KAPA HTP Library Preparation Kit
`Illumina® platforms
`KR0426 – v3.13
`
`This Technical Data Sheet provides product information
`and a detailed protocol for the KAPA HTP Library
`Preparation Kit (Illumina® platforms), product codes
`KK8234 and KK8235.
`
`Contents
`
`Product Description ........................................................2
`
`Product Applications .......................................................2
`
`Product Specifications ....................................................2
`
`Shipping and storage ...............................................2
` Handling ....................................................................2
` Quality control ..........................................................2
`
`Important Parameters .....................................................3
`
`Automated library construction .................................3
`
`Reaction setup ..........................................................3
`
`Safe stopping points .................................................3
`
`Paramagnetic SPRI® beads and reaction cleanups ..4
`
`Input DNA and fragmentation ...................................4
` Cleanups after end repair and A-tailing ....................4
`
`Adapter design and concentration ............................5
`
`Post-ligation cleanup ................................................5
`
`Size selection ............................................................6
`
`Library amplification ..................................................6
`
`Evaluating the success of library construction .........7
`
`Process Workflow ...........................................................9
`
`Library Construction Protocol ........................................10
`
`Appendix 1:
`Library construction guidelines for target enrichment with
`the Roche Nimblegen™ SeqCap EZ system ................17
`
`Technical Data Sheet
`
`Kit Codes and Components
`
`KK8234
`KK8235
`
`96 libraries
`
`KAPA End Repair Buffer (10X)
`KAPA End Repair Enzyme Mix
`KAPA A-Tailing Buffer (10X)
`KAPA A-Tailing Enzyme
`KAPA Ligation Buffer (5X)
`KAPA DNA Ligase
`KAPA PEG/NaCl SPRI® Solution
`KAPA HiFi HotStart ReadyMix (2X)*
`
`1.2 ml
`600 µl
`650 µl
`360 µl
`1.3 ml
`600 µl
`40 ml
`3 ml
`
`*KK8235 is available for PCR-free workflows and does not contain KAPA
`HiFi HotStart ReadyMix for library amplification.
`
`Quick Notes
`
`• The protocol provided in this document is a
`generic prototype, and may require additional
`tailoring and optimization.
`
`• The process workflow (p. 9) provides an overview
`of the library construction process and options
`for size selection.
`
`• The KAPA NGS Library Preparation Technical
`information
`Guide contains more detailed
`about library construction parameters, and may
`facilitate protocol development and optimization.
`
`• Separate, concentrated enzyme formulations
`and reaction buffers for end repair, A-tailing, and
`ligation provide the best combination of product
`stability, convenience, and efficiency.
`
`• Adapters and PCR primers are not supplied with
`this kit, and can be obtained from any reputable
`oligonucleotide vendor.
`
`• SPRI® beads are not included in the kit, but the
`PEG/NaCl SPRI® Solution required for "with-
`bead" reaction cleanups is provided.
`
`• Generous reagent excesses are supplied to
`accommodate the dead volumes required for
`automated liquid handling.
`
`• A single kit per batch of 96 samples simplifies
`reagent handling and inventory control.
`
`Effective date: July 2013
`
`1
`
`00001
`
`EX1015
`
`

`

`KAPA HTP Library Preparation Kit
`Illumina® platforms
`
`Product Description
`The KAPA HTP Library Preparation Kit is designed for high-
`throughput library construction for Illumina® sequencing,
`starting from fragmented, double-stranded DNA. The kit
`provides all of the enzymes and reaction buffers required
`for the following steps of library construction:
`
`1. End repair, which produces blunt-ended, 5'-phosphor-
`ylated fragments.
`
`2. A-tailing, during which dAMP is added to the 3'-ends
`of blunt-ended dsDNA library fragments.
`
`3. Adapter ligation, during which dsDNA adapters with
`3'-dTMP overhangs are ligated to 3'-A-tailed library
`fragments.
`
`4. Library amplification (optional), which employs PCR
`to amplify library fragments carrying appropriate
`adapter sequences on both ends.
