KAPABIOSYSTEMS. Application Note. Introduction. Automated NGS library construction for Illumina sequencing

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1 KAPABIOSYSTEMS KAPA Library Preparation Kits enable the construction of high-quality NGS libraries on the Apollo 324 automated NGS library preparation system. Introduction Kapa Biosystems has developed a DNA library construction kit which enables robust library construction for Illumina sequencing from lower amounts of input DNA, difficult samples such as FFPE and ChIP DNA, and samples from organisms with extreme (AT- or GC-rich) genomes. KAPA Library Preparation Kits contain highquality, optimally formulated enzymes and buffers for end repair, A-tailing and adapter ligation, which result in a high conversion rate of input DNA to adapter-ligated molecules. Combined with the engineered KAPA HiFi DNA Polymerase, which enables highfidelity, low-bias NGS library amplification, these kits yield higher quality, more complex libraries and improved sequence coverage 1,2. The KAPA library construction protocol has been successfully automated on the Sciclone NGS Workstation (PerkinElmer ), Biomek FX p Laboratory Automation Workstation (Beckman Coulter), NGS Workstation Option B (Agilent Technologies) and epmotion 5075 TMX (Eppendorf). The purpose of this study was to demonstrate library construction with the KAPA Library Preparation Kit on the Apollo 324 System (IntegenX, Figure 1), thereby offering the benefits of the KAPA reagents to lower-throughput users with access to this platform. The system uses readily available consumables, thereby enabling users to employ library construction reagents other than those supplied with the instrument. The Apollo 324 System provides two Illumina DNA library preparation protocols. The standard ILM DNA library preparation method includes a size selection step, designed to remove both large and small DNA fragments, and yields adapter-ligated libraries with a fragment distribution of bp, with a peak in the range of bp. In the ILM ChIP-Seq method, all adapterligated library fragments >200 bp are retained. This protocol results in significantly smaller losses of adapter-ligated molecules, and is recommended for lower input, precious and/or challenging samples. When appropriate for the sequencing read length and application, input DNA should be fragmented to a size that obviates the need for size selection, as size selection results in a significant loss of adapter-ligated molecules. Using a protocol without size selection results in higher library yields and complexity, and also limits the number of amplification cycles required to generate sufficient material for the next process. To demonstrate the use of KAPA library preparation and amplification reagents on the Apollo 324 System, libraries were prepared from fragmented human (250 ng and 50 ng) or Figure 1. The Apollo 324 instrument. The Apollo 324 is a benchtop instrument that enables low-throughput, automated construction of NGS libraries. The system uses proprietary magnetic bead technology for cleanups between enzymatic steps and size selection of DNA fragments. Up to eight samples can be processed in less than 2 hours, including setup time. Escherichia coli (100 ng and 10 ng) genomic DNA, using the standard ILM DNA library preparation method. Libraries were constructed from the same DNA samples using the PrepX Next Generation Sequencing Library Prep Kit for the Apollo 324 System (IntegenX). All libraries were amplified with KAPA HiFi HotStart ReadyMix (KAPA Library Amplification Kit). The fragment size distributions of amplified libraries prepared with the KAPA vs. IntegenX reagents were similar, whereas post-ligation yields for libraries prepared with the KAPA Library Preparation Kit were generally higher across the range of input DNA amounts. As expected, the highest yields of adapter-ligated libraries were achieved when libraries were prepared with the KAPA Library Preparation Kit and the Apollo 324 ILM ChIP-Seq method. Experimental details and results, as well as a detailed protocol for using the KAPA Library Preparation Kit to construct DNA libraries for Illumina sequencing on the Apollo 324 System, are included in this document. Version 1.13

