop adaptors, and amplification of the library. Illuminacompatible indexes are also introduced using a high-fidelity, highlyprocessive, low-bias DNA polymerase.
In the final step, the 3’ ends of the genomic DNA are extended to complete
library synthesis and Illumina-compatible indexes are added through ahigh-fidelity amplification. Any remaining free adaptors are destroyed.
Hands-on time and the risk of contamination are minimized by using asingle tube and eliminating intermediate purifications.
Obtained libraries are purified, quantified and sized. The libraries poolingcan be performed as well before sequencing (Figure 1).

7

Kit materials
The MicroPlex Library Preparation Kit v2 contains sufficient reagentsto prepare up to the specified number of reactions. Enough buffers andenzymes are provided for 4 uses or freeze-thaw cycles. Contents of theMicroPlex Library Preparation Kit v2 are not interchangeable with other
Diagenode products.
Table 1. MicroPlex Library Preparation kit v2
Cap colourµl/rxn12 single
indexes*(12 rxns)

12 single
indexes**(48 rxns)

48 single
indexes***(48 rxns)

Template Preparation
Buffer

Red

2

24 µl

96 µl

96 µl

Template Preparation
Enzyme

Red

1

12 µl

48 µl

48 µl

Library Synthesis Buffer

Yellow

1

12 µl

48 µl

48 µl

Library Synthesis Enzyme

Yellow

1

12 µl

48 µl

48 µl

Library Amplification
BufferGreen

25

300 µl1200 µl1200 µl

Library Amplification
Enzyme

Green

1

12 µl

48 µl

48 µl

Nuclease-Free Water

Clear

4

48 µl

192 µl192 µlIndexing Reagents Tube

Blue

5

40 µl

120 µl/Indexing Reagents Plate

/

5

/

/

5 µl***Name

8

MICROPLEX LIBRARY PREPARATION KIT MANUALSHIPPING AND STORAGE: The MicroPlex Library Preparation Kit v2 isshipped on dry ice. The kit should be stored at −20°C upon arrival.
The volumes of buffers and enzymes mentioned above are the minimum
volumes required to complete 12 or 48 reactions. However, an excess is
included in each tube to cover pipetting loss.
* Indexing reagents from MicroPlex   Kit v.2 (12 single indexes (12 rxns)) are
pre-dispensed in 12 individual tubes  with blue caps. Each tube contains sufficientvolume for up to 8 uses. No more than 4 freeze/thaw cycles are recommended
for the Indexing Reagent Tubes.
** Indexing reagents from MicroPlex Kit v.2 (12 single indexes (48 rxns)) are predispensed in 12 individual tubes with blue caps. Each tube contains sufficientvolume for up to 24 uses. No more than 4 freeze/thaw cycles are recommended
for the Indexing Reagent Tubes.
*** Indexing reagents from MicroPlex Kit v.2 (48 single indexes (48 rxns)) are
pre-dispensed and sealed in a linear barcoded index plate. The Indexing Reagent
Plate is sealed with foil that can be pierced with a multichannel pipet tip tocollect the required amount of index to assemble the reactions. Each well of theindex plate contains sufficient volume for a single use. No more than 4 freeze/
thaw cycles are recommended for the Indexing Reagent Plate.

9Required materials not provided• Low binding aerosol barrier tips
• Freshly prepared 80% (v/v) ethanol• Agencourt® AMPure® XP beads (Beckman Coulter, Cat. No. A63880)• Magnetic rack:––Compatible with 0.2 ml PCR tubes if working with tubes or
strips (eg. Diagenode DiaMag 0.2ml , Cat. No. B04000001)––Alternatively: 96 well plate magnetic rack – if working with PCR
plates• Low TE buffer pH 8.0 (10 mM Tris, 0.1 mM EDTA, pH 8.0),
molecular grade• Centrifuge• Thermal cycler
NOTE: Use a thermal cycler equipped with a heated lid that can handle 50 µl
reaction volumes. Set the temperature of the heated lid to 101°C – 105°C toavoid sample evaporation during incubation and cycling. We recommend a ramp
rate of 3°C/s – 5°C/s; higher ramp rates are not recommended and could impact
the quality of the library.

• 0.2 ml PCR tubes or 96-well PCR plates and seals
NOTE: Select appropriate tubes or plates that are compatible with the thermal
cyclers used. Use appropriate caps or sealing films and seal thoroughly toeliminate evaporation during cycling conditions. Evaporation could reducerobustness and reproducibility of the reactions.

• BioAnalyzer® (Agilent) or Fragment Analyzer® (Advanced
Analytical) (for library quantification and sizing)• High sensitivity DNA kit from Agilent or High Sensitivity NGS
Fragment Analysis kit (35 bp-6000 bp) from Advanced Analytical• Qubit 2.0 Fluorometer® (Life Technologies) or QuantiT™PicoGreen® dsDNA Assay Kit (Life Technologies) (for libraryquantification). Optional: KAPA® Library Quantification Kit –Illumina (Kapa Biosystems), specific to a real time PCR systemused) and real time PCR system can be also used.
Additional supplies if working with IP-Star Compact:
10

MICROPLEX LIBRARY PREPARATION KIT MANUAL• IP-Star Compact Automated System (Diagenode, Cat. No.
B03000002)• 200 μl tube strips (8 tubes/strip) + cap strips (Diagenode, Cat. No.
C30020002)• Tips (box) (Diagenode, Cat. No. C30040021)• Tips (bulk) (Diagenode, Cat. No. C30040020)• 2 ml microtube (Diagenode, Cat. No. C30010014)• Medium reagent container (Diagenode, Cat. No. C30020003)• 96 well microplates (Diagenode, Cat. No. C30080030)
Quality Control
The MicroPlex Library Preparation Kit v2 is functionally tested using Next
Generation Sequencing (NGS) to ensure product quality and consistency.
Safety Information
Follow standard laboratory safety procedures and wear a suitable lab coat,protective goggles and disposable gloves to ensure personal safety as wellas to limit potential cross contamination during the sample preparationand subsequent amplification reactions.

11Remarks before startingDNA format
Fragmented double-stranded DNA (gDNA or cDNA), chromatin
immunoprecipitates (ChIP), degraded DNA from sources such as FFPE,
plasma, or other biofluids are suitable. This kit is not for use withsingle-stranded DNA (ssDNA) or RNA.
Input DNA amount
Input DNA in the range of 50 pg to 50 ng can be used as starting material.
For deep whole genome sequencing (WGS) and whole exome sequencing(WES) using human gDNA, FFPE, or plasma DNA, greater than 10 ng of input
DNA is recommended to achieve a highly diverse library. For sequencing
samples with reduced complexity, such as cDNA, immunoprecipitated
DNA from ChIP, bacterial DNA, or targeted genomic regions, lower input
amounts (picogram levels) can be used.
Fragment Size
The optimal DNA fragment size is less than 1,000 bp. The MicroPlex
Library Preparation Kit v2 is a ligation-based technology and adapters
added during the process result in an approximately 140 bp increase in
the size of each DNA template fragment. Library molecules with shorter
inserts (200 – 300 bp) tend to cluster and amplify more efficiently on the
Illumina flow cell. Depending on the application and requirements, the
AMPure purification step following the final step (library amplification)
can be replaced with AMPure size-selection step to remove unwanted
large fragments.
Input Volume
The maximum input sample volume is 10 µl. If a sample is in a larger
volume, the DNA must be concentrated into 10 µl or less. Alternatively,
the sample may be split into 10 µl aliquots, processed in separate tubes,
and the corresponding products pooled prior to the purification step
preceding sequencing.
12

