Ovation Library System for Low Complexity Samples...a PCR amplicon can pose a challenge for the...

U S E R G U I D E

Ovation® Library System for Low Complexity SamplesPART NO. 9092-16, 9092-96, AND 9092-256

Patents, Licensing and Trademarks

© 2013–2019 Tecan Genomics, Inc. All rights reserved. The Encore®, Ovation® and Applause™ families of products and methods of their use are covered by several issued U.S. and International patents and pending applications (www.nugen.com). NuGEN, Allegro, Celero, NuQuant, SoLo, Metaplex, DimerFree, AnyDeplete, Ovation, SPIA, Ribo-SPIA, Encore and Imagine More From Less are trademarks or registered trademarks of Tecan Genomics, Inc. Other marks appearing in these materials are marks of their respective owners.

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Table of Contents

Contents

I. Introduction ......................................................................................................... 1A. Background ....................................................................................................... 1B. Performance Specifications ............................................................................... 4C. Library Quantitation .......................................................................................... 4D. Quality Control ................................................................................................. 4E. Storage and Stability ......................................................................................... 4F. Safety Data Sheet (SDS) .................................................................................... 4

II. Components ........................................................................................................ 5A. Reagents Provided ............................................................................................ 5B. Additional Equipment, Reagents and Labware ................................................ 6

III. Planning the Experiment ..................................................................................... 7A. Input DNA Requirements .................................................................................. 7B. When to Consider Using the Alternate PCR-free Protocol ............................... 7C. Using The Ovation Library System for Low Complexity Samples on Illumina

NGS Platforms................................................................................................... 7D. Amplified Library Storage ................................................................................. 9E. Data Analysis and Parsing Multiplex Libraries .................................................. 9

IV. Protocol ............................................................................................................. 11A. Overview ......................................................................................................... 11B. Protocol Notes ................................................................................................ 11C. Agencourt® Beads ........................................................................................... 12D. Programming the Thermal Cycler ................................................................... 14E. Setting Up an Experiment ............................................................................... 15F. End Repair ....................................................................................................... 15G. Ligation ........................................................................................................... 16H. Ligation Purification ........................................................................................ 17I. Library Amplification ....................................................................................... 18J. Amplified Library Purification .......................................................................... 19K. Quantitative and Qualitative Assessment of the Library ................................. 20

V. PCR-Free Protocol ............................................................................................. 22A. End Repair ....................................................................................................... 22B. Ligation ........................................................................................................... 23C. Final Repair ..................................................................................................... 24D. Library Purification .......................................................................................... 24E. Quantitative Assessment of the Library. .......................................................... 26

VII. Technical Support .............................................................................................. 27

VIII. Appendix ........................................................................................................... 28A. Adaptors Included in Kit Configurations ........................................................ 28B. Sequences of the Barcodes in Index 1 and Index 2 ........................................ 28C. Bioanalyzer Migration Artifacts ....................................................................... 31D. Frequently Asked Questions (FAQs) ............................................................... 32E. Update History ............................................................................................... 33

1 Ovation Library System for Low Complexity Samples

A. Background

The Ovation Library System for Low Complexity Samples provides a simple, fast and scalable solution for producing Illumina sequencing libraries from PCR amplicons or other low sequence diversity samples. The sequencing of low diversity samples such as a PCR amplicon can pose a challenge for the Illumina platforms. The Real Time Analysis (RTA) software works best with high sequence diversity, such as is found in most genomic libraries. Low sequence diversity, especially in the first few sequencing cycles, can result in problems with proper phasing/prephasing estimates, matrix estimates, cluster identification, and even focusing. Until now, the only solution has been to spike a substantial amount of high diversity library, such as the PhiX control library, into the lane. The Ovation Library System for Low Complexity Samples employs Diversity Adaptor core technology, specially designed adaptors that introduce sequence diver-sity, thus enabling the production of high quality sequence from low diversity samples without the need for PhiX control library spike-in. These libraries are suitable for sequencing on Illumina NGS platforms.

As shown in Figure 1, the streamlined workflow consists of three main steps: end repair to generate blunt ends, adaptor ligation and PCR amplification to produce the final library. The entire workflow can be completed in less than four hours, and yields DNA libraries ready for cluster formation and either single read or paired-end sequencing. An alternate, PCR-free protocol is also provided for users who wish to omit the final PCR amplification step (Figure 2).

I. Introduction


I. Introduction

Figure 1. The Ovation Library System for Low Complexity Samples workflow.

Input amplicon

End-repair

5´ 3´P

P

Bead purification

Add FWD+REV diversity adaptors, ligate

Fill in and PCR

Cluster formation and sequencing

AATCGGATCGGTAGGAT …



Bead purification


I. Introduction

Figure 2. The Ovation Library System for Low Complexity Samples PCR-free workflow.

Input amplicon

End-repair

5´ 3´P

P

Final repair

Cluster formation and sequencing




Bead purification

Pool and concentrate

Add FWD+REV diversity adaptors, ligate


I. Introduction

B. Performance Specifications

The Ovation Library System for Low Complexity Samples is designed to produce DNA libraries suitable for either single-read or paired-end sequencing on Illumina NGS platforms without gel-based size selection, using double-stranded amplicons in about four hours.

C. Library Quantitation

Libraries created using the PCR-free workflow must be quantified using qPCR. These libraries cannot be accurately quantified using other means. We recommend using KAPA Library Quantification kits from KAPA Biosystems for quantitation. See Section VII, Quantitative and Qualitative Assessment of the Library, for details.

D. Quality Control

Every lot of the Ovation Library System for Low Complexity Samples undergoes func-tional testing to meet specifications for library generation performance.

E. Storage and Stability

The Ovation Library System for Low Complexity Samples is shipped on dry ice and should be unpacked immediately upon receipt.

Note: This product contains components with multiple storage temperatures.

Vials labeled Agencourt® Beads (clear cap) should be removed from the top of the ship-ping carton upon delivery and stored at 4°C.

All other components should be stored at –20°C on internal shelves of a freezer without a defrost cycle. The kit has been tested to perform to specifications after as many as six freeze/thaw cycles. Kits handled and stored according to the above guidelines will per-form to specifications for at least six months.

Important: Do not warm L2-F and L2-R Adaptor Mixes above room temperature. Heating will severely degrade performance.

F. Safety Data Sheet (SDS)

An MSDS for this product is available on the NuGEN website at www.nugen.com/products/ovation-low-complexity-library-preparation-kit


A. Reagents Provided

Table 1. Ovation Library System for Low Complexity Samples Reagents

(Part No. 9092-256). Please refer to Table 9 in Appendix A for additional

information on adaptors provided for Part Nos. 9092-16 and 9092-96.