2 Materials and Methods Human and E. coli genomic DNA (gdna) were fragmented with a nebulizer to generate fragments with a median size of 250 bp or 300 bp, respectively. Fragmented DNA was purified with a QIAquick PCR Purification Kit (QIAGEN), and quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Fragment size distributions were confirmed using a Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). Two dilutions of each DNA sample were prepared. For the human gdna, these were 16.7 ng/µl and 3.33 ng/µl (250 ng or 50 ng per 15 µl, respectively). The dilutions for the E. coli gdna were 6.67 ng/µl and 0.67 ng/µl, respectively; corresponding to 100 ng or 10 ng per 15 µl aliquot. Duplicate libraries were prepared from each of the four inputs, with the standard ILM DNA library preparation method on the Apollo 324 System, using either the KAPA Library Preparation Kit (Kapa Biosystems) or the PrepX Next Generation Sequencing Library Prep Kit for the Apollo 324 System (IntegenX). IntegenX reactions were set up according to the standard protocol, with the exception that adapter concentrations were scaled down proportionately to maintain Kapa's recommended adapter:insert molar ratio of 10:1 across the range of DNA inputs. Kapa reactions were set up with the same indexed adapters and final adapter concentrations, but using the modified reagent setup described at the end of this document. Adapter-ligated DNA was quantified by qpcr, using the KAPA Library Quantification Kit for Illumina platforms. All libraries were amplified with KAPA HiFi HotStart ReadyMix, according to the recommended protocol. The number of amplification cycles varied for libraries prepared from different amounts of input DNA, and were based on the actual yield of adapter-ligated library fragments for each sample. HPLC-purified adapter oligos and library amplification primers were from Integrated DNA Technologies. Library amplification primers were identical in sequence to those supplied in the KAPA Library Quantification Kit for Illumina platforms. Amplified libraries were analyzed using a Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies), and post-amplification yields were confirmed with the KAPA Library Quantification Kit for Illumina platforms. Library construction was repeated as described above using the Apollo 324 ILM ChIP-Seq method, but with the KAPA Library Preparation Kit only. The resulting adapter-ligated libraries were quantified using the KAPA Library Quantification Kit, but were not amplified. Results Electropherograms for selected amplified libraries, prepared with the standard Apollo 324 ILM DNA library method, are given in Figure 2. All libraries prepared with both the Kapa and IntegenX reagents had the desired fragment size distribution, with a peak in the range of bp. The median fragment size was ~250 bp in all cases. Kapa libraries generated with the ILM ChIP-Seq method where not analyzed electrophoretically after ligation, as unamplified E. coli 100 ng (KAPA_1) E. coli 100 ng (KAPA_2) Human 250 ng (KAPA) Human 50 ng (KAPA) E. coli 100 ng (IntX_1) E. coli 100 ng (IntX_2) Human 250 ng (IntX) Human 50 ng (IntX) Figure 2. Electropherograms of amplified libraries prepared with the KAPA Library Preparation Kit (left) or PrepX Next Generation Sequencing Library Prep Kit for the Apollo 324 System (right). Libraries were prepared, amplified and analyzed as described in Materials and Methods. Both replicates for libraries prepared from 100 ng E.coli gdna are shown, whereas only the replicate with the highest post-amplification peak is shown for libraries prepared from 250 and 50 ng human gdna. Libaries prepared from 10 ng input DNA are not shown, as their concentrations were close or below the detection limit of the assay. adapter-ligated libraries are known to migrate anomalously presumably because the single-stranded "arms" of Illumina Y-adapters get entangled in gel matrices. Electrophoretic analysis of the products generated from these libraries during qpcrbased quantification (not shown) indicated that the average fragment size of libraries prepared with the ILM ChIP-Seq method was similar to that of libraries prepared with the standard ILM DNA library preparation method. This is expected to be the case for DNA fragmented to an average size in the range of bp. Yields of adapter-ligated DNA obtained with the KAPA Library Preparation Kit and the standard ILM DNA library preparation method were generally higher than those obtained with the IntegenX reagent kit (see Table 1 on the next page). Absolute numbers are not given, as the instrument was performing below normal specifications for the duration of the project. As expected, yields of adapter-ligated library fragments obtained with the KAPA Library Preparation Kit and the ILM ChIP-Seq method were higher than those obtained with the same reagents and samples using standard ILM DNA library preparation method.