MICROPLEX LIBRARY PREPARATION KIT MANUAL

Input Buffer
Input DNA must be eluted or resuspended in a low-salt and low-EDTA
buffered solution. The preferred buffer is low TE (10 mM Tris, 0.1 mM
EDTA, pH 8.0). The concentrations of Tris and EDTA must not exceed 10
mM and 0.1 mM, respectively. Avoid phosphate containing buffers.
Positive and Negative Controls
If necessary, include appropriate positive and negative controls in theexperimental design to help verify that reactions proceed as expected.
If the experimental samples contain any carryover contaminant(s) in thebuffer, the downstream reactions may be impacted, and inclusion ofcontrols would help elucidate such problems. A suitable positive control(reference DNA) is Bioruptor-sheared purified genomic DNA (200 – 300 bp)
of comparable input amount. Always prepare fresh dilutions of referenceDNA. Include a negative control (No Template Control, NTC) with low TE
buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) or nuclease-free water. The
positive control and experimental samples should perform equivalently,
while the NTC should not amplify.
Preparation of Master Mixes
A master mix with appropriate buffers and enzymes must be prepared ateach workflow step based on the number of reactions to be performed.
Transfer the enzymes to ice just prior to use and centrifuge briefly tocollect contents at the bottom of the tube. Thaw the buffers, vortex briefly
and centrifuge prior to use. Keep all the components and master mixes
on ice. Once the master mix is prepared, thoroughly mix the contents
several times with a pipette while avoiding introduction of excessive air
bubbles and briefly centrifuge prior to dispensing into the PCR plate or
tube(s).
Indexing ReagentsThe Indexing Reagents consist of amplification primers containing
Illumina-compatible   indexes. Index sequences can be found in the
Appendix or downloaded as CSV files at the MicroPlex product page, under
the Documents tab.

13

For information on index sequences, index plate handling instructions,multiplexing and index pooling guidelines refer also to the Appendix.
Library yield and selection of the optimal number of cycles for library
amplification
The requirements for a final library concentration depend on a sequencer
and may vary between different sequencing service providers. The usual
range is between 5-20 nM in a final volume 10-15 µl but we recommend
inquiring with your sequencing platform.
The number of PCR cycles required at Step 3 of the protocol (Library
Amplification) is dependent on the amount of input DNA, quality,
fragmentation size, and a thermal cycler.  Note that an over-amplification
could result in higher rate of PCR duplicates in the library.
When working with DNA sample of known quantity or quality, use the
Amplification Guide for selecting the number of PCR cycles.
When working with DNA samples of unknown quantity and/or quality (suchas DNA from FFPE tissue or environmental sample, low input samples
below quantification limit etc.), amplify samples for 12 cycles and perform
an intermediate quantification of unpurified libraries as described at
OPTIONAL INTERMEDIATE LIBRARY QUANTIFICATION STEP. Dependingon the yield, libraries can be re-amplified for few additional cycles toachieve a desired yield or purified.
Library purification or size selection
Purification using Agencourt AMPure® XP (Beckman Coulter) is thepreferred method because sequence complexity is conserved. Do not use
silica-based filters for purification. The optimal library size is dictated by
the sequencing application and a sequencer specification. The final librarysize corresponds to the initial size of DNA fragments plus approximately
140 bp due to the ligated adapters. Most part of applications will not requirea size selection if the initial DNA fragment size is less than 1,000 bp. In
this case, amplified products should be purified by Agencourt AMPure
XP (Beckman Coulter) to get rid of primers and adaptors (See section“Library processing, option A”). If the size selection is required, follow the14

MICROPLEX LIBRARY PREPARATION KIT MANUAL

protocol described in the section “Library processing, option B” to get rid
of primers/adaptor and fragments above 600 bp.
Library Quantification
There are several approaches available for library quantification including
real-time PCR, UV absorption, fluorescence detection, or sizing andquantification using the Agilent Bioanalyzer or Fragment Analyzer(Advanced Analytical). It is important to understand the benefits andlimitations of each approach.
Real-time PCR-based approaches (such as the KAPA Library Quantification
Kit from Kapa Biosystems) quantify the library molecules that carry the
Illumina adapter sequences on both ends and, therefore reflect thequantity of the clustering competent library molecules. This approachassumes a relatively uniform size of sheared or fragmented starting gDNA
inserts used for library construction. Quantification by PCR can be doneon unpurified libraries.
The Agilent Bioanalyzer system or Fragment Analyzer (Advanced
Analytical) provide sizing and quantification information about the library
analysed, but not about the clustering competency. Quantification canbe done both on unpurified or purified samples. In a case of unpurified
samples, a region corresponding to libraries should be limited in order todiscriminate between primers/adaptors and a library itself.
UV absorption/fluorescence detection-based methods (i.e., Nanodrop®
(Thermo Scientific), Qubit®2.0 Fluorometer (Life Technologies), or QuantiT™ PicoGreen® dsDNA Assay Kit (Life Technologies) simply quantify total
nucleic acid concentration. These methods do not discriminate adapter
presence and offer no information about the size of the library molecules.
They can be used only on purified libraries. We better recommend
fluorescence-based assays than spectrophotometric measurements (e.g.
NanoDrop) due to higher specificity and sensitivity.

15

PR OTOCO LMANUAL PROCESSING
PROTOCOL FOR LIBRARY PREPARATIONTemplate Preparation

PROTOCOL

STEP 11.1. Dispense 10 µl of fragmented doubled-stranded DNA into each PCR
tube or well of a PCR plate compatible with your thermal cycler.
NOTE: Positive control reactions using reference DNA: If necessary, assemble
reactions using 10 µl of a reference gDNA (e.g., Bioruptor-fragmented DNA,
200-300 bp average size) at an input amount comparable to that of the samples.
NOTE: Negative control reactions (NTCs): If necessary, assemble NTCs with
10 µl of nuclease-free water or TE buffer (e.g., 10 mM Tris, 0.1 mM EDTA, pH8.0).

1.2. Prepare Template Preparation Master Mix as described in the tablebelow for the desired number of reactions. Mix thoroughly with apipette. Keep on ice until  used.
Component

Volume for 1 sample

Template Preparation Buffer (red cap)

2.0 µlTemplate preparation Enzyme (red cap)

1.0 µl

1.3. Add 3 µl of the Template Preparation Master Mix to each 10 µl
sample from step 1.1 above. Mix thoroughly with a pipette.
1.4. Tightly cap the tube(s) or seal the PCR plate using an appropriate
sealing film.
1.5. Centrifuge briefly to ensure the entire volume of the reaction iscollected at the bottom of each tube or well.
1.6. Place the tube(s) or plate in a thermal cycler with heated lid set to
101°C – 105°C. Perform the Template Preparation Reaction using
the following settings:
17

Template Preparation Reaction
Temperature

Time

22°C

25 min55°C

20 min4°C

Hold for � 2 hours

1.7. After the thermal cycler reaches 4°C, remove the plate or tube(s)and centrifuge briefly.
1.8. Continue to Library Synthesis Step in the same tube(s) or plate.

18

MICROPLEX LIBRARY PREPARATION KIT MANUALLibrary Synthesis

PROTOCOL

STEP 22.1. Prepare Library Synthesis Master Mix as described in the tablebelow for the desired number of reactions. Mix thoroughly with apipette.  Keep on ice until used.
Component

Volume for 1 sample

Library Synthesis Buffer (yellow cap)

1.0 µlLibrary Synthesis Enzyme (yellow cap)

1.0 µl

2.2. Add 2 µl of the Library Synthesis Master Mix to each tube or well.
Mix thoroughly with a pipette.
2.3. Tightly cap the tube(s) or seal the PCR plate using an appropriate
sealing film.
2.4. Centrifuge briefly to collect the contents to the bottom of each wellor tube.
2.5. Return the tube(s) or plate to the thermal cycler with heated lid set
to 101°C – 105°C.
2.6. Perform Library Synthesis Reaction using the following settings:
Library Synthesis Reaction
Temperature

Time

22°C

40 min4°C

Hold for � 30 min

19

2.7. After the thermal cycler reaches 4°C, remove the tube(s) or plateand centrifuge briefly.
2.8. Continue to the Library Amplification Step in the same tube(s) orplate maintained at 4°C.