COMPONENT PART NUMBER VIAL CAP VIAL NUMBER

End Repair Buffer Mix S01686 Blue ER1 ver 3

End Repair Enzyme Mix S01687 Blue ER2 ver 4

Ligation Buffer Mix S01689 Yellow L1 ver 4

Forward Adaptor Ligation Mix S01949–S01956

(8 tubes)Yellow L2 ver 16-F

Reverse Adaptor Ligation MixS01957-S01988

(32 tubes)Yellow L2 ver 16-R

Ligation Enzyme Mix S01690 Yellow L3 ver 4

Amplification Buffer Mix S01691 Red P1 ver 2

Amplification Primer Mix S01692 Red P2 ver 5

Amplification Enzyme Mix S01693 Red P3

DMSO S01694 Red P4

Nuclease-free Water S01001 Green D1

Note: SPRI beads (Agencourt® RNAClean® XP or Ampure XP) are required for several steps in the library process and must be obtained separately from Beckman Coulter. NuGEN does not supply SPRI beads in this kit.

II. Components


II. Components

B. Additional Equipment, Reagents and Labware

Required Materials

• Equipment - Agilent 2100 Bioanalyzer or materials and equipment for electrophoretic

analysis of nucleic acids - Microcentrifuge for individual 1.5 mL and 0.5 mL tubes - 0.5–10 µL pipette, 2–20 µL pipette, 20–200 µL pipette, 200–1000 µL pipette - Vortexer - Thermal cycler with 0.2 mL tube heat block, heated lid, and 100 µL

reaction capacity - Appropriate spectrophotometer and cuvettes, or Nanodrop® UV-Vis

Spectrophotometer• Reagents

- Agencourt RNAClean XP Beads or AMPure XP Beads (Beckman Coulter, Cat. #A63987 or A63881)

- Ethanol (Sigma-Aldrich, Cat. #E7023), for purification steps - Low-EDTA TE Buffer, 1X, pH 8.0 (Alfa Aesar, Cat. #J75793) - (Required only for PCR-free protocol) KAPA Library Quantification kit speci-

fied for the Illumina NGS platform and the real-time PCR system to be used• Supplies and Labware

- Nuclease-free pipette tips - 1.5 mL and 0.5 mL RNase-free microcentrifuge tubes - 0.2 mL individual thin-wall PCR tubes or 8 X 0.2 mL strip PCR tubes or

0.2 mL thin-wall PCR plates - Magnetic stand for 0.2 mL strip tubes or plates. (Beckman Coulter Cat.

#A29164 or A32782; Thermo Fisher Scientific Cat. #12331D, 12027, or 12332D; Promega Cat. #V8351). Other mag netic stands may be used as well, although their performance has not been validated by Tecan.

- Disposable gloves - Kimwipes - Ice bucket - Cleaning solutions such as DNA-OFF™ (MP Biomedicals, Cat. #QD0500)

To Order

• Alfa, www.alfa.com• Beckman Coulter, www.beckmancoulter.com • Illumina, www.illumina.com• KAPA Biosystems, www.kapabiosystems.com• MP Biomedicals, www.mpbio.com• QIAGEN, www.qiagen.com• Sigma-Aldrich, Inc., www.sigmaaldrich.com


A. Input DNA Requirements

The Ovation Library System for Low Complexity Samples is designed to work with 40–60 ng of a 200 bp double-stranded amplicon. For other sizes, adjust the amount to keep the moles of amplicon approximately the same. For example, for a 100 bp ampli-con, use 20–30 ng. To date, amplicons as small as 100 bp, and as large as 450 bp have been successfully sequenced using this system.

B. When to Consider Using the Alternate PCR-free Protocol

The final PCR amplification in the standard protocol will result in 1–2 µg of purified library, which is enough library for standard library QC such as Nanodrop, Qubit, Bioanalyzer and agarose gel electrophoresis, in addition to sequencing at low multiplex levels. The PCR-free protocol results in a library that is not suitable for the standard library QC approaches listed above. These libraries must be quantified by real time qPCR. In addition, the PCR-free protocol yields a library concentration of approximately 1nM, which is generally less than needed for most standard Illumina sequencing pro-tocols. However, if you are planning on pooling 8 or more libraries together and you wish to avoid PCR amplification of your library, you may choose the PCR-free protocol. By pooling and vacuum concentrating the pool, you can achieve the recommended concentration needed for sequencing.

C. Using The Ovation Library System for Low Complexity Samples on Illumina NGS Platforms

The Ovation Library System for Low Complexity Samples uses a dual-index barcod-ing strategy. As shown in Figure 3, Index 1 is located within the reverse adaptor, while Index 2 is located within the forward adaptor. The Index 1 + Index 2 pair is then used to identify the sample. The kit includes eight forward adaptors, each with a unique Index 2 barcode, and 32 reverse adaptors, each with a unique Index 1 barcode. This enables a maximum of 256 (8x32) sample multiplexing per run identified by the 256 unique index pairs. Both the Index 1 and the Index 2 barcodes are 8 nt in length, and all indexes are an edit distance of 3 or greater from each other (Faircloth and Glenn, 2012, PLoS ONE 7(8): e42543).

The forward and reverse adaptors are supplied separately to allow maximum flexibility. One forward and one reverse adaptor must be mixed together before ligation to the sample in order to form a functional library.

III. Planning the Experiment



Figure 3. Multiplexing strategy used by the Ovation Library System for Low Complexity Samples.

Flow cell surface

Library Insert

Index 2N2–N8

Insert

Library Molecule Schematic

IlluminaIndex 2Seq Primer

IlluminaForward ReadSeq Primer

IlluminaIndex 1Seq Primer

Illumina ReverseRead Seq Primer

Index 1

N2–N8

Insert

Illumina recommends color-balancing index sequences for highest quality results. For convenience, the eight Index 2 sequences are supplied as color-balanced pairs: F001+F002, F003+F004, etc. In addition, Index 1 sequences are supplied as color balanced sets of eight: R001 to R008, R009 to R0016, etc. Use a minimum of two color-balanced Index 2 adaptors per lane. Index sequences and color balanced set information is located in Appendix B. See the “Index Adapters Pooling Guide” and “Low-Plex Pooling Guidelines for Enrichment Protocols” from Illumina for more infor-mation on color balancing.