3 Table 1. Relative yields of adapter-ligated library fragments obtained with different reagents and methods 1,2. Input DNA Conclusions Automated library construction for Illumina sequencing can be successfully performed on the Apollo 324 instrument, using the KAPA Library Preparation Kit and either the standard or ChIP-Seq ILM DNA library preparation methods. In order to use the KAPA Library Preparation Kit, minor changes to the reagent preparation process and deck setup are required, as outlined in the Detailed Protocol. Lower-throughput users with access to an Apollo 324 System can therefore benefit from the improved performance offered by the KAPA Library Preparation Kit. On the Apollo 324 instrument, the primary benefit is a higher yield of adapter-ligated molecules from a given amount of input DNA. This translates to higher library complexity and improved sequence coverage, or the ability to construct high-quality libraries from lower amounts of input DNA. Higher yields of adapter-ligated DNA also reduces the number of library amplification cycles required to generate sufficient material for downstream processing (sequencing or target enrichment). Acknowledgements We wish to thank Robert (Bob) Steen and Kristin Waraska (Department of Genetics/Biopolymers Facility, Harvard Medical School) and Ryan Kim (DNA Technologies and Expression Analysis Cores, University of California Davis Genome Center) for their valuable contributions to this project. References IntegenX Standard KAPA Standard 1. Quail M.A., et al. Nature Methods 9, (2012). 2. Ross, M.G., et al. Genome Biology 14: R51 (2013). KAPA ChIP-Seq 250 ng human ng E. coli ng human ng E. coli Standard = standard ILM DNA library preparation method (upper and lower size cuts); ChIP-Seq = ILM ChIP-Seq method (lower size cut only). 2 Data are based on the amount (in ng, excluding the mass of the adapters) of library after the post-ligation size selection or cleanup, as quantified with the KAPA Library Quantification Kit, and represent the average from duplicate preparations. Absolute values are not given as the instrument returned suboptimal yields throughout the duration of the project. However, conclusions about the relative performance of library construction reagents can still be drawn. To this end, a value of 1.0 was assigned to the yield of adapter-ligated DNA from the 10 ng E. coli library prepared with the IntegenX reagents. Detailed Protocol Detailed guidelines for the construction of libraries for Illumina sequencing on the Apollo 324 instrument with KAPA library construction reagents are provided below. Please refer to your Apollo 324 instrument user guide and DNA library preparation protocols for all instructions not included in this document. 1. General preparation 1.1 Prepare your instrument and work area as per your usual procedures. Select the appropriate DNA library preparation protocol from the instrument menu: Use the ILM ChIP-Seq method if you do not require size selection (an upper and a lower cut), and wish to achieve the highest yield of adapter-ligated molecules. Use the standard ILM DNA library preparation method if your sequencing application and sample fragment distribution requires size selection, i.e. the removal of both large and small fragments. 1.2 Ensure that the KAPA Library Preparation Kit (KK8200 or KK8201, with Standard Library Amplification; or KK8240 or KK8241, no Library Amplification module) and adapter stock solution(s) are fully thawed. Vortex all reagent tubes, spin down and place on ice. 1.3 Prepare 15 ml of 70% ethanol by mixing 10.5 ml of 100% ethanol and 4.5 ml of PCR-grade water in a sterile tube. 1.4 Ensure that the Agencourt AMPure XP reagent is equilibrated to room temperature and thoroughly mixed. 1.5 Transfer 15 ml PCR-grade water to a sterile tube for the run. 1.6 Original Apollo 324 reagent kits (IntegenX) contain a 2.5 M NaCl solution, which is required for the standard ILM DNA library prep protocol, but not for the ILM ChIP- Seq protocol. This solution can be prepared by dissolving g NaCl (molecular biology grade) in 10 ml PCR-grade water. Filter-sterilize or autoclave the solution before use. You will need 0.8 ml per run of eight samples. 2. Sample preparation 2.1 Fragment your input DNA to the appropriate size for your sequencing read length and application, using your normal procedure. You will need ng of fragmented DNA for each library to be constructed on the Apollo 324 instrument. To ensure the highest library complexity, always use the maximum amount of DNA available, but no more than 300 ng per library. To provide for sample loss during fragmentation and QC, it is recommended that you fragment 20 40% more DNA than the amount you wish to use for library construction. 2.2 Confirm the size distribution of each fragmented DNA sample electrophoretically, e.g. using an Agilent Bioanalyzer or TapeStation, PerkinElmer LabChip GX, or similar instrument. 2.3 Quantify each fragmented DNA sample using a NanoDrop spectrophotometer or a Qubit /PicoGreen dsdna assay.