20

MICROPLEX LIBRARY PREPARATION KIT MANUALLibrary Amplification

PROTOCOL

STEP 3
NOTE: Refer to Appendix for selecting the appropriate indexes to use for yourexperiments.

3.1. Prepare the Indexing Reagents as follows:• Remove the Indexing Reagents from freezer and thaw for 10
minutes on the bench top.
• Spin the Indexing Reagents in a table top centrifuge to collectcontents at the bottom of the well.
• Thoroughly wipe the Indexing Reagent Tubes or Indexing Reagent
Plate foil seal with 70% ethanol and allow it to dry.
3.2. Prepare the Library Amplification Master Mix as described in thetable below for the desired number of reactions. Mix thoroughly witha pipette. Keep on ice until use.
Component

Volume for 1 sample

Library Amplification Buffer (green cap)

25.0 µl

Library Amplification Enzyme (green cap)

1.0 µlNuclease-Free Water (clear cap)

4.0 µl3.3. A dd 30 µl of the Library Amplification Master Mix to each tube orwell containing 15 µl of Library Synthesis Reaction Product.
3.4. Add 5 µl of the appropriate Indexing Reagent to each well or tube.

21

NOTE: if using indexing reagents from MicroPlex Kit v.2 (48 single indexes(48 rxns)):

Make sure the two corner notches of the Indexing Reagent Plate
are on the left and the barcode label on the long side of the Index
Plate is facing you.
• Use a clean pipet tip to pierce the seal above the specificindexing reagent on the Indexing Reagent Plate; discard thetip used for piercing.
• Use a clean pipet tip to collect 5 µl of the indexing reagent
and add to the reaction mixture.
• Follow the Indexing Reagent Plate handling instructions in
the Appendix to avoid cross contamination.
3.5. Mix thoroughly with a pipette. Avoid introducing excessive air bubbles.
3.6. S eal the PCR plate or tube(s) tightly and centrifuge briefly to collectthe contents to the bottom of each well or tube.
3.7. R eturn the tube(s) or plate to thermal cycler with heated lid set to
101°C – 105°C.
3.8. P erform Library Amplification Reaction using the cycling conditionsin the tables below.
Caution: Ensure that the thermal cycler does not have a denaturing step
programmed until Stage 3.

22

MICROPLEX LIBRARY PREPARATION KIT MANUAL

Denaturation

Addition of Indexes

Temperature

Time

Number of Cycles

1

72°C

3 min

1

2

85°C

2 min

1

3

98°C

2 min

1

98°C

20 s

67°C

20 s

72°C

40 s

98°C

20 s

72°C*

50 s

4°C

Hold

4

Library Amplification

5

Hold

6

PROTOCOL

Extension & Cleavage

Stage

4

5 to16*1* If working with DNA samples of known quantity or quality, please use the
Amplification Guide below to select the required number of amplification
cycles.
Amplification guide (for stage 5)
DNA Input (ng)Number of Cycles

50

5

20

6

10

7

5

8

2

10

1

11

0.2

14

0.05

16

If working with DNA samples of unknown quantity and/or low quality (eg.  
FFPE extracted DNA ), amplify for 12 cycles and perform an INTERMEDIATELIBRARY QUANTIFICATION OF UNPURIFIED LIBRARY to estimate thelibrary yield . If the desired yield is not achieved, the libraries can be reamplified for few additional cycles.

3.9. R emove the tube(s) or PCR plate from the thermal cycler andcentrifuge briefly to collect the contents to the bottom of each well.

23

3.10. Proceed to the Library Purification by Agencourt AMPure XP in thesection “Library processing” (recommended for samples of known
quantity or quality) or to the Intermediate Library Quantification
option shown below (recommended for samples with unknown
quantity and/or low quality).
Optional Intermediate Library Quantification(recommended for samples with unknown quantity and/or a low quality)
Quantify unpurified libraries using the BioAnalyzer, the Fragment Analyzer
or similar devices. This intermediate quantification allows estimating thelibrary yield and enabling an additional re-amplification if needed. You willneed a High Sensitivity NGS Fragment Analysis kit (1 bp-6,000 bp) from
Advanced Analytical if using Fragment Analyzer or High Sensitivity DNA kit
from Agilent if using BioAnalyzer. Follow the manufacturer’s instruction
for kit handling and protocol.
NOTE: The quantification of unpurified library by qPCR (e.g. using KAPA® Library
Quantification Kit – Illumina (Kapa Biosystems), is also possible at this step
while it will increase the total protocol duration.

24

1.

Take an aliquot of unpurified libraries: 1 µl if the BioAnalyzer will be
used or 2 µl if the Fragment Analyzer will be used. Keep remaining
libraries at 4°C .

2.

Load the aliquot of unpurified library on the Fragment Analyzer or
BioAnalyzer and run the analysis.

3.

Estimate the library yield using a region selection option todiscriminate between unincorporated primers/adaptors and libraries
themself as it is shown in the Figure 2 below.

MICROPLEX LIBRARY PREPARATION KIT MANUALPROTOCOL
Figure 2. BioAnalyzer trace of unpurified library. A region
corresponding to the amplified library is selected (blue bars) toestimate the yield.
4.

If the library has a sufficient yield, proceed to the Library Purificationor size selection step in the section: Library processing. If the libraryhas a low yield, the remaining library can be further re-amplified for
2 to 3 additional cycles to achieve the desired yield. Spin down the
tube or plate containing the library and return the plate or tube(s) toa thermal cycler.

5.

Use the following cycling settings:
Number of cycles
2-3 cycles
1 cycleTemperature

Time

98°C

20 s

72°C

50 s

4°C

hold

NOTE: Higher yields may come at the expense of reduced sequencing quality,
therefore it is important to avoid an over-amplification.
NOTE: MicroPlex libraries can be further amplified with no extra reagents added
after storage at 4°C for up to 6 hours or -20°C for up to 7 days.

25

PR OTOCO LMANUAL PROCESSING
LIBRARY PROCESSING

Most part of applications will not require a size selection if an initial
DNA fragments size is less than 1,000 bp. Amplified products should be
purified by Agencourt AMPure  XP beads (Beckman Coulter) as describedat Option A. Mixing the sample and AMPure XP beads at a 1:1 ratio is
critical to get rid of primers and adaptors.

PROTOCOL

Library Purification or Size Selection

If your application requires a size-selection, please refer to the Option B.
You will need
• Freshly prepared 80% (v/v) ethanol• DiaMag 0.2ml magnetic rack compatible with 0.2 ml tubes(Diagenode, Cat. No. B04000001) or 96 well plate magnetic rack
• Low TE buffer pH 8.0

Option A: Library Purification using AMPure® XP beads
1. Carefully resuspend the AMPure XP beads by shaking and lightvortexing until no pellet is visible at the bottom of the container.
2. Precisely estimate a library volume and add an equal volume of
AMPure XP beads to get a final 1:1 volume ratio. Mix by pipette 8 – 10
times until the mixture is homogeneous.
3. Incubate at room temperature for 5 minutes.
4. Place the tube (or the plate) on a magnetic rack. Wait until the beads
are completely bound to the magnet (~ 2 minutes).
5. Carefully aspirate by pipette and discard the supernatant without
disturbing the pellet.
6. Wash the beads pellet 2 times as follows:• With the tubes on the magnet, add 150 µl of 80% ethanol without
27

disturbing the bead pellet and wait for 30 seconds.
• Carefully aspirate by pipette and discard the supernatant without
disturbing the pellet.
• Perform a brief low speed spin (~2000 g) to collect all dropletsremaining on the tube walls.
Repeat the wash as described above 1 time and proceed to thenext step.
7. Leaving the tube cap open, let dry the beads on the magnet for 5 minutes.
8. Elute DNA by resuspending the beads in 20 µl of 1x Low TE buffer, pH 8.0.
9. Place the tube (or a plate) on a magnetic rack. Wait until the beads arecompletely bound to the magnet (~ 2 minutes).
10. Without disturbing the pellet, carefully aspirate and transfer thesupernatant containing purified libraries to a new tube.
11. Quantify purified libraries using the method of your choice (for more
information refer to Remarks before starting).