When setting up the sequencing run, Index 1 and Index 2 reads should each be 8 bases. Please note that Illumina follows different protocols for single-end and paired-end flow cells. For paired-end flow cells, the Index 1 read product is removed and the template anneals to the grafted forward primer on the surface of the flow cell. The run proceeds through an additional seven chemistry-only cycles (no images are taken) fol-lowed by eight cycles of sequencing to read Index 2. Take this extra 7 cycles of chemis-try into consideration when planning the sequencing run. For single-read flow cells, the Index 1 read product is removed and the Index 2 sequencing primer is annealed to the same template strand. The run proceeds through 8 cycles of sequencing to read the Index 2. No extra chemistry-only cycles are used in the single-read flow cell protocol.

The 32 Index 1 and 8 Index 2 adaptors allow multiplexing of up to 256 samples per lane. For 32 or fewer samples, Index 2 reads may be omitted. However, Index 2 reads are useful as a control for instrument run-to-run sample carryover. On some Illumina instruments, low levels of library molecules from the previous run have been sequenced



in the current run. See Illumina’s support bulletin dated April 24, 2013, “Best Practices for High Sensitivity Applications: Minimizing Sample Carryover.”

For example, Index 2 barcodes F001 and F002 could be used for sequencing run 1, F003 and F004 for sequencing run 2, and so on. Any reads containing Index 2 bar-codes F001 or F002 observed in runs 2, 3, or 4 can be identified as carryover from run 1 and discarded. There would be three runs performed (runs 2, 3, and 4) between the first and second time barcodes F001 and F002 are used, greatly reducing the potential of carryover affecting results.

D. Amplified Library Storage

Amplified libraries may be stored at –20°C.

E. Data Analysis and Parsing Multiplex Libraries

The first step in the post-sequencing data analysis for the Ovation Library System for Low Complexity Samples reads is to parse the reads by sample, based on the barcode sequence pairs. These sequences are found in Appendix B. Barcode sequences can be parsed on-instrument or off-line. For on-instrument parsing perform the following steps:

1. Using the Illumina Experiment Manager, create a new sample sheet. Select the appropriate instrument as the instrument, and TruSeq HT as the assay. Select the 2 index radio button. Use the pull down menus to select any Index 1 and Index 2 (these will be changed later). Save the .csv file.

2. Open the .csv file in Excel, and add the appropriate NuGEN® barcode names and sequences for each sample. Use a new line for each sample. Save as .csv and use this sample sheet for the sequencing run.

When attempting to identify rare variants, sample misassignment during multiplex sequencing can be problematic. The dual index approach used in this system has been shown to reduce false assignment rates. This rate can be further reduced by applying a quality score filter on the index reads, so that only data from clusters with high quality index read sequence is used. See Kircher, Sawer, and Meyer (2012) Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform, Nucleic Acids Research, 40(1):e3 for further details.

Once the data have been parsed according to sample, additional sample specific data analysis may be employed according to the requirements of the experiment. The adaptors used in the Ovation Library System for Low Complexity Samples add 2–8 random bases at the start of both the forward and reverse reads. This variable length dephases the amplicon sequence and evens out the nucleotide distribution at each cycle during sequencing. When analyzing sequencing results, it may be beneficial to trim the first 8 bases of the read before performing alignment. This will trim any



random bases, in addition to trimming between 2–8 bases from the insert, depending on the length of the particular adaptor molecule ligated to that end.

The portion of the amplicon derived from the PCR primers is typically non-informative, since it represents primer oligonucleotide sequence, and not the sequence of the target. Depending on the purity of these oligonucleotides, a higher level of deletions and substitutions may be observed. Therefore, it may be useful to align sequences based on the non-primer portion of the insert, and then trim away the ends. This will remove the random sequence derived from the Diversity Adaptors as well as the non-informative sequence derived from the PCR primers.


Important: If a PCR-free protocol is desired please refer to Section V.

A. Overview

The library preparation process used in the Ovation Library System for Low Complexity Samples is performed in three stages:

1. DNA end repair 0.75 hours

2. Adaptor ligation and purification 1.5 hours

3. Amplification and purification 1.75 hours

Total time to prepare amplified library ~4 hours

Components in the Ovation Library System for Low Complexity Samples are color coded, with each color linked to a specific stage of the process. Performing each stage requires making a master mix then adding it to the reaction, followed by incubation. Master mixes are prepared by mixing components provided for that stage.

B. Protocol Notes

• We recommend the use of a positive control DNA, especially the first time a reaction is set up, to establish a baseline for performance and provide the opportunity to become familiar with the bead purification steps.

• Use the water provided with the kit (green: D1) or an alternate source of nuclease-free water. We do not recommend the use of DEPC-treated water with this protocol.

• Setting up a minimum of four reactions at a time ensures that very small vol-umes are not being pipetted.

• Thaw components used in each step and immediately place them on ice. Do not thaw all reagents at once.

• Always keep thawed reagents and reaction tubes on ice unless otherwise instructed.

• After thawing and mixing buffer mixes, if any precipitate is observed, re-dissolve it completely prior to use. The buffer mix may be gently warmed for 2 minutes at room temperature followed by brief vortexing. Do not warm the Diversity Adaptors, or any enzyme or primer mixes.

• When placing small amounts of reagents into the reaction mix, pipet up and down several times to ensure complete transfer.

• When instructed to pipet mix, gently aspirate and dispense a volume that is at least half of the total volume of the reaction mix.

• Always allow the thermal cycler to reach the initial incubation temperature prior to placing the tubes or plates in the block.

IV. Protocol


IV. Protocol

• When preparing master mixes, use a minimal amount of extra material to ensure there will be sufficient reagent volumes to perform the full number of reactions specified for the kit.

• Components and reagents from other NuGEN kits should not be used with the Ovation Library System for Low Complexity Samples.

• Use only fresh ethanol stocks to make 70% ethanol used in the purification protocols.

• Make the ethanol mixes fresh, carefully measuring both the ethanol and water with pipettes. Lower concentrations of ethanol in wash solutions will result in loss of yield as the higher aqueous content will dissolve the cDNA and wash it off the beads or column.

Important Note: Libraries created using the PCR-free workflow must be quantified using qPCR. These libraries cannot be accurately quantified using other means. We recommend using KAPA Library Quantification kits from KAPA Biosystems for quantitation. See Section V.E., Quantitative Assessment of the Library, for details.

C. Agencourt® Beads

Tips and Notes

There are significant modifications to the Agencourt beads standard procedure; there-fore, it is important to follow the protocols outlined in this user guide for the use of these beads. However, the Beckman Coulter user guide may be reviewed to become familiar with the manufacturer’s recommendations.