`
`The kit has been validated for library construction from
`100 ng – 5 µg of human genomic DNA for whole-genome
`shotgun sequencing or targeted sequencing by solution
`hybrid selection (capture). For smaller genomes, or lower
`complexity samples, such as ChIP DNA, amplicons, or
`cDNA (for RNA-seq), successful library construction has
`been achieved from low nanogram to picogram quantities
`(≥100 pg) of input DNA.
`
`The kit provides all of the enzymes and buffers required
`for library construction and amplification, but does not
`include adapters, PCR primers or SPRI® beads. Enzyme
`formulations and reaction buffers for end repair, A-tailing
`and ligation are supplied in convenient, concentrated
`formats.
`
`Efficient, cost-effective and automation-friendly reaction
`cleanups and higher recovery of input DNA are achieved
`through implementation of the "with-bead" strategy
`developed at The Broad Institute of MIT & Harvard and
`Foundation Medicine1. The kit includes PEG/NaCl SPRI®
`(Solid Phase Reversible Immobilization) Solution for this
`purpose.
`
`In order to maximize sequence coverage uniformity, it is
`critical to minimize library amplification bias. KAPA HiFi
`DNA Polymerase has been designed for low-bias, high-
`fidelity PCR, and is the reagent of choice for NGS library
`amplification2, 3, 4. The KAPA HTP Library Preparation Kit
`(KK8234) includes KAPA HiFi HotStart ReadyMix (2X), a
`ready-to-use PCR mix comprising all the components for
`library amplification, except primers and template. A kit
`without an amplification module (KK8235) is available for
`PCR-free workflows. These kits can also be combined
`with KAPA HiFi Real-Time Library Amplification Kits
`(KK2701 and KK2702), or with KAPA HiFi HotStart Uracil+
`ReadyMix (KK2801 and KK2802) for the amplification of
`libraries that have undergone bisulfite-treatment.
`1. Fisher, S. et al. Genome Biology 12, R1 (2011).
`2. Oyola, S.O. et al. BMC Genomics 13, 1 (2012).
`3. Quail M.A. et al. Nature Methods 9, 10 – 11 (2012).
`4. Quail M.A. et al. BMC Genomics 13: 341 (2012).
`
`2
`
`Technical Data Sheet
`
`Product Applications
`The KAPA HTP Library Preparation Kit is ideally suited
`for high-throughput NGS library construction workflows
`that involve end repair, A-tailing, adapter ligation, and
`library amplification (optional). The kit has been tailored
`to accommodate the specific requirements of automated
`liquid handling, and the protocol may be adapted for
`incorporation into workflows for a wide range of NGS
`applications, including:
`
`• Whole-genome shotgun sequencing
`• Targeted sequencing by solution hybrid selection
`(i.e. exome or custom capture using the Roche
`Nimblegen™, Agilent SureSelect, Illumina® TruSeq™, or
`IDT xGen™ Lockdown™ Probes systems)
`• ChIP-seq
`• RNA-seq
`• Methyl-seq (in combination with the KAPA HiFi HotStart
`Uracil+ ReadyMix)Library Amplification ReadyMix)
`
`Specific guidelines for the construction of libraries for
`target enrichment using the Roche Nimblegen™ SeqCap
`EZ system may be found in Appendix 1.
`
`Product Specifications
`
`Shipping and storage
`The enzymes provided in this kit are temperature
`sensitive, and appropriate care should be taken during
`shipping and storage. KAPA Library Preparation Kits are
`shipped on dry ice or ice packs, depending on the country
`of destination. Upon receipt, immediately store enzymes
`and reaction buffers (including PEG/NaCl SPRI® Solution)
`at -20 °C in a constant-temperature freezer. When
`stored under these conditions and handled correctly,
`the kit components will retain full activity until the expiry
`date indicated on the kit label.