Option B: Library size selection using AMPure XP beads
1. Carefully resuspend the AMPure XP beads by shaking and lightvortexing until no pellet is visible at the bottom of the container.
2. Precisely estimate   library volume and adjust it to 50 µl using
nuclease-free water.
3. Add 32.5 µl of AMPure XP beads to each sample (to get a final volume
ratio of AMPure beads: library of  0.65). Mix by pipette 8 – 10 times
until the mixture is homogeneous.
4. Incubate at room temperature for 10 minutes.
5. Place the tube (or a plate) on a magnetic rack. Wait until the beads
are completely bound to the magnet (~2 minutes). Transfer the28

MICROPLEX LIBRARY PREPARATION KIT MANUAL

6. Add to the supernatant 12.5 µl of room temperature AMPureXP beads
each. Mix by pipette 8 – 10 times until the mixture is homogeneous.
7. Incubate at room temperature for 10 minutes.

PROTOCOL

supernatant to new tube for further processing.

8. Place the tube (or a plate) on a magnetic rack. Wait until the beads
are completely bound to the magnet (~2 minutes). Carefully aspirate
by pipette and discard the supernatant without disturbing the pellet.
9. Wash the beads pellet 2 x times as follows:• With the tubes on the magnet, add 150 µl of 80% ethanol without
disturbing the bead pellet and wait for 30 seconds.
• Carefully aspirate by pipette and discard the supernatant without
disturbing the pellet.
• Perform a brief low speed spin (~2000 g) to collect all dropletsremaining on the tube walls.
Repeat the wash as described above 1 time and proceed to thenext step.
10. Leaving the tube cap open, let dry the beads on the magnet for
5 minutes.
11. Elute DNA by resuspending the beads in 15 µl of 1x Low TE buffer, pH 8.0.
12. Place the tube (or a plate) on a magnetic rack. Wait until the beads arecompletely bound to the magnet (~2 minutes).
13. Without disturbing the pellet, carefully aspirate and transfer thesupernatant containing purified libraries to a new tube.
14. Quantify purified libraries using the method of your choice (for more
information refer to Remarks before starting).
Individual libraries, quantified and purified according to the above protocol,
can be pooled at desired molar ratios to allow multiplex sequencing.
29

Libraries that are being pooled must have been prepared with different
indexes (for multiplexing and index pooling guidelines refer to Appendix).  
The minimal molar concentration needed to sequence the pool depends
on the requirements of the sequencing platform. The total molarity is thesum of all the individual libraries’ molarities in the final volume, e.g. if you
add 5 μl of a 10 nM library to 5 μl of a 20 nM library, you have 10 μl of a
15 nM pool. If libraries are prepared from similar input amounts, they canbe pooled by combining equal volume aliquots of each library.

30

MICROPLEX LIBRARY PREPARATION KIT MANUALPROTOCO L
AUTOMATED PROCESSING
PROTOCOL FOR LIBRARY PREPARATION

Protocol for library preparation using
IP-Star Compact Automated System
The MicroPlex Library Preparation Kit v2 has been validated on IP-Star
Compact Automated System (Diagenode, Cat. No. B03000002). The below
protocol provides flexibility to prepare 1 to 48 libraries in one run startingwith 50 pg-50 ng of DNA. The whole protocol of library preparation takes
approximately 1h 30 minutes. It allows you to prepare up to 96 libraries
per day with 2 runs. At the end, you will recover ligated products readyfor amplification. Amplified libraries can be further processed on IP-Star
Compact - for libraries purification or size selection.
LIBRARY PREPARATIONLIBRARY PROCESSING

AUTOMATED
Template
preparation

AUTOMATED
Library
synthesis

Library
amplification

Clean-up or
Size selection

ChIP DNA
Sheared DNA
Plasma DNA

Figure 3. Microplex Library preparation workflow using IP-Star Compact
Automated System.

32

MICROPLEX LIBRARY PREPARATION KIT MANUALAUTO PROTOCOL

IP-Star setup
1.1. Switch ON the IP-Star Compact.
1.2. Select “Protocols” icon and then “Library prep” category.
Top menu

Protocols

Sample prep.

Protocols

Maintenance

MeDIP

hMeDIP

MethylCap

Information
ChIP

ChIPmentation MagBisulfite

IPure

Library prep.

RNA IP

1.3. Select “MicroPlex_Library_Preparation” protocol:
1.4. Setup the exact number of samples that you want to process bypressing the black box.

If you plan to run between

1 and 8 samples> “MicroPlex_Library_Preparation_08”

9 and 16 samples> “MicroPlex_Library_Preparation_16”

17 and 24 samples> “MicroPlex_Library_Preparation_24”

25 and 32 samples> “MicroPlex_Library_Preparation_32”

33 and 40 samples> “MicroPlex_Library_Preparation_40”

41 and 48 samples> “MicroPlex_Library_Preparation_48”

NOTE: The Peltier Block 1 is now cooling down to 4°C to keep the enzymes andreagents cold.

1.5. Setup all the plastics on the platform according to the screen layout(press the relevant module to see detailed information).

33

96 plate 1

7 8 9 10 11 12

Reagent
Rack 2

Peltier
Block 1

12
11
10
9
8
7
6
5
4
3
2
1

3 4 5 6

Reagent
Rack 17 8 9 10 11 12

TIP Rack

3 4 5 6

TIP Rack

12
11
10
9
8
7
6
5
4
3
2
1

Peltier
Block 296 plate 2

1.6. Fill TIP Rack 1 (and 2 if processing more than 8 samples) with tips
according to the screen.
1.7. Fill Peltier Blocks 1 and 2 with 200 μl tube strips according to thescreen.

34

MICROPLEX LIBRARY PREPARATION KIT MANUALNOTE: Allow the reagent from MicroPlex Library Preparation kit v2 to come at
4°C. Work on ice from this point.

AUTO PROTOCOL

Reagents &Samples setup
2.1. Prepare the following mixes.
Template Preparation pre-mix:
Number of samples
1

8

16

24

32

40

48

Template Preparation
Buffer (red cap)

2 µl

16 µl

32 µl

48 µl

64 µl

80 µl

96 µl

Template Preparation
Enzyme (red cap)

1 µl

8 µl

16 µl

24 µl

32 µl

40 µl

48 µl

TOTAL

3 µl

24 µl

48 µl

72 µl

96 µl

120 µl144 µl

NOTE: 10 µl of DNA will be added later for each sample.

