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Agilent SureSelect QXT Whole Genome Library Prep Sequencer Protocol Manual PDF
Summary of Content for Agilent SureSelect QXT Whole Genome Library Prep Sequencer Protocol Manual PDF
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing Featuring Transposase-Based Library Prep Technology
Protocol Version F0, January 2021
SureSelect platform manufactured with Agilent SurePrint Technology
For Research Use Only. Not for use in diagnostic procedures.
Agilent Technologies
Notices Agilent Technologies, Inc. 2014, 2015, 2018, 2021
No part of this manual may be reproduced in any form or by any means (including elec- tronic storage and retrieval or translation into a foreign language) without prior agree- ment and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.
Manual Part Number G9682-90000
Edition Version F0, January 2021
Printed in USA
Agilent Technologies, Inc.
Warranty The material contained in this document is provided as is, and is subject to being changed, with- out notice, in future editions. Fur- ther, to the maximum extent permitted by applicable law, Agi- lent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchant- ability and fitness for a particular purpose. Agilent shall not be lia- ble for errors or for incidental or consequential damages in con- nection with the furnishing, use, or performance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this doc- ument that conflict with these terms, the warranty terms in the separate agreement shall control.
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2 SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
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SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing 3
In this Guide...
4 SureSe
This guide describes an optimized protocol for Illumina paired- end multiplexed whole- genome library preparation using the SureSelectQXT system.
If you wish to prepare target- enriched libraries using the SureSelectQXT system, instead see publication part number G9681- 90000 at www.agilent.com.
1
This chapter contains information (such as procedural notes, safety information, required reagents and equipment) that you should read and understand before you start an experiment.
2
This chapter describes the steps to prepare dual- indexed gDNA sequencing libraries for the Illumina platform.
3
This chapter contains reference information, including component kit contents, index sequences, and dual index usage guidelines.
lectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Whats New in Version F0
SureSelectQXT Whole Genome Libra
Updates to thermal cycler and plasticware recommendations (see Table 2 on page 13 and see step 4 on page 17)
Updates to ordering information for AMPure XP Kits and 1X Low TE Buffer (see Table 1 on page 12) and for Qubit Fluorometer (see Table 2 on page 13)
Minor updates to Agilent Bioanalyzer assay use instructions and reference document links (see page 27)
Updates to downstream sequencing support information including sequencing kit selection and seeding concentration updates (see Table 9 on page 31) and support for the NovaSeq platform (see page 31 through page 36 and see page 42)
Updates to instructions for adaptor trimming using Agilents AGeNT Trimmer utility and removal of SureCall adaptor trimming information (see page 36 to page 37)
Removal of reference information for expired SureSelectQXT Reagent Kits p/n G9682A/G9682B, replaced by G9684A/G9684B in 2018 (see Table 1 on page 12 and see Table 21 through Table 23 on page 40 for current Reagent Kit information)
Updates to Technical Support contact information (see page 2)
Updates to Notice to Purchaser (see page 2)
Whats New in Version E0
p/n G9682A/G9682B with p/n G9684A/G9684B for use with Illuminas HiSeq and MiSeq platforms (see Table 1 on page 12, Table 21 on page 40, and Table 19 on page 41 including footnotes for each table)
Support for use of Illuminas HiSeq 3000 and HiSeq 4000 platforms for downstream sequencing steps (see page 31 through page 33)
ry Prep for Illumina Multiplexed Sequencing 5
6 SureSe
Updates to sequencing kit selection and seeding concentration guidelines (see page 31)
Updates to custom sequencing primer dilution instructions (See page 32 to page 34)
Update to sequencing run setup recommendations (seeHiSeq or NextSeq 500 platform sequencing run setup and adaptor trimming guidelines" on page 36)
Updates to dual index multiplexing guidelines (see Table 28 on page 44)
Updates to Agilent 2100 Bioanalyzer system ordering information (see page 13)
Updates to supplier name for materials purchased from Thermo Fisher Scientific (see Table 1 on page 12 and Table 2 on page 13)
Addition of product guarantee and support statement (see Note on page 9)
Updates to Technical Support contact information (see page 2)
lectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Content
1 Before You Begin 9
Procedural Notes 10
Safety Notes 11
Required Reagents 12
Required Equipment 13
2 Sample Preparation 15
Step 1. Fragment and adaptor-tag the genomic DNA samples 16
Step 2. Purify the adaptor-tagged library using AMPure XP beads 20
Step 3. Amplify and index the adaptor-tagged DNA library 22
Step 4. Purify the amplified library with AMPure XP beads 25
Step 5. Assess library DNA quantity and quality using the 2100 Bioanalyzer and High Sensitivity DNA Assay 27
Step 6. Pool samples for multiplexed sequencing 29
Step 7. Prepare sequencing samples 31
Step 8. Set up the sequencing run and trim adaptors from the reads 36
3 Reference 39
Kit Contents 40
Nucleotide Sequences of SureSelectQXT Dual Indexes 41
Guidelines for Multiplexing with Dual-Indexed Samples 43
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing 7
Contents
8 SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing Protocol
1 Before You Begin
Procedural Notes 10
Safety Notes 11
Required Reagents 12
Required Equipment 13
Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment.
Agilent guarantees performance and provides technical support for the SureSelect reagents required for this workflow only when used as directed in this Protocol.
NOTE
9Agilent Technologies
1 Before You Begin Procedural Notes
Procedural Notes
10
The SureSelectQXT system requires high- quality DNA samples for optimal performance. Use best practices for verifying DNA sample quality before initiating the workflow. For best practice, store diluted DNA solutions at 4C to avoid repeated freeze- thaw cycles, which may compromise DNA quality.
Performance of the SureSelectQXT library preparation protocol is very sensitive to variations in amounts of DNA sample and other reaction components. It is important to quantify and dilute DNA samples as described on page 17. Carefully measure volumes for all reaction components, and combine components as described on page 17. Use best- practices for liquid handling, including regular pipette calibration, to ensure precise volume measurement.
Use care in handling the SureSelect QXT Enzyme Mix. After removing the vial from storage at 20C, keep on ice or in a cold block while in use. Return the vial to storage at 20C promptly after use.
For each protocol step that requires removal of tube cap strips, reseal the tubes with a fresh strip of domed caps. Cap deformation may result from exposure of the cap strips to the heated lid of the thermal cycler and from other procedural steps. Reuse of strip caps can cause sample loss, sample contamination, or imprecision in sample temperatures during thermal cycler incubation steps.
Use best- practices to prevent PCR product contamination of samples throughout the workflow:
1 Assign separate pre- PCR and post- PCR work areas and use dedicated equipment, supplies, and reagents in each area. In particular, never use materials designated to post- PCR work areas for pre- PCR segments of the workflow.
