ThruPLEX® DNA-seq Kit
`Instruction Manual
`
`High Performance Library Preparation
`for Illumina® NGS Platforms
`
`Research Use Only
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`Product Use Limitations
`
`ThruPLEX® DNA-seq Kit is intended for Research Use Only. It may not be used for any other purposes
`including, but not limited to, use in diagnostics, forensics, therapeutics, or in humans. ThruPLEX DNA-
`seq may not be transferred to third parties, resold, modified for resale or used to manufacture
`commercial products without prior written approval of Rubicon Genomics, Inc.
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`*Protected by U.S. Patents 7,803,550; 8,071,312; 8,399,199; 8,728,737 and corresponding foreign
`patents. Additional patents are pending.
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`Contents
`
`Product Description ......................................................................................................................................................................................................... 3
`
`Kit Contents ........................................................................................................................................................................................................................ 3
`
`Technical Assistance ........................................................................................................................................................................................................ 4
`
`A.
`
`B.
`
`Introduction ............................................................................................................................................................................................................ 5
`
`ThruPLEX DNA-seq Kit ........................................................................................................................................................................................ 6
`
`I. Overview ............................................................................................................................................................................................................... 6
`
`II. Principle ............................................................................................................................................................................................................... 6
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`III. ThruPLEX DNA-seq Workflow ..................................................................................................................................................................... 7
`
`C.
`
`Getting Started ...................................................................................................................................................................................................... 9
`
`I. Additional Supplies and Equipment Needed ............................................................................................................................................. 9
`
`II. Thermal Cycler Considerations ..................................................................................................................................................................... 9
`
`III. Starting Material ........................................................................................................................................................................................... 10
`
`IV. Positive and Negative Controls ................................................................................................................................................................ 11
`
`V. Preparation of Master Mixes ...................................................................................................................................................................... 11
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`VI. Indexing Reagents ........................................................................................................................................................................................ 12
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`VII. Using Illumina Experiment Manager ..................................................................................................................................................... 12
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`VIII. Target Enrichment ..................................................................................................................................................................................... 12
`
`D.
`
`ThruPLEX DNA-seq Library Preparation Protocol ................................................................................................................................... 13
`
`I. Template Preparation Step .......................................................................................................................................................................... 13
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`II. Library Synthesis Step .................................................................................................................................................................................. 14
`
`III. Library Amplification Step ......................................................................................................................................................................... 15
`
`E.
`
`Library Processing for Illumina Next Generation Sequencing .............................................................................................................. 19
`
`I. Overview ............................................................................................................................................................................................................ 19
`
`II. Library Quantification ................................................................................................................................................................................... 20
`
`III. Additional Amplification ............................................................................................................................................................................. 20
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`IV. Library Pooling for Purification ................................................................................................................................................................ 21
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`V. Library Purification by AMPure XP beads .............................................................................................................................................. 21
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`VI. Library Purification by Gel Size-Selection (Alternate) ....................................................................................................................... 23
`
`VII. Sequencing Recommendations .............................................................................................................................................................. 24
`
`Appendix 1. Indexing Reagents.................................................................................................................................................................................. 25
`
`A. Overview .......................................................................................................................................................................................................... 25
`
`B. ThruPLEX DNA-seq 6S (12 Rxn) and 12S (48 Rxn) Kits ....................................................................................................................... 26
`
`C. ThruPLEX DNA-seq 48S Kit ........................................................................................................................................................................ 27
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`D. ThruPLEX DNA-seq 48D and 96D Kits .................................................................................................................................................... 30
`
`Appendix 2. Troubleshooting Guide......................................................................................................................................................................... 33
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`Product Description
`ThruPLEX® DNA-seq builds on the innovative ThruPLEX chemistry to generate DNA libraries with
`expanded multiplexing capability and with even greater diversity. Kits contain up to 96 Illumina-
`compatible indexes. ThruPLEX DNA-seq can be used in DNA-seq, RNA-seq, or ChIP-seq and offers
`robust target enrichment performance with all of the leading platforms. For more information,
`please visit: http://rubicongenomics.com/products/thruplex-dna-seq-kit/.