`
`Handling
`Always ensure that components have been fully thawed
`and thoroughly mixed before use. Keep all enzyme
`components and master mixes on ice as far as possible
`during handling and preparation. KAPA HiFi HotStart
`ReadyMix (2X) contains isostabilizers and may not freeze
`completely, even when stored at -20 °C. Nevertheless,
`always ensure that the KAPA HiFi HotStart ReadyMix is
`fully thawed and thoroughly mixed before use. PEG/NaCl
`SPRI® Solution does not freeze at -20 °C, but should be
`equilibrated to room temperature and thoroughly mixed
`before use.
`
`Quality control
`All kit components are subjected to stringent functional
`quality control, are free of detectable contaminating exo-
`and endonuclease activities, and meet strict requirements
`with respect to DNA contamination. Please contact
`support@kapabiosystems.com for more information.
`
`00002
`
`

`

`KAPA HTP Library Preparation Kit
`Illumina® platforms
`
`Important Parameters
`High-throughput library construction workflows must
`be tailored and optimized to accommodate specific
`experimental designs, sample characteristics, sequencing
`applications, and equipment. The protocol provided
`in this document is a generic prototype, and there are
`many parameters which may be adjusted to optimize
`performance, efficiency, and cost-effectiveness.
`
`In addition to the information in this section, please
`consult the KAPA NGS Library Preparation Technical
`Guide and/or contact support@kapabiosystems.com
`for further guidelines when designing or optimizing your
`library construction workflow.
`
`Automated library construction
`in this
`The
`library construction protocol described
`document can be carried out manually, and most protocol
`development and validation work is usually done this way.
`Although it is possible to achieve moderately high sample
`throughput by using multi-channel pipettes and 96-well
`plates, automated liquid handling is indispensable for
`high-throughput NGS production lines, and automating
`a validated manual library construction protocol can
`represent a significant challenge.
`
`In addition to increased sample throughput, automation
`may be expected to provide additional advantages
`such as improved reproducibility and process control.
`Nevertheless, automation may result in slightly lower
`yields and/or different size distributions when compared
`with manual library construction performed by a skilled,
`experienced and attentive technician. Most often, these
`discrepancies can be minimized through careful selection
`of appropriate hardware and plasticware, and by
`optimizing liquid handling parameters such as aspiration
`speeds and volumes in automation scripts.
`
`Kapa Biosystems does not supply automated liquid
`handling equipment, but we are constantly working
`in partnership with automation solution providers and
`customers to develop and validate optimized, automated
`methods for liquid handling platforms suitable for use
`in NGS library construction. Please contact support@
`kapabiosystems.com for more information about using
`this kit with your particular automated liquid handling
`system.
`
`Reaction setup
`library
`for high-throughput
`This kit
`is
`intended
`construction, and the protocol is therefore designed to
`be automation-friendly. For this reason, and to enable a
`streamlined “with-bead” strategy, reaction components
`are combined into master mixes, rather than dispensed
`separately into individual reactions. When processing
`multiple samples, prepare 10 – 20% excess of each
`master mix. When processing batches of 48 or more
`samples with an automated liquid handling system,
`20% excess is required for all master mixes. For other
`
`Technical Data Sheet
`
`reagents (adapters, AMPure® XP reagent, SPRI® Solution,
`80% ethanol and elution buffer), the required excess may
`vary from one specific liquid handling system to another.
`Please refer to Section 1 of the Protocol for more details.
`
`96-well PCR plates are typically used for high-throughput
`library construction. The maximum working volume in
`these plates is usually ~200 µl, and this is accounted for in
`this protocol. It may be possible to employ 96-well plates
`with larger working volumes or deep well plates to
`accommodate
`larger
`reaction volumes
`for special
`applications. Always use plastics that are certified to
`be free of DNAses, RNAses, and nucleases. Low DNA-
`binding plastics are recommended. When selecting the
`most appropriate plasticware for your workflow, consider
`compatibility with:
`
`•
`
`the plate gripper and other components of your liquid
`handling system.
`the magnet used during SPRI® bead manipulations.
`•
`• vortex mixers and centrifuges, where appropriate.
`• heating blocks or thermocyclers used for reaction
`incubations and/or library amplification.