Library Synthesis pre-mix:
Number of samples
1

8

16

24

32

40

48

Library Synthesis Buffer(yellow cap)

1 µl

8 µl

16 µl

24 µl

32 µl

40 µl

48 µl

Library Synthesis Enzyme(yellow cap)

1 µl

8 µl

16 µl

24 µl

32 µl

40 µl

48 µl

TOTAL

2 µl

16 µl

32 µl

48 µl

64 µl

80 µl

96 µl

35

Library Amplification pre-mix:
Number of samples
1

8

16

24

32

40

48

Nuclease-Free Water(clear cap)

4 µl

32 µl

64 µl

96 µl

128 µl160 µl192 µlLibrary Amplification
Buffer (green cap)

25 µl

200 µl400 µl600 µl800 µl1,000 µl 1,200 µl

Library Amplification
Enzyme (green cap)

1 µl

8 µl

16 µl

24 µl

32 µl

40 µl

48 µl

TOTAL

30 µl

240 µl

480 µl

720 µl960 µl

1,200 µl

1,440 µl2.2. Fill the Peltier Block 1 with Library Synthesis pre-mix and Library
Amplification pre-mix according to the screen layout.
2.3. Fill the Peltier Block 2 with the Template Preparation pre-mix
according to the screen layout.
Peltier Block 1
12
11
10
9
8
7
6
5
4
3
2
1

4.

Peltier Block 2
Strip tubes
Well 7-12
Library Amplification pre-mix
Nuclease Free Water•Library Amplification Buffer•Library Amplification Enzyme
Well 1-6
Library Synthesis pre-mix•Library Synthesis Buffer•Library Synthesis Enzyme

30 µl
4 µl
25 µl
1 µl

2 µl
1 µl
1 µl

12
11
10
9
8
7
6
5
4
3
2
1

PCR tube
Well 1-6DNA
10 µl
Template Preparation pre-mix 3 µl

•Template Preparation Buffer•Template Preparation Enzyme
Samples 41-48
Samples 33-40
Samples 25-32
Samples 17-24
Samples 9-16
Samples 1-8

Add 10 µl of DNA to each tube containing the Template Preparation
pre-mix (according to the screen layout).

2.5. Check the proper insertion of the racks and consumables.
2.6. Close the door and press “Run” to start.
2.7. MicroPlex is running. The “Remaining time” calculation will give youan estimation of the processing time of your experiment.

36

MICROPLEX LIBRARY PREPARATION KIT MANUAL

2 µl
1 µl

NOTE: Refer to Appendix for selecting the appropriate indexes to use for yourexperiments.

AUTO PROTOCOL

Library
Amplification
Prepare the Indexing Reagents as follows:• Remove the Indexing Reagents from freezer and thaw for 10
minutes on the bench top.
• Spin the Indexing Reagents in a table top centrifuge to collectcontents at the bottom of the well.
• Thoroughly wipe the Indexing Reagent Tubes or Indexing Reagent
Plate foil seal with 70% ethanol and allow it to dry.
3.1. Recover your samples on the Peltier Block 1 in lane 7 to 12 according
to the screen layout.
3.2. Press “OK” and “Back” until the homepage appears on the screen.
3.3. Add 5 μl of the appropriate Indexing Reagent to each sample. Final
volume of your sample is 50 µl.
NOTE: if using indexing reagents from MicroPlex Kit v.2 (48 single indexes(48 rxns)):

Make sure the two corner notches of the Indexing Reagent Plate areon the left and the barcode label on the long side of the Index Plateis facing you.
• Use a clean pipet tip to pierce the seal above the specificindexing reagent on the Indexing Reagent Plate; discard thetip used for piercing.
• Use a clean pipet tip to collect 5 µl of the indexing reagent
and add to the reaction mixture.

37

• Follow the Indexing Reagent Plate handling instructions in
the Appendix to avoid cross contamination.
3.4. Mix 4x with a pipette set to 50 µl. Avoid introducing excessive air
bubbles.
3.5. Seal the tube(s) tightly and centrifuge briefly to collect the contents
to the bottom of each well or tube.
3.6. Return the tube(s) to thermal cycler with heated lid set to 101°C –105°C.
3.7. Perform Library Amplification Reaction using the cycling conditionsin the tables below.
Caution: Ensure that the thermal cycler does not have a denaturing
step programmed until Stage 3.

Extension & Cleavage
Denaturation

Addition of Indexes

Stage

Temperature

Time

Number of Cycles

1

72°C

3 min

1

2

85°C

2 min

1

3

98°C

2 min

1

98°C

20 s

67°C

20 s

72°C

40 s

98°C

20 s

72°C*

50 s

4°C

Hold

4

Library Amplification

5

Hold

6

4

5 to16*1* If working with DNA samples of known quantity or quality, please use the
Amplification Guide below to select the required number of amplification cycles.

38

MICROPLEX LIBRARY PREPARATION KIT MANUALDNA Input (ng)Number of Cycles

50

5

20

6

10

7

5

8

2

10

1

11

0.2

14

0.05

16

AUTO PROTOCOL

Amplification guide (for stage 5)

If working with DNA samples of unknown quantity and/or low quality (eg.  
FFPE extracted DNA ), amplify for 12 cycles and perform an INTERMEDIATELIBRARY QUANTIFICATION OF UNPURIFIED LIBRARY to estimate the libraryyield . If the desired yield is not achieved, the libraries can be re-amplified for
few additional cycles.

3.8. Remove the tube(s) from the thermal cycler and centrifuge briefly tocollect the contents to the bottom of each well.
3.9. Proceed to the Library processing section (recommended forsamples of known quantity or quality) or to the Intermediate Library
Quantification option shown below (recommended for samples withunknown quantity and/or low quality).
Optional Intermediate Library Quantification(recommended for samples with unknown quantity and/or a low quality)
Quantify unpurified libraries using the BioAnalyzer, the Fragment Analyzer
or similar devices. This intermediate quantification allows estimating thelibrary yield and enabling an additional re-amplification if needed. You willneed a High Sensitivity NGS Fragment Analysis kit (1 bp-6,000 bp) from
Advanced Analytical if using Fragment Analyzer or High Sensitivity DNA kit
from Agilent if using BioAnalyzer. Follow the manufacturer’s instruction
for kit handling and protocol.

39

NOTE: The quantification of unpurified library by qPCR (e.g. using KAPA® Library
Quantification Kit – Illumina (Kapa Biosystems), is also possible at this step
while it will increase the total protocol duration.

1.

Take an aliquot of unpurified libraries: 1 µl if the BioAnalyzer will be
used or 2 µl if the Fragment Analyzer will be used. Keep remaining
libraries at 4°C .

2.

Load the aliquot of unpurified library on the Fragment Analyzer or
BioAnalyzer and run the analysis.

3.

Estimate the library yield using a region selection option todiscriminate between unincorporated primers/adaptors and libraries
themself as it is shown in the Figure 2 below.

Figure 2. BioAnalyzer trace of unpurified library. A region
corresponding to the amplified library is selected (blue bars) toestimate the yield.
4.

40

If the library has a sufficient yield, proceed to the Library Purificationor size selection step in the section: Library processing. If the libraryhas a low yield, the remaining library can be further re-amplified for
2 to 3 additional cycles to achieve the desired yield. Spin down the
tube or plate containing the library and return the plate or tube(s) toa thermal cycler.

MICROPLEX LIBRARY PREPARATION KIT MANUAL

5.

Use the following cycling settings:
Number of cycles
2-3 cycles
1 cycleTemperature

Time

98°C

20 s

72°C

50 s

4°C

hold

NOTE: Higher yields may come at the expense of reduced sequencing quality,
therefore it is important to avoid an over-amplification.

41

PR OTOCO L
AUTOMATED PROCESSING
LIBRARY PROCESSING

Library Purification or Size Selection
Most part of applications will not require size selection if initial DNA
fragment size is less than 1,000 bp. Amplified products should be purified
by Agencourt AMPure XP beads (Beckman Coulter) as described at
Option A. If your application requires a size-selection, please refer to the
Option B, which allows to perform size selection from 250bp to 500bp justby changing the amount of beads.