`
`Kit Contents
`
`ThruPLEX DNA-seq Kit contains sufficient reagents to prepare up to the specified number of
`reactions. Enough buffers and enzymes are provided for 4 uses or freeze-thaw cycles. Contents of
`ThruPLEX DNA-seq Kit are not interchangeable with other Rubicon Genomics products.
`
`Table 1: ThruPLEX DNA-seq Kit Contents – Single Index Kits
`
`6S Kit
`(12 Rxn)
`R400523
`6 Single
`Indexes
`12 Reactions
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
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`12S Kit
`(48 Rxn)
`R400428
`12 Single
`Indexes
`48 Reactions
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`
`Cap
`Color
`Red
`Red
`Yellow
`Yellow
`Green
`Green
`Clear
`
`Name
`Template Preparation Buffer
`Template Preparation Enzyme
`Library Synthesis Buffer
`Library Synthesis Enzyme
`Library Amplification Buffer
`Library Amplification Enzyme
`Nuclease-Free Water
`
`Indexing Reagents
`
`Blue
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`6 Tubes
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`12 Tubes
`
`Quick Protocol
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`48S Kit
`R400427
`48 Single
`Indexes
`48 Reactions
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Single Index
`Plate (48S)
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`Table 2: ThruPLEX DNA-seq Kit Contents – Dual Index Kits
`
`Name
`Template Preparation Buffer
`Template Preparation Enzyme
`Library Synthesis Buffer
`Library Synthesis Enzyme
`Library Amplification Buffer
`Library Amplification Enzyme
`Nuclease-Free Water
`
`Indexing Reagents
`
`Cap Color
`Red
`Red
`Yellow
`Yellow
`Green
`Green
`Clear
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`
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`48D Kit
`R400406
`48 Dual Indexes
`48 Reactions
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Tube
`1 Dual Index Plate
`(48B or 48D)
`
`96D Kit
`R400407
`96 Dual Indexes
`96 Reactions
`2 Tubes
`2 Tubes
`2 Tubes
`2 Tubes
`2 Tubes
`2 Tubes
`1 Tube
`1 Dual Index Plate
`(96A or 96D)
`
`Quick Protocol
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` Note: Dual Index Plates (48B) and (48D) are identical; Dual Index Plates (96A) and (96D) are also
`identical.
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`Shipping and Storage
`
`ThruPLEX DNA-seq Kit is shipped on dry ice. The kit should be stored at −20°C upon arrival.
`
`Quality Control
`
`ThruPLEX DNA-seq Kit is functionally tested using Next Generation Sequencing (NGS) to ensure
`product quality and consistency.
`
`Safety Information
`
`Follow standard laboratory safety procedures and wear a suitable lab coat, protective goggles and
`disposable gloves to ensure personal safety as well as to limit potential cross contaminations during
`the sample preparation and subsequent amplification reactions. For more information, please refer
`to
`the
`appropriate Material Safety Data Sheets
`(MSDS)
`available online
`at
`http://rubicongenomics.com/resources/msds/.
`
`
`Technical Assistance
`For technical support with any of the Rubicon Genomics, Inc. products please contact technical
`support by email: support@rubicongenomics.com or call at (734)-677-4845 (9 AM – 5:30 PM EST).
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`A. Introduction
`
`Next Generation Sequencing (NGS) is a dynamic field with rapidly evolving needs. Regardless of
`sample type or application, a DNA library must be prepared from each sample in order to be
`sequenced on Illumina NGS platforms. The process of library preparation (Figure 1) involves placing
`Illumina sequencing adapters on DNA fragments and adding Illumina-compatible indexes to allow
`pooling of multiple samples (multiplexing). This library preparation is a critical step in the NGS
`workflow and has direct impact on the quality of sequencing results.
`
`
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`Figure 1. Illumina NGS workflow: DNA samples are first purified and sheared. Library preparation follows,
`consisting of repair, Illumina adapter addition, and DNA fragment amplification. Indexed libraries are purified,
`pooled, and quantified prior to sequencing on Illumina NGS platforms.