2 Maintain clean work areas. Clean pre- PCR surfaces that pose the highest risk of contamination daily using a 10% bleach solution.
3 Always use dedicated pre- PCR pipettors with nuclease- free aerosol- resistant tips to pipette dedicated pre- PCR solutions.
4 Wear powder- free gloves. Use good laboratory hygiene, including changing gloves after contact with any potentially- contaminated surfaces.
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Before You Begin 1 Safety Notes
SureSelectQXT Who
Possible stopping points, where samples may be stored at 20C, are marked in the protocol. Do not subject the samples to multiple freeze/thaw cycles.
To prevent contamination of reagents by nucleases, always wear powder- free laboratory gloves and use dedicated solutions and pipettors with nuclease- free aerosol- resistant tips.
In general, follow Biosafety Level 1 (BSL1) safety rules.
Safety Notes
Wear appropriate personal protective equipment (PPE) when working in the laboratory.
CAUTION
le Genome Library Prep for Illumina Multiplexed Sequencing 11
1 Before You Begin Required Reagents
Required Reagents
12
Table 1 Required Reagents for SureSelectQXT Library Preparation
Description Vendor and part number
SureSelectQXT Library Prep Kit (for Illumina HiSeq, MiSeq, and NextSeq platforms)
16 Samples 96 Samples
Agilent
p/n G9684A p/n G9684B
AMPure XP Kit 5 mL 60 mL 450 mL
Beckman Coulter Genomics p/n A63880 p/n A63881 p/n A63882
1X Low TE Buffer (10 mM Tris-HCl, pH 7.5-8.0, 0.1 mM EDTA)
Thermo Fisher Scientific p/n 12090-015, or equivalent
100% Ethanol, molecular biology grade Sigma-Aldrich p/n E7023
Qubit dsDNA HS Assay Kit or
Qubit dsDNA BR Assay Kit 100 assays 500 assays
Thermo Fisher Scientific p/n Q32851
Thermo Fisher Scientific p/n Q32850 p/n Q32853
Nuclease-free Water (not DEPC-treated) Thermo Fisher Scientific p/n AM9930
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Before You Begin 1 Required Equipment
Required Equipment
SureSelectQXT Who
Table 2 Required Equipment for SureSelectQXT Library Preparation
Description Vendor and part number
Thermal Cycler with 96-well, 0.2 ml block Various suppliers
Plasticware compatible with the selected thermal cycler:
96-well plates or 8-well strip tubes Tube cap strips
Consult the thermal cycler manufacturers recommendations
DNA Analysis Platform and Consumables
Agilent 2100 Bioanalyzer Instrument
Agilent 2100 Expert SW Laptop Bundle (optional)
High Sensitivity DNA Kit
Agilent p/n G2939BA
Agilent p/n G2953CA
Agilent p/n 5067-4626
Qubit Fluorometer Thermo Fisher Scientific p/n Q33238
Qubit Assay Tubes Thermo Fisher Scientific p/n Q32856
DNA LoBind Tubes, 1.5-ml PCR clean, 250 pieces Eppendorf p/n 022431021 or equivalent
Centrifuge Eppendorf Centrifuge model 5804 or equivalent
Plate or strip tube centrifuge Labnet International MPS1000 Mini Plate Spinner p/n C1000 (requires adapter, p/n C1000-ADAPT, for use with strip tubes) or equivalent
Multichannel pipette Rainin Pipet-Lite Multi Pipette or equivalent
P10, P20, P200 and P1000 pipettes Rainin Pipet-Lite Pipettes or equivalent
Magnetic separator*
* Select a magnetic separator configured to collect magnetic particles on one side of each well. Do not use a magnetic separator configured to collect the particles in a ring formation.
Thermo Fisher Scientific p/n 12331D or equivalent
Vortex mixer general laboratory supplier
Ice bucket general laboratory supplier
Powder-free gloves general laboratory supplier
Sterile, nuclease-free aerosol barrier pipette tips general laboratory supplier
le Genome Library Prep for Illumina Multiplexed Sequencing 13
1 Before You Begin Required Equipment
14
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing Protocol
2 Sample Preparation
Step 1. Fragment and adaptor-tag the genomic DNA samples 16
Step 2. Purify the adaptor-tagged library using AMPure XP beads 20
Step 3. Amplify and index the adaptor-tagged DNA library 22
Step 4. Purify the amplified library with AMPure XP beads 25
Step 5. Assess library DNA quantity and quality using the 2100 Bioanalyzer and High Sensitivity DNA Assay 27
Step 6. Pool samples for multiplexed sequencing 29
Step 7. Prepare sequencing samples 31
Step 8. Set up the sequencing run and trim adaptors from the reads 36
This section contains instructions for genomic DNA sequencing library preparation for Illumina platforms.
15Agilent Technologies
2 Sample Preparation Step 1. Fragment and adaptor-tag the genomic DNA samples
Step 1. Fragment and adaptor-tag the genomic DNA samples
16
In this step, the gDNA is enzymatically fragmented and adaptors are added to ends of the fragments in a single reaction. This step uses the SureSelectQXT Library Prep Kit components listed in Table 3 in addition to some reagents obtained from other suppliers (see Table 1 on page 12).
Before you begin, remove the SureSelect QXT Enzyme Mix ILM and the SureSelect QXT Buffer tubes from storage at 20C and place on ice. Vortex each reagent vigorously to mix before use. Remove the AMPure XP beads from storage at 4C and allow to warm up to room temperature.
Table 3 Reagents for DNA fragmentation and adaptor-tagging
Kit Component Storage Location Where Used
SureSelect QXT Stop Solution SureSelect QXT Library Prep Kit Box 1, Room Temperature
page 16 (below)
SureSelect QXT Buffer SureSelect QXT Library Prep Kit Box 2, 20C page 18
SureSelect QXT Enzyme Mix ILM SureSelect QXT Library Prep Kit Box 2, 20C page 18
NOTE While obtaining components for this step, also remove the DMSO vial from the SureSelect
QXT Library Prep Kit Box 2 in 20C storage. Leave the DMSO vial at room temperature in preparation for use on page 24.
For each DNA sample to be sequenced, prepare 1 library.
1 Verify that the SureSelect QXT Stop Solution contains 25% ethanol, by referring to the container label and the instructions below.
Before the first use of a fresh container, add 1.5 ml of ethanol to the provided bottle containing 4.5 ml of stop solution, for a final ethanol concentration of 25%. Seal the bottle then vortex well to mix. After adding the ethanol, be sure to mark the label for reference by later users.