`
`Safe stopping points
`The library construction process, from end repair to
`final, amplified library, can be performed in 4 – 8 hours,
`depending on the specific workflow and number of
`samples being processed. Automated methods are
`typically designed to complete the process from end repair
`to post-ligation processing in an uninterrupted manner,
`with minimal user intervention. However, the protocol may
`be paused safely after any of the bead cleanup steps, as
`described below:
`
`• After the end repair cleanup (Steps 3.1 – 3.13),
`resuspend the washed beads in 20 µl of 1X A-Tailing
`Buffer (without enzyme; Step 4.1), and store the
`reactions at 4 °C for up to 24 hours.
`
`• After the A-tailing cleanup (Steps 5.1 – 5.13), resuspend
`the washed beads in 20 µl of 1X Ligation Buffer (without
`enzyme or adapter; Step 6.1), and store the reactions
`at 4 °C for up to 24 hours.
`
`• After the first post-ligation cleanup (Steps 7.1 – 7.13),
`resuspend the washed beads in the appropriate
`volume of 10 mM Tris-HCl (pH 8.0) as outlined in Step
`7.14, and store the reactions at 4 °C for up to 24 hours.
`
`DNA solutions containing beads must not be frozen,
`and beads must not be stored dry, as this is likely to
`damage the beads and result in sample loss. To resume
`the library construction process, centrifuge the reaction
`vessels briefly to recover any condensate, and add the
`remaining components required for the next enzymatic
`reaction in the protocol (see Tables 4B and 5B on
`p. 10). If the protocol was paused after the first post-
`ligation cleanup, continue directly with the second post-
`ligation cleanup (Step 7.16), dual-SPRI® size selection
`(Step 8.1), or size selection using an alternative method.
`
`3
`
`00003
`
`

`

`KAPA HTP Library Preparation Kit
`Illumina® platforms
`
`Safe stopping points (continued)
`Adapter-ligated DNA that has been completely cleaned up
`or size-selected can be stored at 4 °C for one week, or at
`-20 °C for at least one month before amplification, target
`enrichment, and/or sequencing. To avoid degradation,
`always store DNA in a buffered solution (10 mM Tris-HCl,
`pH 8.0) and minimize the number of freeze-thaw cycles.
`
`Paramagnetic SPRI® beads and reaction cleanups
`• Cleanups should be performed in a timely manner to
`ensure that enzyme reactions do not proceed beyond
`optimal incubation times.
`
`• This protocol has been validated using Agencourt®
`AMPure® XP reagent (Beckman Coulter, part number
`A63880, A63881, or A63882). Solutions and conditions
`for DNA binding and size selection may differ if other
`beads are used.
`
`• Observe all the manufacturer's storage and handling
`recommendations for AMPure® XP reagent.
`
`• Beads will settle gradually; always ensure that they
`are fully resuspended before aspirating AMPure® XP
`reagent.
`
`• The incubation times provided for reaction cleanups
`and size selection are guidelines only, and should
`be modified/optimized according to your current
`protocols, previous experience, and specific equipment
`and samples in order to maximize library construction
`efficiency and throughput.
`
`• The time required to completely capture magnetic
`beads varies according to the reaction vessel and
`magnet used. It is important to not discard or transfer
`any beads with the removal or transfer of supernatant.
`Capture times should be optimized accordingly.
`
`• The volumes of 80% ethanol used for bead washes
`may be adjusted to accommodate smaller reaction
`vessels and/or limited pipetting capacity, but it is
`important that the beads are entirely submerged during
`the wash steps. Where possible, use a wash volume
`that is equal to the volume of sample plus AMPure® XP
`reagent or PEG/NaCl SPRI® Solution.
`
`•
`
`It is important to remove all ethanol before proceeding
`with subsequent reactions. However, over-drying of
`beads may make them difficult to resuspend, and
`may result in a dramatic loss of DNA. With optimized
`pipetting, drying of beads for 3 – 5 min at room
`temperature should be sufficient. Drying of beads at
`37 °C is not recommended.