`
`As NGS clinical applications emerge and as NGS instruments become more powerful, researchers
`and clinicians are increasingly investigating more difficult and challenging samples which are present
`in limited quantities, used in small amounts, and/or damaged. In many applications, such as cell-free
`DNA from plasma, the sample DNA material is limited and highly degraded. In cases where DNA is
`not limited, such as analysis of tumor tissues, the ability to use low input amounts is important for
`conserving samples for multiple uses. Clinical samples also necessitate careful tracking of samples; a
`protocol in which the sample never leaves the tube is advantageous to ensure accurate sample
`tracking and to avoid contamination. This growing trend requires library preparation kits which
`accurately preserve the complexity of the samples and provide higher sensitivity and greater
`multiplexing capability, with a simple workflow.
`
`The commitment to fulfill these needs is the core of ThruPLEX DNA-seq. It has been developed to
`expand multiplexing capability and provide high-quality, Illumina-compatible NGS libraries from low
`input amounts. ThruPLEX DNA-seq’s three-step, single-tube library preparation workflow (Figure 2)
`is the simplest in the industry and minimizes handling errors and loss of valuable samples.
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`Figure 2. ThruPLEX DNA-seq single-tube library preparation workflow: The ThruPLEX DNA-seq
`workflow consists of 3 simple steps that take place in the same PCR tube or well and eliminates the need
`to purify and transfer the sample material.
`
`B. ThruPLEX DNA-seq Kit
`
`I. Overview
`
`The ThruPLEX DNA-seq Kit is designed to provide up to 96 indexed libraries for higher multiplexing
`capabilities on Illumina NGS platforms. The ThruPLEX DNA-seq chemistry is engineered and
`optimized to generate DNA libraries with high molecular complexity from the lowest input amounts.
`Only 50 pg to 50 ng of fragmented double-stranded DNA is required for library preparation. The
`entire three-step workflow takes place in a single tube or well in about 2 hours. No intermediate
`purification steps and no sample transfers are necessary to prevent handling errors and loss of
`valuable samples. Providing high library diversity, ThruPLEX DNA-seq libraries excel in target
`enrichment performance and deliver high quality sequencing results.
`
`The ThruPLEX DNA-seq Kit includes all necessary reagents including indexes for multiplexing up to
`96 samples. Once purified and quantified, the resulting library is ready for Illumina NGS instruments
`using standard Illumina sequencing reagents and protocols. The kit provides excellent results for
`high-coverage, deep sequencing such as de novo sequencing, whole genome resequencing, whole
`exome sequencing, and/or other enrichment techniques. It is ideally suited for use in ChIP-seq and
`for use with small fragments of DNA such as cell-free plasma DNA.
`
`II. Principle
`
`The ThruPLEX DNA-seq Kit is based on Rubicon Genomics’ patented ThruPLEX technology (Figure
`3). Unlike other NGS library preparation kits, which are based on ligation of Y-adapters, ThruPLEX
`uses stem-loop adapters to construct high quality libraries in a fast and efficient workflow. In the first
`step, Template Preparation, the DNA is repaired and yields molecules with blunt ends. In the next
`step, stem-loop adaptors with blocked 5’ ends are ligated with high efficiency to the 5’ end of the
`genomic DNA, leaving a nick at the 3’ end. The adaptors cannot ligate to each other and do not have
`single-strand tails, both of which contribute to non-specific background found with many other NGS
`preparations. In the final step, the 3’ ends of the genomic DNA are extended to complete library
`synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. Any
`remaining free adaptors are destroyed. Hands-on time and the risk of contamination are minimized
`by using a single tube and eliminating intermediate purifications.
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`Figure 3. ThruPLEX DNA-seq technology: A three-step, single-tube reaction that starts with fragmented
`double-stranded DNA (0.05 ng to 50 ng). Stem-loop adapters are blunt end ligated to repaired input DNA.
`These molecules are extended then amplified to include barcodes using a high fidelity polymerase to yield an
`indexed Illumina NGS library.
`
`III. ThruPLEX DNA-seq Workflow
`
`The ThruPLEX DNA-seq Kit workflow is highly streamlined (Figure 4) and consists of the following
`three steps:
`
` Template Preparation for efficient repair of the fragmented double-stranded DNA input.