Keep the prepared 1X SureSelect QXT Stop Solution at room temperature, tightly sealed, until it is used on page 19.
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Sample Preparation 2 Step 1. Fragment and adaptor-tag the genomic DNA samples
SureSelectQXT Who
2 Prepare reagents for the purification protocols on page 20 and page 25.
a Verify that the AMPure XP beads are being held at room temperature. The beads should be held at room temperature for at least 30 minutes before use. Do not freeze the beads at any time.
b Prepare 800 l of fresh 70% ethanol per sample, plus excess, for use in the purification steps. The 70% ethanol may be used for multiple steps done on the same day, when stored in a sealed container.
3 Quantify and dilute gDNA samples using two serial fluorometric assays:
a Use the Qubit dsDNA BR Assay or Qubit dsDNA HS Assay to determine the initial concentration of each gDNA sample. Follow the manufacturers instructions for the specific assay kit and the Qubit instrument. This step is critical for successful preparation of input DNA at the required concentration to ensure optimal fragmentation.
b Dilute each gDNA sample with nuclease- free water to a final concentration of 50 ng/l in a 1.5- ml LoBind tube.
c Carefully measure the DNA concentration of each of the 50 ng/l dilutions using a second Qubit dsDNA BR or HS Assay.
d Adjust each gDNA sample with nuclease- free water to a final concentration of 25 ng/l in a 1.5- ml LoBind tube.
CAUTION The duration and temperature of incubation for DNA fragmentation must be precisely controlled for optimal results. Make sure to preprogram the thermal cycler as directed in step 4 before setting up the fragmentation reactions. Do not exceed 10 minutes at 45C, as indicated in Table 4.
4 Preprogram a thermal cycler (with the heated lid ON) with the program in Table 4. Immediately pause the program, and keep paused until samples are loaded in step 8.
Table 4 Thermal cycler program for DNA fragmentation
Step Temperature Time
Step 1 45C 10 minutes
Step 2 4C 1 minute
Step 3 4C Hold
le Genome Library Prep for Illumina Multiplexed Sequencing 17
2 Sample Preparation Step 1. Fragment and adaptor-tag the genomic DNA samples
18
5 Before use, vortex the SureSelect QXT Buffer and SureSelect QXT Enzyme Mix ILM tubes vigorously at high speed. Note that the SSEL QXT Buffer is viscous and thorough and vigorous mixing is critical for optimal fragmentation.
These components are in liquid form when removed from 20C storage and should be returned to 20C storage promptly after use in step 6.
CAUTION Minor variations in volumes of the solutions combined in step 6 below may result in DNA fragment size variation.
The SureSelect QXT Buffer and Enzyme Mix solutions are highly viscous. Be sure to follow the dispensing and mixing instructions in the steps below. Thorough mixing of the reagents and reactions is critical for optimal performance.
6 Set up the fragmentation reactions on ice using a PCR plate or strip tube. Components must be added in the order listed below. Do not pre- mix the SureSelect QXT Buffer and Enzyme Mix.
a To each sample well, add 17 l of SureSelect QXT Buffer.
b Add 2 l of each DNA sample to its assigned sample well. While dispensing the DNA, be sure to place the pipette tip at the bottom of the well.
c Add 1 l of SureSelect QXT Enzyme Mix, ILM to each sample well. While dispensing the enzyme mixture, place the pipette tip at the bottom of the well. After dispensing of the 1 l of enzyme mix, pipette up and down 8 to 10 times to ensure complete transfer of the viscous solution to the well.
7 Seal the wells, briefly spin, then mix thoroughly by vortexing the plate or strip tube at high speed for 20 seconds.
8 Briefly spin the samples, then immediately place the plate or strip tube in the thermal cycler and resume the thermal cycling program in Table 4.
9 During the 10- minute incubation of samples in the thermal cycler, vigorously vortex the AMPure XP beads at high speed to ensure homogeneous distribution of beads throughout the solution so that the beads are ready for use on page 20.
10 When the thermal cycler has completed the 1- minute incubation at 4C, immediately place the samples on ice and proceed to step 11.
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Sample Preparation 2 Step 1. Fragment and adaptor-tag the genomic DNA samples
SureSelectQXT Who
11 Add 32 l of 1X SureSelect QXT Stop Solution (containing 25% ethanol) to each fragmentation reaction. Seal the wells with fresh caps, then vortex at high speed for 5 seconds. Briefly spin the plate or strip tube to collect the liquid.
Incubate the samples at room temperature for 1 minute. Proceed directly to the purification protocol on page 20.
le Genome Library Prep for Illumina Multiplexed Sequencing 19
2 Sample Preparation Step 2. Purify the adaptor-tagged library using AMPure XP beads
Step 2. Purify the adaptor-tagged library using AMPure XP beads
20
Before you begin, verify that the AMPure XP beads have been incubated at room temperature for at least 30 minutes and that fresh 70% ethanol has been prepared for use in step 6.
1 Verify that the AMPure XP bead suspension has been well mixed and appears homogeneous and consistent in color.
2 Add 52 l of the homogeneous bead suspension to each sample well containing the 52- l DNA samples. Seal the wells, then vortex for 5 seconds. Briefly spin the samples to collect the liquid, without pelleting the beads.
Check that the beads are in a homogeneous suspension in the sample wells. Each well should have a uniform color with no layers of beads or clear liquid present.
3 Incubate samples for 5 minutes at room temperature.
4 Put the plate or strip tube on the magnetic stand at room temperature. Wait for the solution to clear (approximately 3 to 5 minutes).
5 While keeping the plate or tubes in the magnetic stand, carefully remove and discard the cleared solution from each well. Do not disturb the beads while removing the solution.
6 Continue to keep the plate or tubes in the magnetic stand while you dispense 200 l of fresh 70% ethanol in each sample well.
7 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
8 Repeat step 6 and step 7 once for a total of two washes. Make sure to remove all of the ethanol at each wash step.
9 Dry the samples on the thermal cycler (with lid open) at 37C for 1 to 3 minutes. Do not overdry the samples.
10 Add 11 l of nuclease- free water to each sample well.
11 Seal the sample wells, then mix well on a vortex mixer and briefly spin the plate or tubes to collect the liquid.
12 Incubate for 2 minutes at room temperature.
13 Put the plate or tubes in the magnetic stand and leave for 2 minutes or until the solution in each well is clear.
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Sample Preparation 2 Step 2. Purify the adaptor-tagged library using AMPure XP beads
SureSelectQXT Who
14 Remove each cleared supernatant (approximately 10 L) to wells of a fresh plate or strip tube and keep on ice. You can discard the beads at this time.
le Genome Library Prep for Illumina Multiplexed Sequencing 21
2 Sample Preparation Step 3. Amplify and index the adaptor-tagged DNA library
Step 3. Amplify and index the adaptor-tagged DNA library
22
In this step, the adaptor- tagged DNA libraries are PCR amplified using an appropriate pair of dual indexing primers.