The bead purification processes used in this kit consist of the following steps:

1. Binding of DNA to Agencourt beads

2. Magnetic separation of beads from supernatant

3. Ethanol wash of bound beads to remove contaminants

4. Elution of bound DNA from beads


IV. Protocol

Figure 4. Agencourt Bead purification process overview.

1. Binding 2. Separation

Magnet

3. Ethanol Wash 4. Elution

Magnet Magnet

Reproduced from original picture from Agencourt/Beckman Coulter Genomics

Additional Tips and Notes

• Remove beads from 4°C and leave at room temperature for at least 15 minutes before use, and ensure that they have completely reached room temperature. Cold beads reduce recovery.

• Fully resuspend beads by inverting and tapping before adding to sample. • Note that ratio of Agencourt bead volume to sample volume varies between

the End Repair Product Bead Purification protocol and the Bead Purification of the Amplified Material protocol. The bead:sample ratios used differ from the standard Agencourt protocol.

• It is critical to let the beads separate on the magnet for a full 5 minutes. Removing binding buffer before the beads have completely separated will impact DNA yields.

• After completing the binding step, it is important to minimize bead loss when removing the binding buffer. With the samples placed on the magnet, remove only 160 μL (for Ligation Purification) and 140 μL (for Bead Purification of the Amplified Material) of the binding buffer from each sample. Some liquid will remain at the bottom of the tube but this will minimize bead loss.

• Any significant loss of beads during the ethanol washes will impact DNA yields, so make certain to minimize bead loss throughout the procedure.

• Ensure that the ethanol wash is freshly prepared from fresh ethanol stocks at the indicated concentration. Lower percent ethanol mixes will reduce recovery.

• During the ethanol washes, keep the samples on the magnet. The beads should not be allowed to disperse; the magnet will keep the beads on the walls of sample wells or tubes in a small ring. It is critical that all residual ethanol be removed prior to continuing with the next step. Therefore, when removing the final ethanol wash, first remove most of the ethanol, then allow the excess to collect at the bottom of the tube before removing the remaining ethanol. This reduces the required bead air drying time.

• After drying the beads for the time specified in the protocol, inspect each tube carefully and make certain that all the ethanol has evaporated before proceeding.


IV. Protocol

• It is strongly recommended that strip tubes or partial plates are firmly placed when used with the magnetic plate. We do not advise the use of individual tubes as they are difficult to position stably on the magnetic plates.

D. Programming the Thermal Cycler

Use a thermal cycler with a heat block designed for 0.2 mL tubes, equipped with a heated lid, and with a capacity of 100 μL reaction volume. Prepare the programs shown in Table 4, following the operating instructions provided by the manufacturer. For ther-mal cyclers with an adjustable heated lid, set the lid temperature to 100°C only when sample temperature reaches above 30°C. For thermal cyclers with a fixed temperature heated lid (e.g., ABI GeneAmp® PCR 9600 and 9700 models), use the default settings (typically 100 to 105°C).

Table 2. Thermal Cycler Programming

END REPAIR

Program 1 End Repair

25°C – 30 min, 70°C – 10 min, hold at 4°C

LIGATION

Program 2 Ligation


AMPLIFICATION

Program 3 Amplification

72°C – 2 min, 12 cycles* (94°C – 30 sec, 60°C – 30 sec, 72°C – 1 min), 72°C – 5 min, hold at 10°C

FINAL REPAIR (ALTERNATE PCR-FREE PROTOCOL)

Program 4 Final Repair

72°C – 2 min, hold at 25°C

Important Note: If using DNA input greater than 0.4 picomoles (equivalent to 60 ng of 200 bp amplicon), the number of PCR cycles may be reduced, one cycle for each two-fold increase in input.


IV. Protocol

E. Setting Up an Experiment

It is important when performing multiplex sequencing to produce each library inde-pendently, and not to cross-contaminate adaptors or samples. Before starting, decide which Index 1 and Index 2 will be used for each sample. See Appendix A for informa-tion on the number of adaptors provided for each kit configuration. Multiplexing is achieved by mixing the amplified libraries prior to adding to the flow cell. A list of the adaptors and barcodes is found in Appendix B.

Important: If a PCR-free protocol is desired please refer to Section V.

F. End Repair

Note: Remove P4 (DMSO) for the Library Amplification step and allow to thaw on the bench top at room temperature.

1. Remove the End Repair Buffer Mix (blue: ER1), End Repair Enzyme Mix (blue: ER2) and Nuclease-free Water (green: D1) from –20°C storage.

2. Thaw ER1 at room temperature. Mix by vortexing, spin and place on ice.

3. Spin down contents of ER2 and place on ice.

4. Leave the Nuclease-free Water (green: D1), to thaw at room temperature.

5. Dilute the DNA sample (40–60 ng of 200 bp amplicon, or mole equivalent for other sizes) to 11 µL with water.

6. Prepare a master mix by combining ER1 and ER2 in a 0.5 mL capped tube, accord-ing to the volumes shown in Table 3.

Table 3. End Repair Master Mix (volumes listed are for a single reaction)

END REPAIR BUFFER MIX (BLUE: ER1 ver 3)

END REPAIR ENZYME MIX (BLUE: ER2 ver 4)

3.5 µL 0.5 µL

7. Add 4 μL of the End Repair Master Mix to 11 µL of each sample.

8. Mix by pipetting, cap and spin tubes and place on ice.

9. Place the tubes in a pre-warmed thermal cycler programmed to run Program 1 (End Repair; see Table 2):


Mix by pipetting and spin down the master mix briefly. Place on ice. Use immediately.


IV. Protocol

10. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice.

11. Continue immediately with the Ligation protocol.

G. Ligation

1. Remove the Ligation Buffer Mix (yellow: L1), Forward and Reverse Adaptor Mixes that will be used (yellow: L2 ver 16-F and L2 ver 16-R) and Ligation Enzyme Mix (yellow: L3) from –20°C storage.

2. Thaw L1 and L2 at room temperature. Mix by vortexing, spin and place on ice.


3. Spin L3 and place on ice.

4. Add 1.5 µL of the appropriate L2 ver 16-F Forward Adaptor Mix and 1.5 µL of the appropriate L2 ver 16-R Reverse Adaptor Mix to each sample. Mix thoroughly by pipetting.

Note: All samples intended to share the same sequencing flow cell lane should have unique ligation adaptor pairs.

5. Make a master mix by combining the Nuclease-free Water (green: D1), L1 and L3 in a 0.5 mL capped tube, according to the volumes shown in Table 3. Mix by pipetting slowly, without introducing bubbles, spin and place on ice. Use mix immediately.