`
` Library Synthesis for ligation of Rubicon Genomics’ patented stem-loop adapters.
`
` Library Amplification for extension of the template, cleavage of the stem-loop adaptors,
`and amplification of the library. Illumina-compatible indexes are also introduced using a
`high-fidelity, highly-processive, low-bias DNA polymerase.
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`The three-step ThruPLEX DNA-seq workflow takes place in a single tube or well and is completed in
`about 2 hours.
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`Figure 4. ThruPLEX DNA-seq library preparation workflow overview: Steps involved in ThruPLEX library
`preparation for Illumina NGS starting with fragmented DNA.
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`C. Getting Started
`
`I. Additional Supplies and Equipment Needed
`
`Required Supplies and Equipment
`
` Thermal cycler (real-time instrument recommended)
`
`Note: See Thermal Cycler Considerations below.
`
` Centrifuge
` PCR tubes or 96-well PCR plates and seals
`
`Note: Select appropriate tubes or plates that are compatible with the thermal cyclers and/or real-
`time thermal cyclers used. Use appropriate caps or sealing films and seal thoroughly to eliminate
`evaporation during cycling conditions. Evaporation could reduce robustness and reproducibility
`of the reactions.
`
` Low binding aerosol barrier tips
` Freshly prepared 80% (v/v) ethanol
` Agencourt® AMPure® XP beads (Beckman Coulter, CAT. NO. A63880)
`
`Optional Supplies
`
` KAPA® Library Quantification Kit – Illumina (Kapa Biosystems, CAT. NO. specific to real time
`PCR system used)
` EvaGreen® Dye, 20X in water (Biotium, CAT. NO. 31000-T)
` Fluorescein Calibration Dye (Bio-Rad Laboratories, CAT. NO. 170-8780)
`
`II. Thermal Cycler Considerations
`
`Thermal cycling and heated lid
`
`Use a thermal cycler equipped with a heated lid that can handle 50 µL reaction volumes. Set the
`temperature of the heated lid to 101°C – 105°C to avoid sample evaporation during incubation and
`cycling.
`
`Thermal cycler ramp rates
`
`We recommend a ramp rate of 3C/s – 5C/s; higher ramp rates are not recommended and could
`impact the quality of the library.
`
`Monitoring amplification during the Library Amplification Reaction
`
`Amplification can be monitored using a real-time thermal cycler with the addition of fluorescent dyes
`(not provided with the kit, see Optional Supplies above) to the reaction (Figure 5). If a regular thermal
`cycler is used instead, there is no need to add the dyes; use an appropriate amount of nuclease-free
`water to prepare the Library Amplification Master Mix. In the absence of real-time monitoring, library
`amplification can be analyzed by gel or by analysis of an aliquot of the library using the Agilent®
`Bioanalyzer® (see Library Quantification, Section E.II.).
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`Depending on the real-time instrument used, select an appropriate calibration dye and mix with
`EvaGreen dye to prepare the dye mix (see Library Amplification Step, Section D.III.). For some real-
`time instruments, calibration dye may not be needed; please refer to the real-time thermal cycler
`instrument’s manual.
`
`
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`Figure 5. Example of real-time analysis of library amplification using ThruPLEX DNA-seq: A typical real-
`time amplification analysis of libraries prepared with ThruPLEX DNA-seq Kit using 20 ng, 2 ng, or 200 pg of
`Covaris-sheared human DNA (GM 10851, Coriell Institute, 200 bp) relative to a No Template Control (NTC).
`Results were obtained using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) with EvaGreen as
`the dye for detection and fluorescein as the calibration dye. The red line marks the midpoint of the linear phase
`of the amplification curves and is used to determine the optimal number of amplification cycles at Stage 5 of
`the Library Amplification Reaction (Section D.III.). It is recommended to stay within one cycle above or below
`the optimal number of cycles. For example, for a 2 ng input, the optimal number of amplification cycles is 10±1
`cycles or 9 to 11 cycles. The Relative Fluorescence Unit (RFU) values on the y-axis may vary based on the
`instrument used.