CAUTION To avoid cross-contaminating libraries, set up PCR reactions in a dedicated clean area or PCR hood with UV sterilization and positive air flow.
Before you begin, thaw then vortex to mix the reagents listed in Table 5. Keep all reagents except DMSO on ice.
Table 5 Reagents for PCR amplification and indexing
Kit Component Storage Location Where Used
Herculase II Fusion DNA Polymerase SureSelect QXT Library Prep Kit Box 2, 20C page 23
Herculase II 5 Reaction Buffer SureSelect QXT Library Prep Kit Box 2, 20C page 23
100 mM dNTP Mix (25 mM each dNTP) SureSelect QXT Library Prep Kit Box 2, 20C page 23
SureSelect QXT P7 and P5 dual indexing primers SureSelect QXT Library Prep Kit Box 2, 20C page 23
DMSO Transferred to Room Temperature storage on page 16 page 23
Prepare one indexing amplification reaction for each DNA library.
1 Determine the appropriate index assignments for each sample. See the Reference section for sequences of the index portion of the P7 (page 41) and P5 (page 42) indexing primers used to amplify the DNA libraries in this step.
Use a different indexing primer combination for each sample to be sequenced in the same lane.
For sample multiplexing, Agilent recommends maximizing index diversity on both P7 and P5 primers as required for color balance. For example, when 8-plexing, use eight different P7 index primers with two P5 index primers. See Table 27 on page 43 and Table 28 on page 44 for additional details.
NOTE
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Sample Preparation 2 Step 3. Amplify and index the adaptor-tagged DNA library
SureSelectQXT Who
2 Prepare the appropriate volume of PCR reaction mix, as described in Table 6, on ice. Mix well on a vortex mixer and keep on ice.
Table 6 Preparation of PCR Reaction Mix
3 Add 36 l of the PCR reaction mixture from step 2 to each 10- l purified library DNA sample held on ice from step 14 on page 21.
4 Add 2 l of the appropriate P7 dual indexing primer (P7 i1 to P7 i12) to each PCR reaction well. Add only one of the twelve possible P7 primers to each reaction well.
5 Add 2 l of the appropriate P5 dual indexing primer (P5 i13 to P5 i20) to each PCR reaction well. Add only one of the eight possible P5 primers to each reaction well.
6 Seal the wells and mix by vortexing gently for 5 seconds. Spin samples briefly to collect the liquid.
Reagent Volume for 1 reaction Volume for 16 reactions (includes excess)
Nuclease-free water 22 L 374 L
Herculase II 5 Reaction Buffer 10 L 170 L
100 mM dNTP Mix (25 mM each dNTP) 0.5 L 8.5 L
DMSO 2.5 L 42.5 L
Herculase II Fusion DNA Polymerase 1 L 17 L
Total 36 L 612 L
le Genome Library Prep for Illumina Multiplexed Sequencing 23
2 Sample Preparation Step 3. Amplify and index the adaptor-tagged DNA library
24
7 Incubate the plate or strip tube in the thermal cycler (with the heated lid ON) and run the program in Table 7.
Table 7 Thermal cycler program for PCR indexing
Segment Number Number of Cycles Temperature Time
1 1 68C 2 minutes
2 1 98C 30 seconds
3 5 98C 30 seconds
56C 30 seconds
72C 1 minute
4 1 4C Hold
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Sample Preparation 2 Step 4. Purify the amplified library with AMPure XP beads
Step 4. Purify the amplified library with AMPure XP beads
SureSelectQXT Who
Before you begin, verify that the AMPure XP beads have been kept at room temperature for at least 30 minutes and that fresh 70% ethanol has been prepared for use in step 6.
1 Mix the AMPure XP bead suspension well so that the suspension appears homogeneous and consistent in color.
2 Transfer the samples to room temperature, then add 35 l of the homogeneous bead suspension to each sample well containing the 50- l amplified DNA samples. Seal the wells, then vortex for 5 seconds. Briefly spin the samples to collect the liquid.
Check that the beads are in a homogeneous suspension in the sample wells. Each well should have a uniform color with no layers of beads or clear liquid present.
3 Incubate samples for 5 minutes at room temperature.
4 Put the plate or strip tube on the magnetic stand at room temperature. Wait for the solution to clear (approximately 3 to 5 minutes).
5 While keeping the plate or tubes in the magnetic stand, carefully remove and discard the cleared solution from each well. Do not disturb the beads while removing the solution.
6 Continue to keep the plate or tubes in the magnetic stand while you dispense 200 l of fresh 70% ethanol in each sample well.
7 Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol.
8 Repeat step 6 and step 7 once for a total of two washes. Make sure to remove all of the ethanol at each wash step.
9 Dry the samples on the thermal cycler (with lid open) at 37C for 1 to 3 minutes. Do not overdry the samples.
10 Add 30 l of nuclease- free water to each sample well.
11 Seal the sample wells, then mix well on a vortex mixer and briefly spin the plate or tubes to collect the liquid.
12 Incubate for 2 minutes at room temperature.
13 Put the plate or tubes in the magnetic stand and leave for 2 minutes or until the solution in each well is clear.
14 Remove each cleared supernatant (approximately 29 L) to a fresh LoBind tube. You can discard the beads at this time.
le Genome Library Prep for Illumina Multiplexed Sequencing 25
2 Sample Preparation Step 4. Purify the amplified library with AMPure XP beads
26
15 Remove a 1- l sample of each amplified library for analysis in Step 5. Assess library DNA quantity and quality using the 2100 Bioanalyzer and High Sensitivity DNA Assay on page 27. Dilute each of the 1- l samples with 9 l of nuclease- free water prior to analysis.
Stopping Point
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Sample Preparation 2 Step 5. Assess library DNA quantity and quality using the 2100 Bioanalyzer and High Sensitivity DNA
Assay
Step 5. Assess library DNA quantity and quality using the 2100 Bioanalyzer and High Sensitivity DNA Assay
SureSelectQXT Who
Use the Bioanalyzer High Sensitivity DNA Assay to analyze the amplified indexed DNA. Perform the assay according to the High Sensitivity DNA Kit Guide.
NOTE Do not use Agilents DNA 1000 Assay to analyze the whole genome samples. The expected
distribution of whole genome library fragment sizes is not compatible with the DNA 1000 Assay. See Figure 1, below, for a sample electropherogram.