`
`• Where appropriate, DNA should be eluted from beads
`in elution buffer (10 mM Tris-HCl, pH 8.0). Elution of
`DNA in PCR-grade water is not recommended, as
`DNA is unstable in unbuffered solutions.
`
`4
`
`Technical Data Sheet
`
`Input DNA and fragmentation
`• This protocol has been validated for library construction
`from 100 ng – 5 µg of appropriately fragmented,
`double-stranded DNA. However, libraries can be
`prepared from lower input amounts if the sample
`represents sufficient copies to ensure the requisite
`coverage and complexity in the final library. Successful
`library construction has been achieved from <100 pg
`of ChIP DNA, low nanogram quantities of cDNA or
`microbial DNA, and 1 – 10 ng of high-quality human or
`mouse genomic DNA.
`
`• The above typically refers to the input into the end
`repair reaction. If input DNA is quantified before
`fragmentation, and/or fragmented DNA is subjected to
`cleanup or size selection prior to end repair, the actual
`input into library construction may be significantly
`lower. This should be taken into account when
`evaluating the efficiency of the process and/or during
`optimization of library amplification cycle number.
`
`• The proportion of fragmented DNA that is successfully
`converted to adapter-ligated molecules decreases as
`input is reduced. When starting library construction
`(end repair) with >100 ng fragmented DNA, 15 – 40%
`of input DNA is typically recovered as adapter-ligated
`molecules, whereas the recovery typically ranges
`from 0.5 to 15% for libraries constructed from 100 pg
`– 100 ng DNA. These figures apply to high quality
`DNA and can be significantly lower for DNA of lower
`quality, e.g. FFPE samples. Workflows that contain
`additional SPRI® cleanups or size selection prior to
`library amplification are likely to result in a lower yield
`of adapter-ligated molecules.
`
`• Solutions containing high concentrations of EDTA and
`strong buffers may negatively affect the end repair
`reaction, and should be avoided. If fragmented DNA
`will not be processed (i.e. subjected to cleanup or size
`selection) prior to end repair, DNA should be fragmented
`in 10 mM Tris-HCl (pH 8.0 or 8.5) with 0.1 mM EDTA.
`Fragmentation in water is not recommended.
`
`•
`
`In some circumstances it may be convenient to
`fragment input DNA in 1X KAPA End Repair Buffer,
`in which case the end repair reaction setup should
`be adjusted accordingly. Please contact support@
`kapabiosystems.com for more information.
`
`Cleanups after end repair and A-tailing
`• This protocol provides for 1.7X – 1.8X cleanups after
`end repair and A-tailing. This ratio of PEG/NaCl SPRI®
`Solution to sample volume will retain most DNA
`fragments larger than ~75 bp. If you wish to retain very
`small DNA fragments, the PEG/NaCl SPRI® Solution
`to sample ratio can be increased to 2X – 3X for all
`cleanups prior to adapter ligation.
`
`00004
`
`

`

`KAPA HTP Library Preparation Kit
`Illumina® platforms
`
`Cleanups after end repair and A-tailing (continued)
`•
`If a >2X SPRI® bead cleanup is desired after end repair,
`the end repair reaction must be scaled down when
`performing library construction in standard PCR plates
`(maximum working volume of ~200 ul). Please contact
`support@kapabiosystems.com if your workflow or
`sample type requires modified SPRI® bead cleanups.
`
`Adapter design and concentration
`• This protocol has been validated using standard,
`indexed Illumina® TruSeq™ "forked" adapters, but the
`kit is compatible with other adapters of similar design.
`
`• Adapter concentration affects ligation efficiency, as
`well as adapter and adapter-dimer carry-over in post-
`ligation cleanups. The optimal adapter concentration
`for your workflow represents a compromise between
`cost and the above factors. Your choice of post-
`ligation cleanup and size-selection options should be
`informed by your choice of adapter concentration.
`Please refer to Important Parameters: Post-ligation
`cleanup for more details.