`
`III. Starting Material
`
`Nucleic Acid
`
`Source
`
`Type
`
`Molecular Weight
`Input Amount
`Input Volume
`Input Buffer
`
`DNA Sample Requirements
`Fragmented double-stranded DNA or cDNA
`Cells, plasma, urine, other biofluids, FFPE, tissues,
`fresh tissues, frozen tissues
`Mechanically sheared; enzymatically fragmented;
`ChIP DNA; low molecular weight cell-free DNA
`< 1,000 bp
`50 pg to 50 ng
`10 µL
`≤ 10 mM Tris, ≤ 0.1 mM EDTA
`
`DNA Format
`
`Fragmented double-stranded DNA (gDNA or cDNA), chromatin immunoprecipitates (ChIP),
`degraded DNA from sources such as FFPE, plasma, or other biofluids are suitable. This kit is not for
`use with single-stranded DNA (ssDNA) or RNA.
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`Input DNA Amount
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`Input DNA in the range of 50 pg to 50 ng can be used as starting material. For deep Whole Genome
`Sequencing (WGS) and Whole Exome Sequencing (WES) using human gDNA, FFPE, or plasma DNA,
`greater than 10 ng of input DNA is recommended to achieve a highly diverse library. For sequencing
`samples with reduced complexity, such as cDNA, ChIP DNA, bacterial DNA, or targeted genomic
`regions, lower input amounts (picogram levels) can be used.
`
`Fragment Size
`
`The optimal DNA fragment size is less than 1,000 bp. The ThruPLEX DNA-seq Kit is a ligation-based
`technology and adapters added during the process result in an approximately 140 bp increase in the
`size of each DNA template fragment. Library molecules with shorter inserts (200 – 300 bp) tend to
`cluster and amplify more efficiently on the Illumina flow cell. Depending on the application and
`requirements, the AMPure purification step following the final step (Library Amplification) can be
`replaced with a size-selection step to remove unwanted fragments.
`
`Input Volume
`
`The maximum input sample volume is 10 µL. If a sample is in a larger volume, the DNA must be
`concentrated into 10 µL or less. Alternatively, the sample may be split into 10 µL aliquots, processed
`in separate tubes, and the corresponding products pooled prior to the purification step preceding
`sequencing.
`
`Input Buffer
`
`Input DNA must be eluted or resuspended in a low-salt and low-EDTA buffered solution. The
`preferred buffer is low TE (10 mM Tris, 0.1 mM EDTA, pH 8.0). The concentrations of Tris and EDTA
`must not exceed 10 mM and 0.1 mM, respectively. Avoid phosphate containing buffers.
`
`IV. Positive and Negative Controls
`
`If necessary, include appropriate positive and negative controls in the experimental design to help
`verify that reactions proceed as expected. If the experimental samples contain any carryover
`contaminant(s) in the buffer, the downstream reactions may be impacted, and inclusion of controls
`would help elucidate such problems. A suitable positive control (reference DNA) is Covaris-sheared
`purified genomic DNA (200 – 300 bp) of comparable input amount. Always prepare fresh dilutions of
`reference DNA. Include a negative control (No Template Control, NTC) with low TE buffer (10 mM
`Tris, 0.1 mM EDTA, pH 8.0) or nuclease-free water. The positive control and experimental samples
`should perform equivalently, while the NTC should not amplify.
`
`V. Preparation of Master Mixes
`
`A master mix with appropriate buffers and enzymes must be prepared at each workflow step based
`on the number of reactions to be performed. Transfer the enzymes to ice just prior to use and
`centrifuge briefly to collect contents at the bottom of the tube. Thaw the buffers, vortex briefly and
`centrifuge prior to use. Keep all the components and master mixes on ice. Once the master mix is
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`prepared, thoroughly mix the contents several times with a pipette while avoiding introduction of
`excessive air bubbles and briefly centrifuge prior to dispensing into the PCR plate or tube(s).
`
`VI. Indexing Reagents
`
`ThruPLEX DNA-seq Kit includes all necessary reagents including Indexing Reagents for multiplexing
`samples. The Indexing Reagents consist of amplification primers containing Illumina-compatible
`indexes. Index sequences can be downloaded as CSV files at the ThruPLEX DNA-seq Product Page,
`under the Resources tab: http://rubicongenomics.com/products/thruplex-dna-seq-kit/.