The presence of magnetic beads in the samples may adversely impact the Bioanalyzer results. If you suspect bead contamination in the samples, place the plate or strip tube on the magnetic rack before withdrawing samples for analysis.
1 Set up the 2100 Bioanalyzer as instructed in the reagent kit guide.
2 Prepare the chip, samples and ladder as instructed in the reagent kit guide, using 1 l of a 1:10 dilution of each sample for the analysis.
3 Load the prepared chip into the instrument and start the run within five minutes after preparation.
4 Analyze the results, using the guidelines below:
Typical whole genome library electropherograms show a broad distribution of DNA fragments. A sample electropherogram is shown in Figure 1.
Check the Average Size [bp] of DNA fragments reported in the Bioanalyzer results. Sequencing data may be acquired from libraries with a broad range of average fragment sizes. The protocol has been optimized, however, to produce whole genome libraries with average DNA fragment sizes between approximately 600 and 1000 bp.
An average fragment size significantly less than 600 bp may indicate too little gDNA in the fragmentation reaction and may be associated with increased duplicates in the sequencing data. In contrast, libraries with an unusually large average fragment size may indicate too much gDNA in the fragmentation reaction and may require higher DNA concentrations for optimal cluster density in the sequencing reaction.
NOTE
5 Measure the concentration of each library by integrating under the entire peak. For accurate quantification, make sure that the concentration falls within the linear range of the assay.
le Genome Library Prep for Illumina Multiplexed Sequencing 27
2 Sample Preparation Step 5. Assess library DNA quantity and quality using the 2100 Bioanalyzer and High Sensitivity DNA
Assay
Stopping Point
28
If you do not continue to the next step, seal the plate and store at 4C overnight or at 20C for prolonged storage.
Figure 1 Analysis of amplified library DNA using a High-Sensitivity DNA Assay.
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Sample Preparation 2 Step 6. Pool samples for multiplexed sequencing
Step 6. Pool samples for multiplexed sequencing
SureSelectQXT Who
The number of indexed libraries that may be multiplexed in a single sequencing lane is determined by the output specifications of the platform used, together with the amount of sequencing data required for your research design. Calculate the number of indexes that can be combined per lane, according to the capacity of your platform and the amount of sequencing data required per sample.
Guidelines for optimal low- level multiplexing of samples indexed using the SureSelectQXT dual indexes are provided on page 43.
1 Combine the libraries such that each index- tagged sample is present in equimolar amounts in the pool. For each library, use the formula below to determine the amount of indexed sample to use.
Volume of Index V f C f # C i
--------------------------------=
where V(f) is the final desired volume of the pool,
C(f) is the desired final concentration of all the DNA in the pool
# is the number of indexes, and
C(i) is the initial concentration of each indexed sample.
Table 8 shows an example of the amount of 4 index- tagged samples (of different concentrations) and Low TE needed for a final volume of 20 l at 10 nM.
2 Adjust the final volume of the pooled library to the desired final concentration.
Table 8 Example of indexed sample volume calculation for total volume of 20 L
Component V(f) C(i) C(f) # Volume to use (L)
Sample 1 20 L 20 nM 10 nM 4 2.5
Sample 2 20 L 10 nM 10 nM 4 5
Sample 3 20 L 17 nM 10 nM 4 2.9
Sample 4 20 L 25 nM 10 nM 4 2
Low TE 7.6
le Genome Library Prep for Illumina Multiplexed Sequencing 29
2 Sample Preparation Step 6. Pool samples for multiplexed sequencing
30
If the final volume of the combined index- tagged samples is less than the desired final volume, V(f), add Low TE to bring the volume to the desired level.
If the final volume of the combined index- tagged samples is greater than the final desired volume, V(f), lyophilize and reconstitute to the desired volume.
3 If you store the library before sequencing, add Tween 20 to 0.1% v/v and store at - 20C short term.
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Sample Preparation 2 Step 7. Prepare sequencing samples
Step 7. Prepare sequencing samples
SureSelectQXT Who
Proceed to cluster amplification using the appropriate Illumina Paired- End Cluster Generation Kit. See Table 9 for kit configurations compatible with the recommended read length plus reads for the SureSelectQXT 8- bp dual indexes.
The optimal seeding concentration for SureSelectQXT whole genome libraries varies according to sequencing platform, run type and Illumina kit version. See Table 9 for guidelines. Seeding concentration and cluster density may also need to be optimized based on the DNA fragment size range for the library and on the desired output and data quality.
To do this step, refer to the manufacturers instructions, using the modifications described on page 32 for use of the SureSelectQXT Read Primers with the Illumina Paired- End Cluster Generation Kits. Follow Illuminas recommendation for a PhiX control in a low- concentration spike- in for improved sequencing quality control.
Table 9 Illumina Kit Configuration Selection Guidelines
Platform Run Type Read Length* SBS Kit Configuration Chemistry Seeding Concentration
HiSeq 2500 Rapid Run 2 100 bp 200 Cycle Kit v2 1420 pM
HiSeq 2500 High Output 2 100 bp 250 Cycle Kit v4 1420 pM
MiSeq All Runs 2 100 bp or
2 150 bp
300 Cycle Kit v2 1420 pM
MiSeq All Runs 2 76 bp 150 Cycle Kit v3 1420 pM
NextSeq 500/550 All Runs 2 100 bp or
2 150 bp
300 Cycle Kit v2.5 2 pM
HiSeq 3000/4000 All Runs 2 100 bp or
2 150 bp
300 Cycle Kit v1 200300 pM
NovaSeq 6000 Standard Workflow Runs
2 100 bp or
2 150 bp
300 Cycle Kit v1.0 or v1.5 300600 pM
NovaSeq 6000 Xp Workflow Runs
2 100 bp or
2 150 bp
300 Cycle Kit v1.0 or v1.5 200400 pM
* If your application requires a different read length, verify that you have sufficient sequencing reagents to complete Reads 1 and 2 in addition to the dual 8-bp index reads.
le Genome Library Prep for Illumina Multiplexed Sequencing 31
2 Sample Preparation Step 7. Prepare sequencing samples
32
Using the SureSelectQXT Read Primers with Illuminas Paired-End Cluster Generation Kits
To sequence the SureSelectQXT libraries on Illuminas sequencing platforms, you need to use the following custom sequencing primers, provided in SureSelect QXT Library Prep Kit Box 2: SureSelect QXT Read Primer 1 SureSelect QXT Read Primer 2 SureSelect QXT Index 1 Read Primer SureSelect QXT Index 2 Read Primer (this primer is used only for
HiSeq 3000, HiSeq 4000, and NextSeq platforms and for NovaSeq platform runs using v1.5 chemistry)
These SureSelectQXT custom sequencing primers are provided at 100 M and must be diluted in the corresponding Illumina primer solution, using the platform- specific instructions below:
For the HiSeq 2500 platform, combine the primers as shown in Table 10 or Table 11 on page 33.