AUTO PROTOCOL

Library processing using IP-Star
Compact Automated System

You will need
• Freshly prepared 80% (v/v) ethanol• Low TE buffer pH 8.0• Agencourt AMPure XP beads (Beckman Coulter)

Option A: Library Purification using AMPure XP beads
Use the room temperature AMPure XP beads for the clean-up.

1. Select “Protocols” icon and then “IPure” category.
2. Select “AMPure_XP_Purification” protocol:

If you plan to run between

1 and 8 samples> “AMPure_XP_Purification_08”

9 and 16 samples> “AMPure_XP_Purification_16”

17 and 24 samples > “AMPure_XP_Purification_24”

3. Setup the exact number of samples that you want to process bypressing the black box.
NOTE: The Peltier Block 1 is now cooling down to 4°C to keep your samples cold.

4. Set up all the plastics on the platform according to the screen layout.

43• Fill TIP Rack 1 (and 2 if processing more than 8 samples) with tips
according to the screen.
• Fill Reagent Rack 1 & 2 with reagent containers according to thescreen.
• Fill 96 plate 1 with a 96-well microplate.
• Fill Peltier Block 1 with 200 µl tube strips according to the screen

12
11
10
9
8
7
6
5
4
3
2
1

G
H

C

96 plate 2DEF

1 2 3 4 5 6

7 8 9 10 11 12

Peltier
Block 2

A
B

G
H

A
B

96 plate 1

C

Reagent
Rack 2D
E
F

Reagent
Rack 1

Peltier
Block 17 8 9 10 11 12

TIP Rack 2

1 2 3 4 5 6

TIP Rack 1

12
11
10
9
8
7
6
5
4
3
2
1

5. Fill the robot with all reagents.
• Peltier Block 1:
Put your samples in row 1 - if processing 1 to 8 samples
in row 2 - if processing 9 to 16 samples
in row 3 – if processing 17 to 27 samples• 96-well microplate:Distribute room temperature AMPure XP Beads:
in row 1 - if processing 1 to 8 samples
in row 5 - if processing 9 to 16 samples
in row 9 - if processing 17 to 24 samples
The amount of beads must be 1x the volume of sample (e.g. for45 μl of samples use 45 μl of beads).
NOTE: Resuspend the beads with pipetting up and down several times
before dispense them.

44

MICROPLEX LIBRARY PREPARATION KIT MANUAL

6. Close the door and press “Run” to start.

AUTO PROTOCOL• Fill the container of the Reagent Rack 1 with freshly prepared
80% ethanol according to the screen.
• Fill the container of Reagent Rack 2 with Low TE buffer pH 8.0(“Resuspension Buffer” on the screen) according to the screen.
• Check the proper insertion of the racks and the consumables.

7. Purification is running. The “Remaining time” calculation will give youan estimation of the processing time of your experiment.
8. After the run, recover your samples on the upper row of the Peltier
Block 1 as shown on the screen layout. The final volume of eachsample is 20 µl.
9. Press “OK” and “Back” until the homepage appears on the screen.
Press “Shutdown” and wait until the screen is black before switching
off the IP-Star.
NOTE: Remove all the plastics from the IP-Star platform, empty the waste
shuttle, and clean the inner side of the IP-Star with 70% ethanol.

Option B: Library size selection using AMPure XP beads
NOTE: Use room temperature AMPure XP beads for the size selection.

1. Select “Protocols” icon and then “Library prep.” category.
2. Select “AMPure_XP_Size_Selection” protocol:
If you plan to run between

1 and 8 samples> “AMPure_XP_ Size_Selection_08”

9 and 16 samples> “AMPure_XP_ Size_Selection_16”

3. Setup the exact number of samples that you want to process bypressing the black box.
NOTE: The Peltier Block 1 is now cooling down to 4°C to keep your samples cold.

45

4. Setup all the plastics on the platform according to the screen layout.
• Fill TIP Rack 1 (and 2 if processing more than 8 samples) with tips
according to the screen.
• Fill Reagent Rack 1 & 2 with reagent containers according to thescreen.
• Fill 96 plate 1 with a 96 well microplate.
• Fill Peltier Block 1 with 200 μl tube strips according to the screen.

12
11
10
9
8
7
6
5
4
3
2
1

G
H

C

96 plate 2DEF

1 2 3 4 5 6

7 8 9 10 11 12

Peltier
Block 2

A
B

G
H

A
B

96 plate 1

C

Reagent
Rack 2D
E
F

Reagent
Rack 1

Peltier
Block 17 8 9 10 11 12

TIP Rack 2

1 2 3 4 5 6

TIP Rack 1

12
11
10
9
8
7
6
5
4
3
2
1

5. Fill the robot with all reagents.
• Fill 20 μl of samples in lane 1 (and 2 if processing more than 8
samples) of the Peltier Block 1.
• Add 80 μl of nuclease-free water (“Resuspension buffer” on thescreen) to each sample to have a final volume of 100 μl.
• Distribute AMPure XP Beads in lane 1 on 96 Plate 1 (and 7 ifprocessing more than 8 samples) according to the required size
following recommendations from the table shown below (and on
the screen).
NOTE: Resuspend the beads with pipetting up and down several times before
dispensing them.

46

MICROPLEX LIBRARY PREPARATION KIT MANUAL

Well 1 & 7

Ampure XP

Final size

110 µl100 µl
90 µl
80 µl
70 µl

250 bp280 bp325 bp400 bp500 bp

G
H

D
E
F

(1-8)
C

A
B

1 2 3 4 5 6(9-16)

AUTO PROTOCOL

7 8 9 10 11 12

MTP• Fill the container of the Reagent Rack 1 with freshly prepared
80% ethanol according to the screen.
• Fill the container of Reagent Rack 2 with Low TE buffer pH 8.0(“Resuspension Buffer” on the screen) according to the screen.
6. Check the proper insertion of the racks and the consumables.
7. Close the door and press “Run” to start.
8. Library size selection is running. The “Remaining time” calculation
will give you an estimation of the processing time of your experiment.
9. After the run, recover your samples on the upper row of the Peltier
Block 1. The final volume is 20 μl for each sample.
10. Press “OK” and “Back” until the homepage appears on the screen.
Press “Shutdown” and wait until the screen is black before switching
off the IP-Star.
NOTE: Remove all the plastics from the platform, empty the waste shuttle andclean the inner side of the IP-Star with 70% ethanol.

Individual libraries, quantified and purified according to the above
protocol, can be pooled at desired molar ratios to allow multiplex
sequencing. Libraries that are being pooled must have been preparedwith different indexes (for multiplexing and index pooling guidelines refer47

to the Appendix). The minimal molar concentration needed to sequence
the pool depends on the requirements of the sequencing platform. The
total molarity is the sum of all the individual libraries’ molarities in the
final volume, e.g. if you add 5 μl of a 10 nM library to 5 μl of a 20 nM library,
you have 10 μl of a 15 nM pool. If libraries are prepared from similar input
amounts, they can be pooled by combining equal volume aliquots of eachlibrary.

48

MICROPLEX LIBRARY PREPARATION KIT MANUAL

Appendix: Indexing ReagentsThe MicroPlex Library Preparation Kit v2 contains all necessary reagents togenerate amplified and indexed NGS libraries, including indexing reagentsfor multiplexing up to 48 samples. Table 3 summarizes the characteristicsof the included indexing reagents, which consist of amplification primers
containing Illumina-compatible indexes. Indexing reagents should be stored
at –20°C and should not be subjected to more than 4 freeze/thaw cycles.
NOTE: Indexing reagents provided with the  MicroPlex Library Preparation Kit v2
cannot be substituted with indexing reagents from any other sources.