4 2.4 If required, adjust the concentration of each fragmented DNA sample with PCR-grade water, to fall in the range of ng/µl (i.e ng per 15 µl). Place samples on ice. 2.5 Label the tubes in a strip of 8 x 0.2 ml PCR tubes (Axygen, ) with the numbers 1 8 from left to right, to ensure that the correct DNA sample is matched with the correct adapter solution when reagents are set up on the deck. Transfer 15 µl of diluted, fragmented DNA into the appropriate tube. Cap the tubes with a strip of caps (Axygen, ) and place on ice. 3. Adapter preparation For optimal results, an adapter:insert molar ratio of ~10:1 should be maintained across the range of DNA inputs. For each sample, you will require 15 µl of adapter solution, at 2X the concentration needed to achieve the desired ~10:1 molar ratio once combined with 15 µl DNA. Please contact support@kapabiosystems.com for assistance if needed. 3.1 Label the tubes in a strip of 8 x 0.2 ml PCR tubes (Axygen, ) with the numbers 1A 8A from left to right, to ensure that the correct adapter solution is matched with the correct DNA sample when reagents are set up on the deck. 3.2 Transfer the calculated volume of the correct indexed or nonindexed adapter stock solution into each of the eight tubes of the adapter strip. Adjust the volume in each tube to 15 µl with PCR-grade water, and mix well by carefully pipetting up and down. Cap the tubes with a strip of caps and place on ice. 4. Preparation of reagents for End Repair, A-Tailing and Ligation 4.1 A master mix (buffer and enzyme) is prepared for Ligation, while End Repair and A-Tailing Buffers and Enzymes are prepared separately. Note that the KAPA End Repair Enzyme Mix does not require dilution. For each reagent, add the components listed in Table 2 together in a sterile reagent tube, in the order as listed. Vortex and spin tubes down. Always prepare enough reagents for the number of samples to be processed Once completed, distribute each mix into an appropriately labelled 8 x 0.2 ml tube strip, as follows: Ligation Master Mix: 15.0 µl per tube End Repair Buffer: 15.0 µl per tube A-Tailing Enzyme: 5.0 µl per tube A-Tailing Buffer: 15.0 µl per tube 4.3 Pipette 5.0 µl of KAPA End Repair Enzyme Mix into each of the eight tubes of a fifth tube strip. 4.4 Cap all the strips and place on ice. 5. Deck setup and running the protocol 5.1 Follow the instructions in your Apollo 324 protocol to initiate the instrument and prepare the deck for the run. In short: Place 24 (3 x 8 strips) of 1.1 ml tubes (Axygen, ) in Rows 1 3 of Block 1. Place a clean, 96-well microtiter plate (Eppendorf, ) in Block 2, with well A1 correctly positioned (lower left corner). Table 2. Preparation of End Repair, A-Tailing and Ligation reagents. For Reaction component Per sample For the standard ILM DNA library preparation samples method only: transfer 100 µl of 2.5 M NaCl solution into each of the End Repair Buffer eight wells in Row 1 of the microtiter plate. PCR-grade water 10.5 µl 94.5 µl 10X KAPA End Repair Buffer 4.5 µl 40.5 µl A-Tailing Enzyme PCR-grade water 2.0 µl 18.0 µl KAPA A-Tailing Enzyme 3.0 µl 27.0 µl A-Tailing Buffer PCR-grade water 10.5 µl 94.5 µl 10X KAPA A-Tailing Buffer 4.5 µl 40.5 µl Ligation Master Mix PCR-grade water 4.0 µl 36.0 µl 5X KAPA Ligation Buffer 6.0 µl 54.0 µl KAPA Ligase 5.0 µl 45.0 µl Place the piercing tips and dispensing pipette tips in Block 5, as indicated in the Apollo 324 protocol. Place four empty reservoirs in Block 6, as indicated in the Apollo 324 protocol. Place the strip tube with fragmented DNA into Row 8 of Block 3. Make sure that the strip is correctly orientated. Mix the AMPure XP reagent to ensure that the solution is homogenous. Carefully transfer 200 µl AMPure XP reagent to each of the tubes of a clean strip of 8 x 0.2 ml tubes. Place the strip in Row 6 of Block 3. Place a clean, empty strip of 8 x 0.2 ml tubes in Row 5 of Block 3. Libraries will be collected in this strip. Place the tube strip with KAPA End Repair Enzyme Mix (prepared in step 4.3) in Row 4 of Block 3. Place the tube strip with End Repair Buffer (prepared in step 4.2) in Row 3 of Block 3. Place the tube strip with A-Tailing Enzyme (prepared in step 4.2) in Row 2 of Block 3. Place the tube strip with A-Tailing Buffer (prepared in step 4.2) in Row 1 of Block 3. Place the tube strip with 2X adapter solutions (prepared in step 3.2) in Row 1 of Block 4. Make sure that the strip is correctly orientated, to match the orientation of the DNA samples in Row 8 of Block 3.