For the HiSeq 3000 or HiSeq 4000 platform, combine the primers as shown in Table 12 on page 33.
For the MiSeq platform, combine the primers as shown in Table 13 on page 34.
For the NextSeq platform, combine the primers as shown in Table 14 or Table 15 on page 34.
For the NovaSeq platform, combine the primers as shown in Table 16 through Table 19 on page 35.
NOTE It is important to combine the primers precisely in the indicated ratios. Carefully follow the instructions indicated in Table 10 to Table 19. Where specified, add the custom primer volume directly to the solution already in cBot reagent plate wells. Otherwise, combine measured volumes of each solution; do not rely on volumes reported on vial labels or in Illumina literature. Vortex each mixture vigorously to ensure homogeneity for proper detection of the indexes using the custom read primers.
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Sample Preparation 2 Step 7. Prepare sequencing samples
SureSelectQXT Who
Table 10 HiSeq 2500 High Output custom sequencing primer preparation
Sequencing Read
Volume of SureSelectQXT Primer Volume of Illumina TruSeq Primer Total Volume
Read 1 6 l SureSelect QXT Read Primer 1 (brown cap) 1194 l HP10 1.2 ml*
Index 15 l SureSelect QXT Index 1 Read Primer (clear cap) 2985 l HP12 3 ml
Read 2 15 l SureSelect QXT Read Primer 2 (black cap) 2985 l HP11 3 ml
* Aliquot the mixture as directed for HP6 or HP10 in Illuminas cluster generation protocol.
Table 11 HiSeq 2500 Rapid Mode custom sequencing primer preparation
Sequencing Read
Volume of SureSelectQXT Primer Volume of Illumina TruSeq Primer Total Volume
Read 1 8.8 l SureSelect QXT Read Primer 1 (brown cap) 1741.2 l HP10 1.75 ml
Index 8.8 l SureSelect QXT Index 1 Read Primer (clear cap) 1741.2 l HP12 1.75 ml
Read 2 8.8 l SureSelect QXT Read Primer 2 (black cap) 1741.2 l HP11 1.75 ml
Table 12 HiSeq 3000 and HiSeq 4000 custom sequencing primer preparation
Sequencing Read
Volume of SureSelectQXT Primer Volume of Illumina TruSeq Primer
Total Volume Reagent Rack Position
Read 1 1.5 l SureSelect QXT Read Primer 1 (brown cap) 298.5 l HP10* 0.3 ml per well
cBot Column 11
Read 2 15 l SureSelect QXT Read Primer 2 (black cap) 2985 l HP11 3 ml 16
Index 1+ Index 2
22.5 l SureSelect QXT Index 1 Read Primer (clear cap) + 22.5 l SureSelect QXT Index 2 Read Primer (purple cap)
4455 l HP14 4.5 ml 17
* Use cBot recipe HiSeq_3000_4000_HD_Exclusion_Amp_v1.0. Add 1.5 l SureSelect QXT Read Primer 1 to the 298.5 l of HP10 in each well of column 11 in the cBot reagent plate.
le Genome Library Prep for Illumina Multiplexed Sequencing 33
2 Sample Preparation Step 7. Prepare sequencing samples
Table 13 MiSeq platform custom sequencing primer preparation
Sequencing Read
Volume of SureSelectQXT Primer Volume of Illumina TruSeq Primer
Total Volume
Final Cartridge Position
Read 1 3 l SureSelect QXT Read Primer 1 (brown cap) 597 l HP10 (well 12) 0.6 ml well 18
Index 3 l SureSelect QXT Index 1 Read Primer (clear cap) 597 l HP12 (well 13) 0.6 ml well 19
Read 2 3 l SureSelect QXT Read Primer 2 (black cap) 597 l HP11 (well 14) 0.6 ml well 20
34
Table 14 NextSeq 500/550 High-Output v2 Kit custom sequencing primer preparation
Sequencing Read
Volume of SureSelectQXT Primer Volume of Illumina Primer Total Volume
Final Cartridge Position
Read 1 3.9 l SureSelect QXT Read Primer 1 (brown cap) 1296.1 l BP10 (from well 20) 1.3 ml well 7
Read 2 4.2 l SureSelect QXT Read Primer 2 (black cap) 1395.8 l BP11 (from well 21) 1.4 ml well 8
Index 1+ Index 2
6 l SureSelect QXT Index 1 Read Primer (clear cap) + 6 l SureSelect QXT Index 2 Read Primer (purple cap)
1988 l BP14 (from well 22) 2 ml well 9
Table 15 NextSeq 500/550 Mid-Output v2 Kit custom sequencing primer preparation
Sequencing Read
Volume of SureSelectQXT Primer Volume of Illumina Primer Total Volume
Final Cartridge Position
Read 1 2.7 l SureSelect QXT Read Primer 1 (brown cap) 897.3 l BP10 (from well 20) 0.9 ml well 7
Read 2 3.3 l SureSelect QXT Read Primer 2 (black cap) 1096.7 l BP11 (from well 21) 1.1 ml well 8
Index 1+ Index 2
4.8 l SureSelect QXT Index 1 Read Primer (clear cap) + 4.8 l SureSelect QXT Index 2 Read Primer (purple cap)
1590.4 l BP14 (from well 22) 1.6 ml well 9
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Sample Preparation 2 Step 7. Prepare sequencing samples
SureSelectQXT Who
Table 16 NovaSeq 6000 using SP/S1/S2 flowcell with v1.0 chemistry custom sequencing primer preparation
Sequencing Read
Volume of SureSelectQXT Primer Volume of Illumina Primer Total Volume
Final Cartridge Position
Read 1 12 l SureSelect QXT Read Primer 1 (brown cap) 3988 l BP10 (well 24) 4 ml 5
Index 15 l SureSelect QXT Index 1 Read Primer (clear cap) 4985 l BP14 (well 23) 5 ml 7
Read 2 6 l SureSelect QXT Read Primer 2 (black cap) 1994 l BP11 (well 13) 2 ml 6
Table 17 NovaSeq 6000 using S4 flowcell with v1.0 chemistry custom sequencing primer preparation
Sequencing Read
Volume of SureSelectQXT Primer Volume of Illumina Primer Total Volume
Final Cartridge Position
Read 1 21.9 l SureSelect QXT Read Primer 1 (brown cap) 7278.1 l BP10 (well 24) 7.3 ml 5
Index 15 l SureSelect QXT Index 1 Read Primer (clear cap) 4985 l BP14 (well 23) 5 ml 7
Read 2 10.5 l SureSelect QXT Read Primer 2 (black cap) 3489.5 l BP11 (well 13) 3.5 ml 6
Table 18 NovaSeq 6000 using SP/S1/S2 flowcell with v1.5 chemistry custom sequencing primer preparation
Sequencing Read
Volume of SureSelectQXT Primer Volume of Illumina Primer
Total Volume
Final Cartridge Position
Read 1 12 l SureSelect QXT Read Primer 1 (brown cap) 3988 l BP10 (well 24) 4 ml 5
Index 1+ Index 2
15 l SureSelect QXT Index 1 Read Primer (clear cap) + 15 l SureSelect QXT Index 2 Read Primer (purple cap)
4970 l VP14 (well 23) 5 ml 7