`
`for adapter:insert
`robust
`is
`• Ligation efficiency
`molar ratios ranging from 10:1 to 50:1. As a general
`guideline, we recommend an adapter:insert molar
`ratio of ~10:1, for libraries constructed from ≥100 ng
`fragmented DNA. This translates to different final
`adapter concentrations for libraries with different size
`distributions (see Table 1 below). An adapter:insert
`molar ratio >10:1 may be beneficial for libraries
`constructed from lower amounts of input DNA.
`
`to adjust adapter
`is not necessary
`it
`• While
`concentrations to accommodate moderate sample-
`to-sample variations, we recommend using an adapter
`concentration that is appropriate for the range of input
`DNA concentrations.
`
`• The best way to accommodate different adapter
`concentrations within a batch of samples processed
`together, is to vary the concentration of adapter stock
`solutions, and dispense a fixed volume (5 µl) of each
`
`Table 1. Recommended adapter concentrations.
`
`Technical Data Sheet
`
`adapter. The alternative – using a single stock solution,
`and dispensing variable volumes of adapter into
`ligation reactions – is not recommended for automated
`workflows.
`
`Post-ligation cleanup
`•
`It is important to remove excess unligated adapter
`and adapter-dimer molecules from the library prior to
`library amplification or cluster generation.
`
`• While a single SPRI® bead cleanup removes most
`unligated adapter and adapter-dimer, a second
`SPRI® bead cleanup is recommended to eliminate
`any remaining adapter species from the library.
`The amount of adapter and adapter-dimer carried
`through the first cleanup is dependent on the adapter
`concentration in the ligation reaction.
`
`•
`
`If size selection is carried out between adapter ligation
`and library amplification (or clustering), a single post-
`ligation cleanup with SPRI® beads (1X) is usually
`sufficient prior to size selection. If no post-ligation size
`selection is carried out, two consecutive 1X SPRI®
`bead cleanups are recommended.
`
`• The volume in which washed beads are resuspended
`after the post-ligation cleanup(s) should be adjusted to
`suit your chosen workflow:
`
`•
`
`library amplification,
`If proceeding directly to
`determine an appropriate final volume in which to
`elute the library DNA, keeping in mind that you may
`wish to divert and/or reserve some of this library
`material for archiving and/or QC purposes. Since
`an optimized 50 µl library amplification reaction
`should yield ~1 µg of DNA, and can accommodate
`a maximum of 20 µl template DNA, an elution
`volume of 22 – 32 µl is recommended.
`
`•
`
`If proceeding with size selection, elute the library
`DNA in an appropriate volume according to the size
`selection method of choice. For the dual-SPRI® size
`selection procedure described here, beads have to
`be resuspended in a final elution volume of 100 µl.
`
`Insert DNA per 50 µl
`end repair reaction
`
`Recommended adapter concentration for DNA sheared to an average size of
`
`175 bp
`
`350 bp
`
`500 bp
`
`3 – 5 µg
`
`1 µg
`
`500 ng
`
`100 ng
`
`10 ng
`
`Stock
`
`60 µM
`
`20 µM
`
`10 µM
`
`2 µM
`
`200 nM
`
`Final
`
`6 µM
`
`2 µM
`
`1 µM
`
`200 nM
`
`20 nM
`
`Stock
`
`30 µM
`
`10 µM
`
`5 µM
`
`1 µM
`
`100 nM
`
`Final
`
`3 µM
`
`1 µM
`
`500 nM
`
`100 nM
`
`10 nM
`
`Stock
`
`21 µM
`
`7 µM
`
`3.5 µM
`
`700 nM
`
`70 nM
`
`Final
`
`2.1 µM
`
`0.7 µM
`
`350 nM
`
`70 nM
`
`7 nM
`
`5
`
`00005
`
`

`

`KAPA HTP Library Preparation Kit
`Illumina® platforms
`
`Size selection
`• Size selection requirements vary widely according
`to specific applications. Depending on preference,
`the dual-SPRI® size selection procedures presented
`in this protocol may be omitted entirely, modified, or
`replaced with alternative size selection procedures.