Note: The L1 Ligation Buffer Mix is very viscous. Please be sure to pipet this reagent slowly.

Table 4. Ligation Master Mix (volumes listed are for a single reaction)

WATER (GREEN: D1)

LIGATION BUFFER MIX (YELLOW: L1 ver 4)

LIGATION ENZYME MIX (YELLOW: L3 ver 4)

4.5 µL 6.0 µL 1.5 µL

6. Add 12 μL Ligation Master Mix to 18 µL of each reaction. Mix thoroughly by pipetting slowly and gently, spin and place on ice. Proceed immediately with the incubation.

Do not warm L2 Adaptor Mixes above room tempera-ture. Heating will severely degrade performance.



IV. Protocol

7. Place the tubes in a pre-warmed thermal cycler programmed to run Program 2 (Ligation; see Table 2):



9. Continue immediately with the Ligation Purification protocol.

H. Ligation Purification

1. Retrieve the Agencourt Beads and 70% ethanol set aside previously and ensure they are at room temperature.

2. Resuspend the beads by inverting and tapping the tube. Ensure the beads are fully resuspended before adding to the sample. After resuspending, do not spin the beads. (An excess of beads is provided; therefore, it is not necessary to recover any trapped in the cap.)

3. In a fresh plate, aliquot 80 μL of the bead suspension for each reaction.

4. Add 70 μL of room-temperature Nuclease-free Water (D1) to each ligation reac-tion. Mix thoroughly by pipetting 10 times.

5. Transfer the entire 100 μL volume to the 80 μL of bead suspension prepared above. Mix thoroughly by pipetting 10 times. Incubate at room temperature for 10 minutes.

6. Transfer the tubes to the magnet plate and let stand 5 minutes to completely clear the solution of beads.

7. Carefully remove the binding buffer and discard it.

Note: The beads should not disperse; instead, they will stay on the walls of the tubes. Significant loss of beads at this stage will impact the amount of DNA carried into PCR amplification, so ensure beads are not removed with the binding buffer or the wash.

8. With the tubes still on the magnet, add 200 μL of freshly prepared 70% ethanol and allow to stand for 30 seconds.

9. Remove the 70% ethanol wash using a pipette.

10. Repeat the 70% ethanol wash one more time, for a total of two washes.

Note: With the final wash, it is critical to remove as much of the ethanol as pos-sible. Use at least two pipetting steps and allow excess ethanol to collect at the bottom of the tubes after removing most of the ethanol in the first pipetting step.

11. Air dry the beads on the magnet for 10 minutes. Inspect each tube carefully to ensure that all the ethanol has evaporated. It is critical that all residual ethanol be removed prior to continuing.


IV. Protocol

12. Remove the tubes from the magnet.

13. Add 40 μL 1X TE buffer (low EDTA) to the dried beads. Mix thoroughly by pipet-ting to ensure all the beads are resuspended. Let stand on the bench top for 3 minutes.

14. Transfer the tubes to the magnet and let stand for 3 minutes to completely clear the solution of beads.

15. Carefully remove 36 μL of the eluate, ensuring as few beads as possible are carried over. Transfer to a fresh set of PCR tubes and place on ice.

16. Set aside the Agencourt RNAClean XP beads and 70% ethanol at room tempera-ture for use in the Amplified Library Purification protocol (section IV, J).

17. Continue immediately with the Library Amplification protocol (section IV, I).

I. Library Amplification

1. Remove the Amplification Buffer Mix (red: P1), Amplification Primer Mix (red: P2 ver 5) and Amplification Enzyme Mix (red: P3) from –20°C storage. Obtain the DMSO (red: P4) set aside earlier at room temperature.

2. Thaw P1 and P2 at room temperature. Mix by vortexing, spin and place on ice.

3. Spin P3 and place on ice.

4. Make a master mix by sequentially combining P1, P2 and P4 in an appropriately sized capped tube according to the volumes shown in Table 5. Add P3 at the last moment and mix well by pipetting, taking care to avoid bubbles, spin and place on ice.

Table 5. Amplification Master Mix (volumes listed are for a single reaction)

AMP BUFFER MIX (RED: P1 ver 2)

AMP PRIMER MIX (RED: P2 ver 5)

DMSO (RED: P4)

AMP ENZYME MIX (RED: P3)

35 µL 4.0 µL 4.0 µL 1.0 µL

5. On ice, add 44 μL of the Amplification Master Mix to 36 µL of each sample.



IV. Protocol

6. Place the tubes in a pre-warmed thermal cycler programmed to run Program 3 (Library Amplification; see Table 2):

72°C – 2 min, 12 cycles (94°C – 30 sec, 60°C – 30 sec, 72°C – 1 min), 72°C – 5 min, hold at 10°C

Important Note: If using DNA input greater than 0.4 picomoles (equivalent to 60 ng of 200 bp amplicon), the number of PCR cycles may be reduced, one cycle for each two-fold increase in input.


8. Continue with the Amplified Library Purification protocol.

J. Amplified Library Purification

1. Retrieve the Agencourt Beads and 70% ethanol set aside previously and ensure they are still at room temperature.


3. At room temperature, add the entire volume of each reaction to 80 μL (1 volume) of the bead suspension. (We have found that adding the reaction mixture to the beads results in tighter bead pellets that are much easier to resuspend, and better DNA recovery.)

4. Mix thoroughly by pipetting 10 times. It may be helpful to use a multichannel pipettor to ensure the incubation times are uniform.

5. Incubate at room temperature for 10 minutes.

6. Transfer the tubes to the magnet and let stand 5 minutes to completely clear the solution of beads.


Note: The beads should not disperse; instead, they will stay on the walls of the tubes. Significant loss of beads at this stage will impact the amount of DNA carried into ligation, so ensure beads are not removed with the binding buffer or the wash.

8. With the plate still on the magnet, add 200 μL of freshly prepared 70% ethanol and allow to stand for 30 seconds.


The purification beads should be removed from 4°C and left at bench top to reach room temperature well before the start of purification.


IV. Protocol



11. Air dry the beads on the magnet for a minimum of 10 minutes. Inspect each tube carefully to ensure that all the ethanol has evaporated. It is critical that all residual ethanol be removed prior to continuing.


13. Add 33 μL 1X TE buffer (low EDTA) to the dried beads. Mix thoroughly to ensure all the beads are resuspended.

14. Transfer the tubes to the magnet and let stand for 2 minutes.

15. Carefully remove 30 μL of the eluate, ensuring as few beads as possible are carried over, and transfer to a fresh set of tubes. When pipetting any portion of this eluted library downstream, be sure to let stand briefly on a magnet to minimize bead carryover.