Read 2 6 l SureSelect QXT Read Primer 2 (black cap) 1994 l BP11 (well 13) 2 ml 6
Table 19 NovaSeq 6000 using S4 flowcell with v1.5 chemistry custom sequencing primer preparation
Sequencing Read
Volume of SureSelectQXT Primer Volume of Illumina Primer
Total Volume
Final Cartridge Position
Read 1 21.9 l SureSelect QXT Read Primer 1 (brown cap) 7278.1 l BP10 (well 24) 7.3 ml 5
Index 1+ Index 2
15 l SureSelect QXT Index 1 Read Primer (clear cap) + 15 l SureSelect QXT Index 2 Read Primer (purple cap)
4970 l VP14 (well 23) 5 ml 7
Read 2 10.5 l SureSelect QXT Read Primer 2 (black cap) 3489.5 l BP11 (well 13) 3.5 ml 6
le Genome Library Prep for Illumina Multiplexed Sequencing 35
2 Sample Preparation Step 8. Set up the sequencing run and trim adaptors from the reads
Step 8. Set up the sequencing run and trim adaptors from the reads
36
Refer to Illumina protocols to set up custom sequencing primer runs. Before aligning reads to the reference genome, SureSelectQXT adaptor sequences must be trimmed from the reads using the additional platform- specific guidelines below.
For SureSelectQXT dual index sequence information, see page 41.
MiSeq platform sequencing run setup and adaptor trimming guidelines
Use the Illumina Experiment Manager (IEM) software to generate a custom primer Sample Sheet.
Set up the run to include adapter trimmingusing the IEM Sample Sheet Wizard. When prompted by the wizard, select the Use Adapter Trimming option, and specify CTGTCTCTTGATCACA as the adapter sequence. This enables the MiSeq Reporter software to identify the adaptor sequence and trim the adaptor from reads.
HiSeq/NextSeq/NovaSeq platform sequencing run setup and adaptor trimming guidelines
Set up sequencing runs using the settings shown in Table 20. For HiSeq runs, select Dual Index on the Run Configuration screen of the instrument control software interface. Since custom primers are spiked into the standard sequencing primer tubes, no additional specialized settings are required to accommodate the use of custom primers in the run.
For the NextSeq or NovaSeq platform, Cycle Number and custom sequencing primer settings can be specified on the Run Configuration screen of the instrument control software interface.
Table 20 Run Configuration screen Cycle Number settings
Run Segment Cycle Number
Read 1 100
Index 1 (i7) 8
Index 2 (i5) 8
Read 2 100
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Sample Preparation 2 Step 8. Set up the sequencing run and trim adaptors from the reads
SureSelectQXT Who
After the sequencing run is complete, generate demultiplexed FASTQ data following Illuminas instructions and then trim adaptor sequences from the reads using the Trimmer utility of the Agilent Genomics NextGen Toolkit (AGeNT). For additional information and to download this toolkit free- of- charge, visit the AGeNT page at www.agilent.com.
le Genome Library Prep for Illumina Multiplexed Sequencing 37
2 Sample Preparation Step 8. Set up the sequencing run and trim adaptors from the reads
38
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing Protocol
3 Reference
Kit Contents 40
Nucleotide Sequences of SureSelectQXT Dual Indexes 41
Guidelines for Multiplexing with Dual-Indexed Samples 43
This chapter contains reference information, including component kit contents and reference information for use during the downstream sample sequencing steps.
39Agilent Technologies
3 Reference Kit Contents
Kit Contents
40
SureSelectQXT Library Prep Kits contain the following component kits:
Table 21 SureSelectQXT Library Prep Kit Contents
Component Kits Storage Condition G9684A (16 Samples) G9684B (96 Samples)
SureSelect QXT Library Prep Kit, Box 1 Room Temperature 5500-0119 5500-0119
SureSelect QXT Library Prep Kit, Box 2 20C 5500-0126 5500-0127
The contents of each of the component kits listed in Table 21 are described in Table 22 and Table 23.
Table 22 SureSelect QXT Library Prep Kit, Box 1 Content
Kit Component 16 Reactions 96 Reactions
SureSelect QXT Stop Solution bottle bottle
Table 23 SureSelect QXT Library Prep Kit Box 2 Content
Kit Component 16 Reactions 96 Reactions
SureSelect QXT Buffer tube with white cap bottle
SureSelect QXT Enzyme Mix ILM tube with orange cap tube with orange cap
Herculase II Fusion DNA Polymerase tube with red cap tube with red cap
Herculase II 5 Reaction Buffer tube with clear cap tube with clear cap
100 mM dNTP Mix (25 mM each dNTP) tube with green cap tube with green cap
DMSO tube with green cap tube with green cap
SureSelect QXT Read Primer 1 tube with amber cap tube with amber cap
SureSelect QXT Read Primer 2 tube with black cap tube with black cap
SureSelect QXT Index 1 Read Primer tube with clear cap tube with clear cap
SureSelect QXT Index 2 Read Primer tube with purple cap tube with purple cap
SureSelect QXT P7 dual indexing primers
P7 i1 through P7 i8 provided in 8 tubes with yellow caps (one tube per primer)
P7 i1 through P7 i12 provided in 12 tubes with yellow caps (one tube per primer)
SureSelect QXT P5 dual indexing primers
P5 i13 and P5 i14 provided in 2 tubes with blue caps (one tube per primer)
P5 i13 through P5 i20 provided in 8 tubes with blue caps (one tube per primer)
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Reference 3 Nucleotide Sequences of SureSelectQXT Dual Indexes
Nucleotide Sequences of SureSelectQXT Dual Indexes
SureSelectQXT Who
The nucleotide sequence of each SureSelectQXT index is provided in the tables below.
Note that some index number assignments of the SureSelectQXT P5 and P7 indexes differ from the index numbers assignments used by Illumina for indexes of similar or identical sequence.
Each index is 8 bases in length. Refer to Illuminas sequencing run setup instructions for sequencing libraries using 8- base indexes.