`
`Before starting the ThruPLEX DNA-seq Library Preparation Protocol (Section D), refer to Appendix
`I for information on index sequences, Index Plate handling instructions, and multiplexing and index
`pooling guidelines.
`
`ThruPLEX DNA-seq 6S (12 Rxn) Kits
`
`Indexing Reagents are pre-dispensed in 6 individual tubes with blue caps. Each tube contains
`sufficient volume for up to 8 uses. No more than 4 freeze/thaw cycles are recommended for the
`Indexing Reagent Tubes.
`
`ThruPLEX DNA-seq 12S (48 Rxn) Kits
`
`Indexing Reagents are pre-dispensed in 12 individual tubes with blue caps. Each tube contains
`sufficient volume for up to 8 uses. No more than 4 freeze/thaw cycles are recommended for the
`Indexing Reagent Tubes.
`
`ThruPLEX DNA-seq 48S, 48D, and 96D Kits
`
`Indexing Reagents are pre-dispensed and sealed in a linear barcoded Index Plate. The Index Plate is
`sealed with foil that can be pierced with a multichannel pipet tip to collect the required amount of
`index to assemble the reactions. Each well of the Index Plate contains sufficient volume for a single
`use. No more than 4 freeze/thaw cycles are recommended for the Index Plate.
`
`VII. Using Illumina Experiment Manager
`
`Make sure the latest version of the Illumina Experiment Manager (IEM) is installed (version 1.8 or
`later). Prior to starting the ThruPLEX DNA-seq Library Preparation Protocol (Section D), create a
`Sample Sheet in the IEM to select and validate appropriate indexes to use in your experiments.
`Refer to Appendix 1 for guidelines on using the IEM to validate your index combinations.
`
`VIII. Target Enrichment
`
`ThruPLEX DNA-seq is compatible with the major exome and target enrichment products, including
`Agilent SureSelect®, Roche NimbleGen® SeqCap® EZ and custom panels. ThruPLEX DNA-seq target
`enrichment protocols and application notes can be assessed through the Applications section of the
`Rubicon Genomics website at: http://rubicongenomics.com/applications/enrichment/.
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`D. ThruPLEX DNA-seq Library Preparation Protocol
`
`I. Template Preparation Step
`
`Template Preparation Reagents
`
`Template Preparation Reagents
`Reagent
`Cap color
`Template Preparation Buffer
`Red
`Template Preparation Enzyme
`Red
`
` Note: Assemble all reactions in thin wall 96-well PCR plates or PCR tube(s) that are compatible
`with the thermal cycler and or real-time thermal cycler used.
`
`Template Preparation Protocol
`
`1. Prepare samples as described below.
`
` Samples: Dispense 10 µL of fragmented doubled-stranded DNA into each PCR tube or
`well of a PCR plate.
`
` Positive control reactions using reference DNA: If necessary, assemble reactions using
`10 µL of a reference gDNA (e.g., Covaris-fragmented DNA, 200-300 bp average size) at
`an input amount comparable to that of the samples.
`
` Negative control reactions/No Template Controls (NTCs): If necessary, assemble
`NTCs with 10 µL of nuclease-free water or TE buffer (e.g., 10 mM Tris, 0.1 mM EDTA, pH
`8.0).
`
` Note: The maximum volume of DNA cannot exceed 10 µL.
`
`2. Prepare Template Preparation Master Mix as described in the table below for the desired
`number of reactions. Mix thoroughly with a pipette. Keep on ice until used.
`
`Template Preparation Master Mix
`Component
`Cap color Volume/Reaction
`Template Preparation Buffer
`Red
`2.0 µL
`Template Preparation Enzyme
`Red
`1.0 µL
`
`3. Assemble the Template Preparation Reactions Mixture as shown in the table below. To each
`10 µL sample from step 1 above, add 3 µL of the Template Preparation Master Mix.