16. Proceed to Quantitative and Qualitative Assessment of the Library.

Important Note: After quantitation, barcoded libraries that will be run in the same flow cell should be mixed in equimolar ratios prior to sequencing.

K. Quantitative and Qualitative Assessment of the Library

1. Quantify the library. Any dsDNA quantification method should be acceptable, however we recommend using Qubit™ dsDNA Broad Range Assay Kits (Life Technologies) or a similar assay based on intercalating dyes.

2. Run the samples on a Bioanalyzer DNA Chip. Note that the library made from an amplicon will produce a very sharp peak relative to most other sequencing librar-ies, therefore, much less DNA should be loaded in order to prevent overloading artifacts. For example, when using the High Sensitivity DNA chip, load 250 pg of amplicon library. For the DNA 1000 chip, load 25 ng.


IV. Protocol

Figure 5. Fragment distribution on Bioanalyzer High Sensitivity DNA Chip. Red trace is a 224-bp amplicon used to make two libraries, shown as green and blue traces. Adaptors add 140–152 bp to the insert, representing the adaptors (140 bp) and the variable random insert (4–16 bp), consisting of 2–8 bases at each end.

3. Prepare libraries for sequencing following the Illumina “Denature and Dilute Libraries Guide” for your specific sequencer.


This alternate PCR-free protocol results in a library that is not suitable for standard library QC approaches, and can only be analyzed by real time qPCR. In addition, the PCR-free protocol yields a library concentration of approximately 1 nM, which is gener-ally less than needed for most standard Illumina sequencing protocols. However, if you are planning on pooling 8 or more libraries together and you wish to avoid library PCR amplification, you may choose the PCR-free protocol. By pooling and vacuum concentrating the pool, you can achieve the recommended concentration needed for sequencing.

A. End Repair

Note: Remove P4 (DMSO) and allow to thaw on the bench top at room temperature.

1. Remove the End Repair Buffer Mix (blue: ER1), End Repair Enzyme Mix (blue: ER2) and Nuclease-free Water (green: D1) from –20°C storage.

2. Thaw ER1 at room temperature. Mix by vortexing, spin and place on ice.

3. Spin down contents of ER2 and place on ice.

4. Leave the Nuclease-free Water (green: D1), to thaw at room temperature.

5. Dilute the DNA sample (40–60 ng of 200 bp amplicon, or mole equivalent for other sizes) to 11 µL with water.

6. Prepare a master mix by combining ER1 and ER2 in a 0.5 mL capped tube, accord-ing to the volumes shown in Table 6.

Table 6. End Repair Master Mix (volumes listed are for a single reaction)

END REPAIR BUFFER MIX (BLUE: ER1 ver 3)

END REPAIR ENZYME MIX (BLUE: ER2 ver 4)

3.5 µL 0.5 µL

7. Add 4 μL of the End Repair Master Mix to 11 µL of each sample.

8. Mix by pipetting, cap and spin tubes and place on ice.

9. Place the tubes in a pre-warmed thermal cycler programmed to run Program 1 (End Repair; see Table 2):



11. Continue immediately with the Ligation protocol.


V. PCR-Free Protocol



B. Ligation

1. Remove the Ligation Buffer Mix (yellow: L1), Forward and Reverse Adaptor Mixes that will be used (yellow: L2 ver 16-F and L2 ver 16-R) and Ligation Enzyme Mix (yellow: L3) from –20°C storage.

2. Thaw L1 and L2 at room temperature. Mix by vortexing, spin and place on ice.


3. Spin L3 and place on ice.

4. Add 1.5 µL of the appropriate L2 ver 16-F Forward Adaptor Mix and 1.5 µL of the appropriate L2 ver 16-R Reverse Adaptor Mix to each sample. Mix thoroughly by pipetting.

Note: All samples intended to share the same sequencing flow cell lane should have unique ligation adaptor pairs.

5. Make a master mix by combining the Nuclease-free Water (green: D1), L1 and L3 in a 0.5 mL capped tube, according to the volumes shown in Table 6. Mix by pipetting slowly, without introducing bubbles, spin and place on ice. Use mix immediately.

Note: The L1 Ligation Buffer Mix is very viscous. Please be sure to pipet this reagent slowly.

Table 7. Ligation Master Mix (volumes listed are for a single reaction)

WATER (GREEN: D1)

LIGATION BUFFER MIX (YELLOW: L1 ver 4)

LIGATION ENZYME MIX (YELLOW: L3 ver 4)

4.5 µL 6.0 µL 1.5 µL

6. Add 12 μL Ligation Master Mix to 18 µL of each sample.. Mix thoroughly by pipetting slowly and gently, spin and place on ice. Proceed immediately with the incubation.

7. Place the tubes in a pre-warmed thermal cycler programmed to run Program 2 (Ligation; see Table 2):



9. Continue immediately with the Final Repair protocol.

Do not warm L2 Adaptor Mixes above room tempera-ture. Heating will severely degrade performance.




C. Final Repair

1. Remove the Amplification Buffer Mix (red: P1) and Amplification Enzyme Mix (red: P3) from –20°C storage. Obtain the DMSO (red: P4) set aside earlier at room temperature.

2. Thaw P1 at room temperature. Mix by vortexing, spin and place on ice.

3. Spin P3 and place on ice. Make a master mix by combining P1, P3 and P4 in a 0.5 mL capped tube, according to the volumes shown in Table 8. Mix by pipetting slowly, without introducing bubbles, spin and place on ice. Use the master mix immediately.

Table 8. Final Repair Master Mix (volumes listed are for a single reaction)

AMP BUFFER MIX (RED: P1 ver 2)

DMSO (RED: P4)

AMP ENZYME MIX (RED: P3)

16.5 µL 2.5 µL 1.0 µL

4. Add 20 μL Final Repair Master Mix to 30 µL of each sample. Mix thoroughly by pipetting slowly and gently, spin and place on ice. Proceed immediately with the incubation.

5. Place the tubes in a pre-warmed thermal cycler programmed to run Program 4 (Final Repair; see Table 2):

72°C – 2 min, hold at 25°C

Important Note: If the library purification step (Step D, below) cannot be performed within 60 minutes after the final repair step, change the hold step to 4°C.