Table 3. MicroPlex Library Prepration Kit v2 - Indexing reagents
12 single indexes(12 rxns)

12 single indexes(48 rxns)

48 single indexes*(48 rxns)

Number of Reactions

12

48

48

Number of Indexes

12

12

48

Index Type

SingleSingleSingleLength of Indexes

8 nt

8 nt

8 nt

Format12 Tubes

12 Tubes

96-Well Plate

Number of Uses

Up to 8Up to 24Single

TruSeq LT or
Manual Input

TruSeq LT or
Manual Input

Manual Input only

Name

Illumina® ExperimentManager Kit Selection*The MicroPlex v2 (48 single indexes (48 rxns)) Kit is designed for high throughput
applications; therefore, the experiment should be designed to pool and sequence
the full set of 48 libraries using the entire plate of indexing reagents. If indexing
reagents from the entire plate are not used at the same time, it is critical tofollow the instructions below to avoid cross contamination:49• After removing indexing reagents of choice, cover any pierced or used index
wells with scientific tape (e.g., VWR, Cat. No. 89097-920, General-Purpose
Laboratory Labelling Tape, 0.5”).
• Thoroughly wipe the seal with 70% ethanol and allow it to dry completely.
• Replace the plastic lid, return the Indexing Reagent Plate to its sleeve andstore at −20°C.

The Indexing Reagent Plate should not be frozen and thawed more than
4 times.
Single index - library structure
The MicroPlex Library Preparation Kit v2 uses Illumina-compatible 8nt
sequences developed by the Wellcome Trust Sanger Institute in Cambridge,
UK (Nature Methods 7, 111-118 (2010)).  Each indexing reagent tube (12single indexes, 12 rxns and 48 rxns) or well (48 single indexes (48 rxns))
contains a unique single index sequence. The 12 MicroPlex v2 indexes sharethe same sequences in the first 6 bases as the Illumina TruSeq LT indexes
AD001 through AD012 (Table4 and Table5). The prepared library contains
the 8nt Sanger index on 3’ end and has the structure shown below.
1. Library structure

P5

MicroPlex V2 Single-Indexed library structure

5’ AATGATACGGCGACCACCGAGATCTACACAGGCGAAGACACTCTTTCCCTACACGACGCTCTTCCGATCT----Insert-------Insert----AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG 3’
P7
Sanger index

50

MICROPLEX LIBRARY PREPARATION KIT MANUAL

2. Single index sequences

Table 4. Single index sequences for the kits with 12 single indexes (12 rxns
and 48 rxns)
Tube

Sanger Tag

Sequence

TruSeq LT Index

TruSeq LT
Sequence

1

iPCRtagT1

ATCACGTT

AD001

ATCACG

2

iPCRtagT2

CGATGTTT

AD002

CGATGT

3

iPCRtagT3

TTAGGCAT

AD003

TTAGGC

4

iPCRtagT4

TGACCACT

AD004

TGACCA

5

iPCRtagT5

ACAGTGGT

AD005

ACAGTG

6

iPCRtagT6

GCCAATGT

AD006

GCCAAT

7

iPCRtagT7

CAGATCTG

AD007

CAGATC

8

iPCRtagT8

ACTTGATG

AD008

ACTTGA

9

iPCRtagT9GATCAGCG

AD009

GATCAG

10

iPCRtagT10

TAGCTTGT

AD0010TAGCTT

11

iPCRtagT11

GGCTACAG

AD0011

GGCTAC

12

iPCRtagT12

CTTGTACTAD0012

CTTGTA

Table 5. Single index sequences for the kit with 48 single indexes (48 rxns)
MicroPlex v2 x48 (48 indices)
Well

Sanger Tag

Sequence

Well

Sanger Tag

Sequence

A1

iPCRtagT1

ATCACGTT

B1

iPCRtagT13

TGGTTGTTA2

iPCRtagT2

CGATGTTT

B2

iPCRtagT14

TCTCGGTTA3

iPCRtagT3

TTAGGCAT

B3

iPCRtagT15

TAAGCGTT

A4

iPCRtagT4

TGACCACT

B4

iPCRtagT16

TCCGTCTT

A5

iPCRtagT5

ACAGTGGT

B5

iPCRtagT17

TGTACCTT

A6

iPCRtagT6

GCCAATGT

B6

iPCRtagT18

TTCTGTGT

A7

iPCRtagT7

CAGATCTG

B7

iPCRtagT19

TCTGCTGT

A8

iPCRtagT8

ACTTGATG

B8

iPCRtagT20

TTGGAGGTA9

iPCRtagT9GATCAGCG

B9

iPCRtagT21

TCGAGCGT

A10

iPCRtagT10

TAGCTTGT

B10

iPCRtagT22

TGATACGT

A11

iPCRtagT11

GGCTACAG

B11

iPCRtagT23

GTGCTACC

A12

iPCRtagT12

CTTGTACTB12

iPCRtagT101

GGTTGGAC
51

C1

iPCRtagT25

TGCGATCT

D1

iPCRtagT102

GGCACAACC2

iPCRtagT26

TTCCTGCT

D2

iPCRtagT38

TCTCACGG

C3

iPCRtagT27

TAGTGACT

D3

iPCRtagT39

TCAGGAGG

C4

iPCRtagT28

TACAGGATD4

iPCRtagT40

TAAGTTCG

C5

iPCRtagT29

TCCTCAAT

D5

iPCRtagT41

TCCAGTCG

C6

iPCRtagT30

TGTGGTTG

D6

iPCRtagT42

TGTATGCG

C7

iPCRtagT31

TAGTCTTG

D7

iPCRtagT43

TCATTGAG

C8

iPCRtagT32

TTCCATTG

D8

iPCRtagT44

TGGCTCAG

C9

iPCRtagT33

TCGAAGTG

D9

iPCRtagT45

TATGCCAG

C10

iPCRtagT34

TAACGCTG

D10

iPCRtagT46

TCAGATTC

C11

iPCRtagT35

TTGGTATG

D11

iPCRtagT47

TACTAGTCC12

iPCRtagT36

TGAACTGG

D12

iPCRtagT48

TTCAGCTC

3. Library pooling

Figure 3. Single Index Plate maps with well locations. The colored wells
indicate well positions containing indexing reagents.
When libraries with less than the full set of MicroPlex v2 indexes will be
prepared and pooled, it is critical that compatible index combinations areused to fulfill Illumina requirements. Illumina sequencers use a green
laser to sequence G/T nucleotides and a red laser to sequence A/C
nucleotides. At each sequencing cycle of the index read, at least one of

52

MICROPLEX LIBRARY PREPARATION KIT MANUAL

the two nucleotides for each colored laser should be present to ensure
proper image registration and ensure accurate demultiplexing of thepooled samples.
Follow the steps below before beginning the MicroPlex Library Preparation
Kit v2 protocol if using less than the full set of indexes included with the kit:
• Determine the number of libraries that will be pooled forsequencing.
• Select the appropriate index combinations for multiplexing
and pooling. The following sets of indexes for low-level poolingexperiments are recommended.
Plexity2

12 indexes format (tube number)

48 indexes format (well number)Index 6GCCAATGT

Index A6

Index 12

CTTGTACT

Index A12

Index 4TGACCACT

Index A4

Index 6GCCAATGT

Index A6

Index 12

CTTGTACT

Index A12
Index A1
Index A7
Index B12
Index A2

3

Index A5
Index D1
Index A8
Index B11
Index D10
Index A12
Index D11
Index D12

4 and upUse 3-plex sets and add any other indexes from the kit

53For additional low plex (2- to 11-plex) pooling guidelines, please referto Illumina’s   TruSeq Sample Preparation Pooling Guide (Illumina Part
# 15042173 V.01, 2015).
NOTE: Illumina Experiment Manager (IEM), a desktop tool that allows you tocreate and edit Sample Sheets for Illumina sequencers, can be used to detectand give caution of sub-optimal index combinations, allowing re-design before
library preparation starts. To use this tool with MicroPlex indexes, ensure thatthe latest version of IEM is installed. Prior to starting the MicroPlex Library
Preparation Protocol, create a Sample Sheet in the IEM to select and validate
appropriate indexes to use in your experiments. There are two options forcreating the Sample Sheet:
Option 1:
In the IEM, on the “Workflow Parameters” page, select “TruSeq LT” in the
dropdown menu for “Sample Prep Kit”. Add indexes to be used on the “Sample
Selection” page by clicking “Add Blank Row” and then choosing the appropriate
indexes from the “index 1 (I7)” dropdown menu. If TruSeq LT is selected, index
combinations may be validated using the IEM; however, only the first 6 bases of
the 8nt sequence will be sequenced.
Option 2:
Manually copy and paste the appropriate 8nt single index sequences to be used
to the CSV file of the Sample Sheet. The 8nt single index sequences can be
downloaded as a CSV file at the MicroPlex v2 product page.The IEM will notcheck for colour-balanced index combinations when index sequences areentered manually from the CSV file.