`
`Template Preparation Reaction Mixture
`Component Volume/Reaction
`10 µL
`3 µL
`13 µL
`
`Sample
`Template Preparation Master Mix
`Total Volume
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`4. Mix thoroughly with a pipette.
`
`5. Seal the PCR plate using an appropriate sealing film or tightly cap the tube(s).
`
`6. Centrifuge briefly to ensure the entire volume of the reaction is collected at the bottom of each
`well.
`
`7. Place the plate or tube(s) in a thermal cycler with heated lid set to 101°C – 105°C. Perform the
`Template Preparation Reaction using the conditions in the table below:
`
`Template Preparation Reaction
`Temperature
`Time
`22°C
`25 min
`55°C
`20 min
`4°C
`Hold for ≤ 2 hours
`
`8. After the thermal cycler reaches 4°C, remove the plate or tube(s) and centrifuge briefly.
`
`9. Proceed to the Library Synthesis Step.
`
` Note: Following the Template Preparation Step, continue to Library Synthesis Step in the same
`plate or tube(s).
`
`II. Library Synthesis Step
`
`Library Synthesis Reagents
`
`Library Synthesis Protocol
`
`Library Synthesis Reagents
`Reagent
`Cap Color
`Library Synthesis Buffer
`Yellow
`Library Synthesis Enzyme
`Yellow
`
`1. Prepare Library Synthesis Master Mix as described in the table below for the desired number of
`reactions. Mix thoroughly with a pipette. Keep on ice until used.
`Library Synthesis Master Mix
`Component
`Cap Color Volume/Reaction
`Library Synthesis Buffer
`Yellow
`1.0 µL
`Library Synthesis Enzyme
`Yellow
`1.0 µL
`
`
`
`
`
`2. Remove the seal on the plate or open the tube(s).
`
`
`
`
`
`
`
`QAM-108-003
`
`
`
`14
`
`15
`
`00015
`
`

`

`
`
`3. Assemble the Library Synthesis Reaction Mixture as shown in the table below. To each well or
`tube, add 2 µL of the Library Synthesis Master Mix.
`
`
`
`
`
`
`
`
`
`
`
`Library Synthesis Reaction Mixture
`Component
`Volume/Reaction
`Template Preparation Reaction Product
`13 µL
`Library Synthesis Master Mix
` 2 µL
`Total Volume
`15 µL
`
`4. Mix thoroughly with a pipette.
`
`5. Seal the PCR plate using an appropriate sealing film or tightly cap the tube(s).
`
`6. Centrifuge briefly to collect the contents to the bottom of each well or tube.
`
`7. Return the plate or tube(s) to the thermal cycler with heated lid set to 101°C – 105°C. Perform
`Library Synthesis Reaction using the cycling conditions in the table below:
`
`
`
`
`
`
`
`
`
`Library Synthesis Reaction
`Temperature
`Time
`22°C
`40 min
` 4°C
`Hold for ≤30 min
`
`8. After the thermal cycler reaches 4°C remove the plate or tube(s) and centrifuge briefly.
`
`9. Continue to the Library Amplification Step.
`
` Note: Following the Library Synthesis step, continue Library Amplification Reaction in the same
`plate or tube(s) maintained at 4°C.
`
`III. Library Amplification Step
`
`Multiple stages occur during the Library Amplification Reaction (see table in step 8 below). Stage
`1 and Stage 2 extend and cleave the stem loop adapters. Proper programming of the thermal
`cycler is critical for these steps to be completed correctly, with no denaturation step until Stage
`3. Illumina-compatible indexes are incorporated into the template library in Stage 4 using 4
`amplification cycles. In Stage 5, the resulting template is amplified; the number of cycles required
`at this stage is dependent on the amount of input DNA used. Samples are cooled to 4C in Stage 6,
`after which they are pooled and purified or stored at −20C.
`
` Note: Refer to Appendix 1 for selecting the appropriate indexes to use for your experiments.
`
`Selection of the optimal number of cycles for library amplification (Stage 5 ▲):
`
`The number of PCR cycles required at Stage 5 of the Library Amplification Reaction is dependent
`on the amount of input DNA and thermal cycler used. Use the table below as a guide for selecting
`the number of PCR cycles.
`
`QAM-108-00