6. Continue immediately with the Library Purification protocol.

D. Library Purification

1. Retrieve the Agencourt Beads and 70% ethanol set aside previously and ensure they are at room temperature.


3. In a fresh plate, aliquot 50 μL of the bead suspension for each reaction.




4. Transfer the entire 50 μL Final Repair reaction volume to the 50 μL of bead suspen-sion prepared above. Mix thoroughly by pipetting 10 times. Incubate at room temperature for 10 minutes.

5. Transfer the tubes to the magnet plate and let stand 5 minutes to completely clear the solution of beads.


Note: The beads should not disperse; instead, they will stay on the walls of the tubes. Significant loss of beads at this stage will impact the amount of DNA carried into PCR amplification, so ensure beads are not removed with the binding buffer or the wash.

7. With the tubes still on the magnet, add 200 μL of freshly prepared 70% ethanol and allow to stand for 30 seconds.




10. Air dry the beads on the magnet for 10 minutes. Inspect each tube carefully to ensure that all the ethanol has evaporated. It is critical that all residual ethanol be removed prior to continuing.


12. Add 33 μL nuclease-free water to the dried beads. Mix thoroughly by pipetting to ensure all the beads are resuspended. Let stand on the bench top for 3 minutes.

13. Transfer the tubes to the magnet and let stand for 3 minutes to completely clear the solution of beads.

14. Carefully remove 30 μL of the eluate, ensuring as few beads as possible are carried over. Transfer to a fresh set of PCR tubes and place on ice.

15. Optional: quantify libraries by quantitative real-time PCR. Based on the recom-mended input as measured by Qubit, this protocol should produce purified librar-ies at approximately 1 nM concentration.

16. Pool libraries and speed vac concentrate to the desired final concentration. Monitor the concentration process carefully to ensure that the sample does not become dry, as this may cause DNA to irreversibly adsorb to the tube resulting in sample loss.

Water is used in step 12 instead of TE buffer so that the vacuum concentration performed in step 16 does not result in high buffer concentrations.



E. Quantitative Assessment of the Library.

Libraries created using the PCR-free protocol must be quantified using qPCR. These libraries cannot be accurately quantified using other means. We recommend using KAPA Library Quantification kits from KAPA Biosystems for quantitation.

Use the KAPA Library Quantification kit specific for the Illumina NGS platform and your available qPCR instrument. Follow the instructions described by KAPA BioSystems in the Technical Data Sheet for the KAPA Library Quantitation kit you are using.

Note: For the library qPCR reactions, we recommend loading at least duplicates of 1:5000 dilutions per library on the same plate as the KAPA Biosystems concentration standards (run in triplicate).

To estimate the size of your library add 144 bp to the amplicon length. This value is required for calculating concentration from the qPCR results and includes the adaptors (136 bp) plus the 0–8 additional bases added to each end.


For help with any of our products, please contact NuGEN Technical Support at 650.590.3674 (direct) or 888.654.6544, option 2 (toll-free, U.S. only). Send faxes to 888.296.6544 (toll-free) or email [email protected].

In Europe contact NuGEN at +31.13.5780215 (Phone) or +31.13.5780216 (Fax) or email [email protected].

In all other locations, contact your NuGEN distributor for technical support.

VII. Technical Support


A. Adaptors Included in Kit Configurations

Table 9. Adaptors included in kit configurations.

PRODUCT NUMBER

NUMBER OF UNIQUE FORWARD ADAPTORS

NUMBER OF UNIQUE REVERSE ADAPTORS

TOTAL NUMBER OF UNIQUE LIBRARIES

9092-162 (sufficient quantity for 8 reactions per adaptor)

8 (sufficient quantity for 2 reactions per adaptor)

16



96



256

B. Sequences of the Barcodes in Index 1 and Index 2

Barcode sequences produced by sequencing Index 1 on the reverse adaptor, and Index 2 on the forward adaptor, are shown in Table 10 and Table 11, respectively. The bar-code sequences shown in the table are the index sequences that will be generated and reported by the sequencer. Barcodes are grouped in color balanced sets as indicated. Note that all adapters are included in 9092-256, whereas 9092-96 and 9092-16 will have a subset of the adapters listed below. Refer to the part numbers on the tubes that are received in the kit to determine which barcodes to use.

Table 10. Barcodes for Index 1 Reverse Adaptors

LIGATION ADAPTOR MIXBARCODE SEQUENCE

COLOR BALANCED SETS

R001 L2V16 Reverse Diversity Adaptor CACGTCTA 1

R002 L2V16 Reverse Diversity Adaptor AGCTAGTG 1

R003 L2V16 Reverse Diversity Adaptor ACTATCGC 1

R004 L2V16 Reverse Diversity Adaptor GTAAGGTG 1

R005 L2V16 Reverse Diversity Adaptor ACTCTCCA 1

R006 L2V16 Reverse Diversity Adaptor CGTCCATT 1

R007 L2V16 Reverse Diversity Adaptor AGCCGTAA 1

VIII. Appendix


VIII. Appendix

Barcodes for Index 1 Reverse Adaptors, continued.


COLOR BALANCED SETS

R008 L2V16 Reverse Diversity Adaptor GAGTAGAG 1

R009 L2V16 Reverse Diversity Adaptor ACGTCGTT 2

R010 L2V16 Reverse Diversity Adaptor GTCCTGTT 2

R011 L2V16 Reverse Diversity Adaptor CCTTCCAT 2

R012 L2V16 Reverse Diversity Adaptor GAAGATCC 2

R013 L2V16 Reverse Diversity Adaptor ATGGCGAT 2

R014 L2V16 Reverse Diversity Adaptor ACGTCCAA 2

R015 L2V16 Reverse Diversity Adaptor CACACATC 2

R016 L2V16 Reverse Diversity Adaptor CGGATCAA 2

R017 L2V16 Reverse Diversity Adaptor TCAGCCTT 3

R018 L2V16 Reverse Diversity Adaptor AAGGCTCT 3

R019 L2V16 Reverse Diversity Adaptor TGTTCCGT 3

R020 L2V16 Reverse Diversity Adaptor GGAATGTC 3

R021 L2V16 Reverse Diversity Adaptor AACGCCTT 3

R022 L2V16 Reverse Diversity Adaptor ACGAATCC 3

R023 L2V16 Reverse Diversity Adaptor TCGCTATC 3

R024 L2V16 Reverse Diversity Adaptor AGCCTATC 3

R025 L2V16 Reverse Diversity Adaptor TCGGATTC 4

R026 L2V16 Reverse Diversity Adaptor CGGAGTAT 4

R027 L2V16 Reverse Diversity Adaptor GATGGAGT 4

R028 L2V16 Reverse Diversity Adaptor AGAGGATG 4

R029 L2V16 Reverse Diversity Adaptor ACGCTTCT 4

R030 L2V16 Reverse Diversity Adaptor CACAGGAA 4

R031 L2V16 Reverse Diversity Adaptor TGTCGACT 4


VIII. Appendix

Barcodes for Index 1 Reverse Adaptors, continued.