Table 24 SureSelectQXT P7 Indexes 1 to 12
Index Name with Number Sequence
P7 Index 1 (P7 i1) TAAGGCGA
P7 Index 2 (P7 i2) CGTACTAG
P7 Index 3 (P7 i3) AGGCAGAA
P7 Index 4 (P7 i4) TCCTGAGC
P7 Index 5 (P7 i5) GTAGAGGA
P7 Index 6 (P7 i6) TAGGCATG
P7 Index 7 (P7 i7) CTCTCTAC
P7 Index 8 (P7 i8) CAGAGAGG
P7 Index 9 (P7 i9) GCTACGCT
P7 Index 10 (P7 i10) CGAGGCTG
P7 Index 11 (P7 i11) AAGAGGCA
P7 Index 12 (P7 i12) GGACTCCT
le Genome Library Prep for Illumina Multiplexed Sequencing 41
3 Reference Nucleotide Sequences of SureSelectQXT Dual Indexes
42
Table 25 SureSelectQXT P5 Indexes 13 to 20 for HiSeq 2500, MiSeq, or NovaSeq (v1.0 chemistry) platform
Index Number Sequence
P5 Index 13 (P5 i13) TAGATCGC
P5 Index 14 (P5 i14) CTCTCTAT
P5 Index 15 (P5 i15) TATCCTCT
P5 Index 16 (P5 i16) AGAGTAGA
P5 Index 17 (P5 i17) GTAAGGAG
P5 Index 18 (P5 i18) ACTGCATA
P5 Index 19 (P5 i19) AAGGAGTA
P5 Index 20 (P5 i20) CTAAGCCT
Table 26 SureSelectQXT P5 Indexes 13 to 20 for HiSeq 3000/4000, NextSeq, or NovaSeq
(v1.5 chemistry) platform*
* When doing runs on these platforms through BaseSpace, use the reverse comple- ment sequences provided in Table 25.
Index Number Sequence
P5 Index 13 (P5 i13) GCGATCTA
P5 Index 14 (P5 i14) ATAGAGAG
P5 Index 15 (P5 i15) AGAGGATA
P5 Index 16 (P5 i16) TCTACTCT
P5 Index 17 (P5 i17) CTCCTTAC
P5 Index 18 (P5 i18) TATGCAGT
P5 Index 19 (P5 i19) TACTCCTT
P5 Index 20 (P5 i20) AGGCTTAG
SureSelectQXT Whole Genome Library Prep for Illumina Multiplexed Sequencing
Reference 3 Guidelines for Multiplexing with Dual-Indexed Samples
Guidelines for Multiplexing with Dual-Indexed Samples
SureSelectQXT Who
Agilent recommends following the dual index sample pooling guidelines shown in Table 27 for 16 reaction kits and shown in Table 28 for 96 reaction kits. These are designed to maintain color balance at each cycle of the index reads on both ends. They also provide flexibility of demultiplexing as single or dual indexed samples in low- plexity experiments. One- base mismatches should be allowed during demultiplexing.
Table 27 Dual index sample pooling guidelines for 16 Reaction Kits
Plexity of Sample Pool
Recommended SureSelectQXT P7 Indexes Recommended SureSelectQXT P5 Indexes
1-plex Any P7 index (i1 to i8) Either P5 index (i13 or i14)
2-plex P7 i1 and P7 i2 OR
P7 i2 and P7 i4
P5 i13 and P5 i14
3-plex P7 i1, P7 i2 and P7 i4 OR
P7 i3, P7 i4 and P7 i6 OR
P7 i5, P7 i7 and P7 i8
P5 i13 and P5 i14 (as needed)
4- or 5-plex P7 i1, P7 i2, P7 i4 and any additional P7 index(es) OR
P7 i3, P7 i4, P7 i6 and any additional P7 index(es) OR
P7 i5, P7 i7, P7 i8 and any additional P7 index(es)
P5 i13 and P5 i14 (as needed)
6- to 8-plex Any combination of 6, 7, or 8 different P7 indexes P5 i13 and P5 i14 (as needed)
9-to 16-plex All eight P7 indexes (i1 to i8) P5 i13 and P5 i14 (as needed)
le Genome Library Prep for Illumina Multiplexed Sequencing 43
3 Reference Guidelines for Multiplexing with Dual-Indexed Samples
Table 28 Dual index sample pooling guidelines for 96 Reaction Kits
Plexity of Sample Pool
Recommended SureSelectQXT P7 Indexes Recommended SureSelectQXT P5 Indexes
1-plex Any P7 index i1 to i12 Any P5 index (i13 to i20)
2-plex P7 i1 and P7 i2 OR
P7 i2 and P7 i4
P5 i13 and P5 i14 OR
P5 i15 and P5 i16 OR
P5 i17 and P5 i18
3-plex P7 i1, P7 i2 and P7 i4 OR
P7 i3, P7 i4 and P7 i6 OR
P7 i5, P7 i7 and P7 i8
P5 i13 and P5 i14 OR
P5 i15 and P5 i16 OR
P5 i17 and P5 i18 (as needed)
4-plex P7 i1, P7 i2, P7 i3* and P7 i4 OR
P7 i3, P7 i4, P7 i5* and P7 i6 OR
P7 i5, P7 i6*, P7 i7 and P7 i8
P5 i13 and P5 i14 OR
P5 i15 and P5 i16 OR
P5 i17 and P5 i18 (as needed)
5-plex P7 i1, P7 i2, P7 i3*, P7 i4 and P7 i5* OR
P7 i3, P7 i4, P7 i5*, P7 i6 and p7 i7* OR
P7 i5, P7 i6*, P7 i7, P7 i8 and p7 i9*
P5 i13 and P5 i14 OR
P5 i15 and P5 i16 OR
P5 i17 and P5 i18 (as needed)
6- to 12-plex Any combination of P7 indexes i1 to i12 using each index only once
P5 i13 and P5 i14 OR
P5 i15 and P5 i16 OR
P5 i17 and P5 i18 (as needed)
13-to 96-plex All twelve P7 indexes (i1 to i12) P5 i13 and P5 i14 and any other P5 index OR
P5 i15 and P5 i16 and any other P5 index OR
P5 i17 and P5 i18 and any other P5 index
(as needed)
* The indicated indexes may be substituted with another index, as long as the substitute index differs from all others used in the sample pool.
44
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In This Book
This guide contains information to run the SureSelectQXT Whole Genome Library Prep protocol for Illumina paired- end multiplexed sequencing.
Agilent Technologies, Inc. 2014, 2015, 2018, 2021
Version F0, January 2021
*G9682-90000 * p/n G9682-90000
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