5 Place the tube strip with Ligation Master Mix (prepared in step 4.2) in Row 2 of Block 4. Dispense the 15 ml 70% ethanol (prepared in step 1.3) in Reservoir 3 of Block 6. Dispense the 15 ml PCR-grade water (set aside in step 1.5) in Reservoir 1 of Block 6. Check that Reservoirs 2 and 4 of Block 6 are empty, and that there are no consumables or reagents in Blocks 7 and 8. Refer to the Apollo 324 protocol for specific instructions relating to the deck setup for a run of <8 samples. Verify that the deck and reagents have been set up correctly (see Figure 3 on the next page), and start the run. 6. Processing of prepared libraries 6.1 Once the run has been completed, retrieve the samples from the instrument. Since these unamplified, adapter-ligated libraries contain single-stranded DNA ends and were eluted in an unbuffered solution (PCR-grade water), they should be processed (amplified or sequenced) as soon as possible, or stored at -20 C until they can be processed. 6.2 Quantify libraries by qpcr, using the KAPA Library Quantification Kit (for Illumina platforms) with the appropriate reference dye for your qpcr instrument. Convert the calculated library concentrations to the amount (in ng) of adapter-ligated molecules in each library, to allow you to determine how much input DNA was converted to adapter-ligated DNA. Contact support@kapabiosystems.com to obtain a spreadsheet that can be used to process quantification data and do the above conversion. 6.3 Use the data from step 6.2 to calculate the number of amplification cycles needed (if any) to generate sufficient library material for your downstream process (cluster amplification and sequencing or target enrichment/capture). Contact support@kapabiosystems.com for a calculator that can be used for this purpose. 6.4 Amplify each library with the KAPA HiFi HotStart Library Amplification ReadyMix supplied in the KAPA Library Preparation Kit, as per the instructions supplied in the KAPA Library Preparation Technical Data Sheet. In short: Each amplification reaction contains 25 µl of 2X KAPA HiFi HotStart ReadyMix, library amplification primers at a final concentration of 0.5 µm each, ~15 µl template DNA (adapter-ligated library) and PCR-grade water up to 50 µl. Cycling is performed with the parameters given in Table Purify the amplified libraries as described in the KAPA Library Preparation Kit Technical Data Sheet. A 1X cleanup with Agencourt AMPure XP reagent is recommended to eliminate primer dimers, and concentrate libraries appropriately for the next step in the process. Amplified libraries should be eluted in 10 mm Tris-HCl (ph 8.0) and can be stored at -20 C for up to 6 months. Table 3. Library amplification cycling parameters. Cycling step Temperature and time Number of cycles Initial denaturation 95 C for 45 sec 1 Denaturation Annealing Extension 98 C for 15 sec 60 C 1 for 15 sec 72 C for 30 sec Final extension 72 C for 60 sec 1 1 Optimal annealing temperature when using the following library amplification primers: F primer = 5 -AAT GAT ACG GCG ACC ACC GA-3 ; R primer = 5 -CAA GCA GAA GAC GGC ATA CGA-3. The annealing temperature may have to be optimized if different library amplification primers are used. 2 The optimal number of amplification cycles depends on the amount of adapter-ligated library used as template in the amplification reaction, as well as the amount of amplified library needed for the next process (capture or sequencing). Contact support@kapabiosystems.com for assistance if needed. 6.6 Quantify purified, amplified libraries by qpcr, using the KAPA Library Quantification Kit (for Illumina platforms) with the appropriate reference dye for your qpcr instrument. 6.7 Confirm the size distribution of the final libraries electrophoretically, e.g. using an Agilent Bioanalyzer or TapeStation, PerkinElmer LabChip GX, or similar instrument. The size distribution of the libraries should be in the range of bp, with a peak between 300 and 400 bp.

6 Figure 3. Reagent and deck layout for the preparation of DNA libaries for Illumina sequencing on the Apollo 324 instrument, using the KAPA Libary Preparation Kit (KK8200, KK8201, KK8240 or KK8241). KAPA library construction reagents, prepared as described in Section 4 of the Detailed Protocol, are indicated in green. The layout is identical for the standard ILM DNA libary preparation and ILM ChIP-Seq methods, with the exception of the 2.5 M NaCl in Row 1 of Block 2 (highlighted in gray), which is not required for the ILM ChIP-Seq method. Technical support: support@kapabiosystems.com Ordering information: sales@kapabiosystems.com