COLOR BALANCED SETS

R032 L2V16 Reverse Diversity Adaptor ACTCTGAG 4

Table 11. Barcodes for Index 2 Forward Adaptors


COLOR BALANCED SETS

F001 L2V16 Forward Diversity Adaptor AATGCAGC 1

F002 L2V16 Forward Diversity Adaptor GGACGTAT 1

F003 L2V16 Forward Diversity Adaptor GAACTGCT 2

F004 L2V16 Forward Diversity Adaptor AGGTCCTA 2

F005 L2V16 Forward Diversity Adaptor CAGCCGCA 3

F006 L2V16 Forward Diversity Adaptor GTCTTAGT 3

F007 L2V16 Forward Diversity Adaptor GTGAAGGA 4

F008 L2V16 Forward Diversity Adaptor CACGTACG 4


VIII. Appendix

C. Bioanalyzer Migration Artifacts

In some instances, PCR enrichment may result in the appearance of a shoulder to the right of the main library peak in Bioanalyzer electropherograms. This phenomenon is due to the ability of the amplicon to be ligated in two orientations relative to the forward adaptor. In the later cycles of library PCR, during the annealing step, a product strand from orientation 1 will anneal to the complementary insert from orientation 2, resulting in a duplex insert, but single stranded ends. This molecule migrates more slowly on the Bioanalyzer, resulting in the appearance of a shoulder. This does not affect sequencing, nor does it affect qPCR quantification, but it may result in a slight underestimation of library concentration when using Qubit fluorescence quantification.

In order to demonstrate that the appearance of this shoulder is a migration artifact and not a property of the library itself, a library with a shoulder was diluted into fresh PCR reagents then subjected to 0 or 1 thermal cycles, followed by purification and HS DNA chip analysis. The blue curve in Figure 6 represents the no-cycle control, while the red curve is the reaction with one PCR cycle. The polymerase extends newly-primed strands, resulting in perfectly duplexed library products that display no migration artifacts.

Figure 6. Fragment distribution on HS DNA Bioanalyzer chip with (blue) or without (red) migration artifacts.

If desired, performing a single round of PCR in the presence of excess primer will resolve the material to a single peak of the correct library size.

When quantifying libraries that may have been subject to migration artifacts, use the lower molecular weight peak to estimate library size and qPCR to determine concentration.


VIII. Appendix

D. Frequently Asked Questions (FAQs)

Q1. What kind of sequencing primers can I use with your library?The Ovation Library System for Low Complexity Samples uses a dual-index barcoding strategy and needs standard sequencing and dual index read primers. Please follow Illumina’s recommendations for the appropriate setup of your sequencing run for dual-indexed samples.

Q2. Can the Ovation Library System for Low Complexity Samples be used with paired-end sequencing?Yes, they can be used for both single end and paired-end sequencing. Special consideration should be given to the expected insert size in the paired-end assay. The expected distances between the 5´-most and 3´-most coordinates of paired-end reads will depend on the average fragment size of the insert.

Q3. How much material should I load into the sequencer?Please follow manufacturer’s recommendations for library QC, quantitation, balancing and loading of the amplified library on the sequencer.

Q4. How can gel purification be eliminated from the workflow and still pre-vent adaptor dimer formation?The Ovation Library System for Low Complexity Samples workflow uses bead-based purification and efficient primer design, thus eliminating the need for gel-based purification.

Q5. Can I modify the number of PCR amplification cycles recommended by the Ovation Library System for Low Complexity Samples workflow when using different DNA input amounts?Generally speaking, fewer PCR cycles will be needed when working with larger input amounts. Reduce one cycle for each two-fold increase in input.

Q6. What kind of error correction is used to minimize the impact of sequenc-ing errors in the barcodes?Each eight-base barcode is a minimum edit distance of 3 from any other barcode. This means that a minimum of three edits (replacement, insertion, or deletion) must occur before one barcode becomes a different barcode. For further details on the barcode design strategy, please refer to Faircloth BC, Glenn TC (2012), Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels. PLoS ONE 7(8): e42543. doi:10.1371/journal.pone.0042543.

Q7. Can I combine the barcoded libraries prior to amplification?The stoichiometry of barcoded libraries may be adversely affected by this modification to the Ovation Library System for Low Complexity Samples workflow. We suggest that the libraries be amplified and quantitated inde-pendently before being balanced and pooled for sequencing.

VIII. Appendix

Tecan Genomics, Inc.

Headquarters USA

900 Chesapeake DriveRedwood City, CA 94063 USA Toll Free Tel: 888.654.6544 Toll Free Fax: 888.296.6544 [email protected]@tecan.com

Europe

P.O. Box 1099350 AC LeekThe Netherlands Tel: +31-13-5780215Fax: [email protected]

Worldwide

For our international distributors contact information, visit our website

www.nugen.com

Q8. What is the expected yield of the amplified DNA library using the Ovation Library System for Low Complexity Samples?The expected yield is 1–2 μg, depending on the quality and quantity of the source DNA and the number of PCR cycles employed. This amount is in excess of the amount of DNA required for sequencing.

Q9. What is the expected yield of the PCR-free protocol?The expected yield is approximately 1 nM using recommended inputs. This yield may be increased with additional input material up to 120 ng of a 200 bp amplicon or mole equivalent (0.8 pmol).

E. Update History

This document, the Ovation Library System for Low Complexity Samples user guide (M01369 v4.1), is an update to address the following topics.

Description Section Page(s)

Updated legal information, emails, address, trademarks, and logo.

Throughout Throughout

Updated Agencourt bead reagent information. Throughout Throughout

Updated purification protocol language to be consistent. IV.H., IV.J.,

V.D.17, 19, 24-25

M01369 v4.1For research use only. Not for use in diagnostic procedures.

©2019 Tecan Genomics, Inc. All rights reserved. The Encore®, Ovation® and Applause™ families of Products and methods of their use are covered by several issued U.S. and International patents and pending applications (www.nugen.com). NuGEN, Allegro, Celero, NuQuant, SoLo, Metaplex, DimerFree, AnyDeplete, Ovation, SPIA, Ribo-SPIA, Encore and Imagine More From Less are trademarks or registered trademarks of Tecan Genomics, Inc. Other marks appearing in these materials are marks of their respective owners.

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