`Size selection may be carried out at alternative points
`in the overall workflow, for example:
`• prior to end repair of fragmented DNA.
`•
`immediately before library amplification (as outlined
`in this protocol).
`
`• after library amplification.
`
`• Size selection inevitably leads to a loss of sample
`material. Depending on the details, these losses can
`be dramatic (>80%), and may significantly increase
`the number of amplification cycles needed to generate
`sufficient material for the next step in the process
`(capture or sequencing). The potential advantages of
`one or more size selection steps in a library construction
`workflow should be weighed against the potential loss
`of library complexity, especially when input DNA is
`limited. A carefully optimized fragmentation protocol,
`especially for shorter insert libraries and/or read
`lengths, may eliminate the need for size selection,
`thereby simplifying the library construction process
`and limiting sample losses.
`
`in
`results
`libraries often
`• Over-amplification of
`the observation of secondary, higher molecular
`weight peaks when amplified libraries are analyzed
`electrophoretically. These higher molecular weight
`peaks are artefacts, and typically contain authentic
`library molecules of the appropriate length. To eliminate
`these artefacts, optimization of library amplification
`reaction parameters (cycle number and/or primer
`concentration), rather than post-amplification size
`selection, is recommended. Please refer to the Library
`amplification subsection for more information.
`
`• KAPA Ligation Buffer contains high concentrations of
`PEG 6000, which will interfere with efficient dual-SPRI®
`size selection and can affect the efficiency of other
`size selection techniques if not removed. It is therefore
`important to perform at least one post-ligation SPRI®
`bead cleanup (1X) prior to dual-SPRI® or any other size
`selection method.
`
`• The dual-SPRI® size selection procedure described in
`Section 8 of the protocol is designed for selection of
`adapter-ligated fragments approximately 250 – 450 bp
`in size. Consult the KAPA NGS Library Preparation
`Technical Guide if you wish to select a different range
`of fragment sizes.
`
`• "Forked" adapters with long single-stranded arms
`(such as the TruSeq™ design) noticeably affect the
`size-dependent binding of DNA to SPRI® beads, as
`well as the apparent size of fragments determined
`by some electrophoresis instruments (e.g. those
`
`6
`
`Technical Data Sheet
`
`tapes). Size
`employing microfluidic chips and
`selection parameters will therefore require optimization
`depending on a number of factors, including:
`
`• The design of the adapters used.
`
`• The point at which size selection is applied in the
`protocol; size selection of dsDNA fragments prior
`to library construction or after amplification may
`require different parameters than those used for
`post-ligation size selection of fragments carrying
`forked adapter ends.
`
`• Dual-SPRI® size selection is sensitive to multiple
`factors that are beyond the scope of this document.
`The KAPA NGS Library Preparation Technical Guide
`contains additional guidelines for the optimization of
`dual-SPRI® size selection parameters. Any dual-SPRI®
`size selection protocol should be carefully optimized
`and validated before it is used for precious samples.
`
`Library amplification
`• KAPA HiFi HotStart, the enzyme provided in the
`KAPA HiFi HotStart ReadyMix,
`is an antibody-
`based hot start formulation of KAPA HiFi DNA
`Polymerase, a novel B-family DNA polymerase
`engineered for increased processivity and high fidelity.
`KAPA HiFi HotStart DNA Polymerase has 5'g3'
`polymerase and 3'g5' exonuclease (proofreading)
`activities, but no 5'g3' exonuclease activity.
`The strong 3'g5' exonuclease activity results in
`superior accuracy during DNA amplification. The
`error rate of KAPA HiFi HotStart DNA Polymerase is
`2.8 x 10-7 errors/base, equivalent to 1 error in 3.5 x 106
`nucleotides incorporated.
`
`•
`
`In library amplification reactions (set up according to the
`recommended protocol), primers are typically depleted
`before dNTPs. When DNA synthesis can no longer
`take place due to substrate depletion, subsequent
`rounds of DNA denaturation and annealing result in the
`separation of complementary DNA strands, followed by
`imperfect annealing to non-complementary partners.