54

MICROPLEX LIBRARY PREPARATION KIT MANUALFAQs
Are the green buffers of the MicroPlex Library Preparation Kit and theMicroPlex Library Preparation Kit v2 exchangeable?
The MicroPlex Library Preparation Kit and MicroPlex Library Preparation
Kit v2 are not the same. So, the buffers are not exchangeable between
these kits.
Can I use the available Illumina primers and validate them with theMicroPlex Kit v2?
Although the final flanking sequences of MicroPlex are the same as thoseused by Illumina, the PCR primers are not identical and part of them is
supplied with the buffer.  For this reason Illumina primers will not work
as substitute.
The BioAnalyzer profile of purified library shows the presence of low
molecular weight peaks (primers/adaptors) in the samples. Should I repurify the samples or they can be used directly to the sequencing? If
the second purification is recommended, which ratio sample/AMPurebeads should I use?
You can do a second round of purification using 1:1 ratio of AMPure beads
to sample and this should get rid of the majority of the dimers.
I am going to use the MicroPlex Library Preparation Kit v2 on ChIP
samples . Our thermocycler has ramp rate 1.5°/s max while the protocol
recommends using a ramp rate 3 to 5°/s. How would this affect thelibrary prep?
We have not used a thermocycler with a ramp rate of 1.5 °C, which seems
faster than most of thermocyclers.  Too fast of a ramp rate may affect theprimer annealing and ligation steps.
What is the function of the replication stop site in the adapter loops?
The replication stop site in the adaptor loops function to stop the polymerase55

from continuing to copy the rest of the stem loop.
I want to do ChIP-seq. Which ChIP-seq kit can I use for sample preparation
prior to Microplex Library Preparation Kit v2?
In our portfolio there are several ChIP-seq kits compatible with Microplex
Library Preparation Kit v2. Depending on your sample type and target
studied you can use the following kits: iDeal ChIP-seq Kit for Transcription
Factors (Cat. No. C01010055), iDeal ChIP-seq Kit for Histones (Cat. No.
C01010051), True MicroChIP kit (Cat. No. C01010130), Universal Plant
ChIP-seq Kit (Cat. No. C01010152). All these kits exist in manual andautomated versions.
Is Microplex Library Preparation Kit v2 compatible with exome
enrichment methods?
Microplex Library Preparation Kit v2 is compatible with major exome
and target enrichment products, including Agilent SureSelect®, Roche
NimbleGen® SeqCap® EZ and custom panels.
What is the nick that is mentioned in the kit method overview?
The nick is simply a gap between a stem adaptor and 3’ DNA end, as
shown on the schema in the kit method overview.

56

MICROPLEX LIBRARY PREPARATION KIT MANUALRelated products
Product

Cat. No.

iDeal ChIP-seq kit for Transcription Factors

C01010055

Auto iDeal ChIP-seq Kit for Transcription Factors

C01010172

iDeal ChIP-Seq Kit for Histones

C01010051

Auto iDeal ChIP-seq Kit for Histones

C01010171

True MicroChIP Kit

C01010130

Auto True MicroChIP Kit

C01010140

Universal Plant ChIP-seq Kit

C01010152

Auto Universal Plant ChIP-seq kit

C01010153

Bioruptor Pico

B01060010

IP-Star Compact Automated System

B03000002

57

Technical support
For technical support contact custsupport@diagenode.com
The index sequences correspond to Illumina® Index sequences for
multiplexing and are copyrighted to Illumina, Inc. Oligonucleotide
sequences© 2007-2012 Illumina, Inc. All rights reserved.
Agencourt® and AMPure® are registered trademarks of Beckman Coulter,
Inc.
Agilent®, Bioanalyzer®, is registered trademarks of Agilent Technologies,
Inc. CFX96 Touch™ is a trademark of Bio-Rad Laboratories, Inc.
Illumina®, HiSeq®, MiSeq®, and TruSeq® are registered trademarks ofIllumina, Inc. KAPA® is a registered trademark of Kapa Biosystems, Inc.
NanoDrop® is a trademark of Thermo Fisher Scientific, Inc. NimbleGen
and SeqCap® are registered trademarks of Roche.
PicoGreen®, Quant-iT®, and Qubit® are registered trademarks of Life
Technologies.
Fragment Analyzer® is registered trademark of Advanced Analytical.

58

MICROPLEX LIBRARY PREPARATION KIT MANUALFOR RESEARCH USE ONLY.
Not intended for any animal or human therapeutic or diagnostic use.
© 2018 Diagenode SA. All rights reserved. No part of this publication may be reproduced, transmitted,
transcribed, stored in retrieval systems, or translated into any language or computer language, in any
form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise,
without prior written permission from Diagenode SA (hereinafter, “Diagenode”) . The informationin this guide is subject to change without notice. Diagenode and/or its affiliates reserve the rightto change products and services at any time to incorporate the latest technological developments.
Although this guide has been prepared with every precaution to ensure accuracy, Diagenode and/or its affiliates assume no liability for any errors or omissions, nor for any damages resulting fromthe application or use of this information. Diagenode welcomes customer input on corrections andsuggestions for improvement.
NOTICE TO PURCHASER LIMITED LICENSE
The information provided herein is owned by Diagenode and/or its affiliates. Subject to the terms andconditions that govern your use of such products and information, Diagenode and/or its affiliates
grant you a nonexclusive, nontransferable, non-sublicensable license to use such products andinformation only in accordance with the manuals and written instructions provided by Diagenodeand/or its affiliates. You understand and agree that except as expressly set forth in the terms andconditions governing your use of such products, that no right or license to any patent or otherintellectual property owned or licensable by Diagenode and/or its affiliates is conveyed or implied
by providing these products. In particular, no right or license is conveyed or implied to use theseproducts in combination with any product not provided or licensed to you by Diagenode and/or its
affiliates for such use. Limited Use Label License: Research Use Only The purchase of this productconveys to the purchaser the limited, non-transferable right to use the product only to perform
internal research for the sole benefit of the purchaser. Noright to resell this product or any of its
components is conveyed expressly, by implication, or by estoppel. This product is for internal research
purposes only and is not for use in commercial applications of any kind, including, without limitation,
quality control and commercial services such as reporting the results of purchaser’s activities fora fee or other form of consideration. For information on obtaining additional rights, please contact
info@diagenode.com.
TRADEMARKSThe trademarks mentioned herein are the property of Diagenode or their respective owners.
Bioanalyzer is a trademark of Agilent Technologies, Inc. Agencourt and AMPure® are registeredtrademarks of Beckman Coulter, Inc. Illumina® is a registered trademark of Illumina®Inc; Qubit is a
registered trademark of Life Technologies Corporation.

59

www.diagenode.com