`This presumably results in the formation of so-called
`"daisy-chains" or "tangled knots", comprising large
`assemblies of improperly annealed, partially double-
`stranded, heteroduplex DNA. These species migrate
`slower and are observed as secondary, higher
`molecular weight peaks during the electrophoretic
`analysis of amplified libraries. However, they are
`typically comprised of library molecules of the desired
`length, which are individualized during denaturation
`prior to cluster amplification or probe hybridization.
`Since
`these heteroduplexes contain significant
`portions of single-stranded DNA, over-amplification
`leads to the under-quantification of library molecules
`with assays employing dsDNA-binding dyes. qPCR-
`based library quantifications methods, such as the
`KAPA Library Quantification assay, quantify DNA by
`denaturation and amplification, thereby providing an
`
`00006
`
`

`

`KAPA HTP Library Preparation Kit
`Illumina® platforms
`
`Library amplification (continued)
`
`accurate measure of the amount of adapter-ligated
`molecules in a library, even if the library was over-
`amplified.
`
` Please refer to the KAPA NGS Library Preparation
`Technical Guide for a more detailed discussion
`of factors that can affect the efficiency of library
`amplification, and the impact of over-amplification on
`library quantification.
`
`• Excessive library amplification can result in other
`unwanted artefacts such as amplification bias, PCR
`duplicates, chimeric library inserts, and nucleotide
`substitutions. The extent of
`library amplification
`should therefore be limited as much as possible, while
`ensuring that sufficient material is generated for QC
`and downstream processing (e.g. target enrichment or
`sequencing).
`
`•
`
`•
`
`(not recommended) a
`If cycled to completion
`single 50 µl library amplification PCR, performed
`as described
`in Section 9,
`can produce
`8 – 10 µg (160 – 200 ng/µl) of amplified library.
`To minimize over-amplification and associated
`undesired artefacts, the number of amplification
`cycles should be optimized to produce an amplified
`library with a concentration
`in
`the
`range of
`10 – 30 ng/µl (0.5 – 1.5 µg of PCR product per 50 µl
`reaction).
`
`libraries prior
` Quantification of adapter-ligated
`to library amplification can greatly facilitate the
`optimization of
`library amplification parameters,
`particularly when a library construction workflow is
`first established. With the KAPA Library Quantification
`Kit, the amount of template DNA (adapter-ligated
`molecules) available for library amplification can be
`determined accurately. Using a simple calculation
`
`Table 2. Theoretical number of cycles required to achieve
`0.5 – 1.5 µg (10 – 30 ng/µl) library in a standard 50 µl KAPA HiFi
`HotStart ReadyMix library amplification reaction, starting from
`different amounts of template DNA.
`
`Template DNA
`
`Number of cycles
`
`1 ng
`
`5 ng
`
`10 ng
`
`25 ng
`
`50 ng
`
`100 ng
`
`250 ng
`
`500 ng
`
`10 – 11
`
`7 – 8
`
`6 – 7
`
`5 – 6
`
`4 – 5
`
`3 – 4
`
`2 – 3
`
`0 – 2
`
`Technical Data Sheet
`
`the number of
`(for exponential amplification),
`amplification cycles needed to achieve a specific yield
`of amplified library may be predicted theoretically
`(Table 2).
`• The actual optimal number of amplification cycles
`may be 1 – 3 cycles higher, particularly for libraries
`constructed from FFPE DNA or other challenging
`samples, or libraries with a broad fragment size
`distribution.
`
`Evaluating the success of library construction
`• Your specific library construction workflow should be
`tailored and optimized to yield a sufficient amount
`of adapter-ligated molecules of the desired size
`distribution for the next step in the process (e.g. target
`enrichment or sequencing), as well as for QC and
`archiving purposes.
`
`• The size distribution of the final or pre-capture
`library should be confirmed with an electrophoretic
`method, whereas KAPA Library Quantification Kits for
`Illumina® platforms are recommended for qPCR-based
`quantification of libraries. These kits employ primers
`based on the Illumina® flow ce