UNIVERSAL SEQUENCING 100035 TELL Seq Target Enrichment User Guide

The TELL-SeqTM Target Enrichment Kit is designed for genomic
sequencing applications. It is essential to follow the instructions
provided in this user guide for proper and safe use of the kit.

2. Kit Contents

The TELL-SeqTM Library Prep Kit contains two boxes of
reagents:

Box 1: Contains various reagents including
Barcoding Enzyme, Exonuclease, Tagging Enzyme, and more. Do not
freeze and thaw these reagents more than 6 times.
Box 2: Contains TELL Bead Plex, Wash Solution,
and Stop Solution. Ensure proper handling of the Stop Solution as
per the instructions provided.

3. Genomic DNA Input Recommendations

For successful sequencing, 5ng of Genomic DNA is required. High
molecular weight DNA is crucial for optimal results with TELL-SeqTM
sequencing.

4. TELL-SeqTM Human Exome Capture Workflow

Follow the detailed workflow provided in the user guide for
capturing the human exome using the TELL-SeqTM kit.

5. Protocol

Refer to the protocol section of the user guide for step-by-step
instructions on using the TELL-SeqTM Target Enrichment Kit.

FAQ:

Q: What should I do if the Stop Solution in Box 2 is not clear
or has white precipitates?

A: If the Stop Solution is not clear or has
precipitates, warm the tube up at 37°C, vortex to dissolve any
precipitate, and store the resuspended solution at room temperature
for future use.

“`

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TELL-SeqTM Target Enrichment User Guide
For Research Use Only. Not for use in diagnostic procedures. Document # 100032-USG v5.0 February 2025

This document is proprietary to Universal Sequencing Technology Corporation and is intended solely for the use of its customer in connection with the use of the products described herein and for no other purposes.
The instructions in this document must be followed precisely by properly trained personnel to ensure the proper and safe use of the TELL-SeqTM kit.
UNIVERSAL SEQUENCING TECHNOLOGY CORPORATION DOES NOT ASSUME ANY LIABILITY OCCURING AFTER INCORRECT USE OF THE TELL-SEQTM KIT.
©2023 Universal Sequencing Technology Corporation. All rights reserved.
TELL-SeqTM is trademark of Universal Sequencing Technology Corporation. All other names, logos and other trademarks are the property of their respective owners.

Revision History Doc #100032-USG v1.0 Doc #100032-USG v2.0 Doc # 100032-USG v3.0
Doc # 100032-USG v4.0
Doc # 100032-USG v5.0

November 2021 August 2022 August 2023
August 2024 February 2025

Initial Release Protocol update to work with updated TELLSeqTM Library Prep kit V1 with Suspension Buffer EZ and TELL Bead Plex option Removed TELL Bead option. Only TELL Bead Plex is used moving forward. Added a Note and a picture with recommended mixing systems for a critical step of proper tube rotation during barcoding process to preserve high molecular weight DNA properties. Changed user guide for general Target Enrichment. Added additional information about using TELL-SeqTM Library Prep with other Target Enrichment systems
Doubled the volumes of primers in TELLSeqTM Illumina® Sequencing Primer Kit (PN 100004 and PN 100013)

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Table of Contents
1. Introduction …………………………………………………………………………………………………………………… 4 Genomic DNA Input Recommendations………………………………………………………………………………. 4
2. Kit Contents …………………………………………………………………………………………………………………… 5 TELL-SeqTM Library Prep Kit, Standard Size ………………………………………………………………………….. 5 Box 1 of 2: TELL-SeqTM Library Reagent Box 1 V1 ……………………………………………………………… 5 Box 2 of 2: TELL-SeqTM Library Reagent Box 2 V1 ……………………………………………………………… 5 TELL-SeqTM Library Multiplex Primer (1-8) Kit ………………………………………………………………………. 6 TELL-SeqTM Library Multiplex Primer (9-16) Kit …………………………………………………………………….. 7 TELL-SeqTM Library Multiplex Primer (17-24) Kit …………………………………………………………………… 7 TELL-SeqTM Illumina® Sequencing Primer Kit ……………………………………………………………………….. 7 TELL-SeqTM Illumina® Sequencing Primer Kit, HT ………………………………………………………………….. 8 TELL-SeqTM Target Blocker (PN 100019) ………………………………………………………………………………. 8
3. Consumables and Equipment (not provided) ………………………………………………………………………. 9 Consumables………………………………………………………………………………………………………………….. 9 *Depends on which system is available in the user facility. …………………………………………………….. 9 Equipment …………………………………………………………………………………………………………………….. 9
4. TELL-SeqTM Human Exome Capture Workflow ……………………………………………………………………. 10 5. Protocol ………………………………………………………………………………………………………………………. 11
A. TELL-SeqTM WGS Library Prep…………………………………………………………………………………… 11 Barcode DNA………………………………………………………………………………………………………………… 11 Stabilize DNA ……………………………………………………………………………………………………………….. 14 Tagment DNA……………………………………………………………………………………………………………….. 14 Wash Beads …………………………………………………………………………………………………………………. 15 Amplify Library …………………………………………………………………………………………………………….. 17 Clean Up Library……………………………………………………………………………………………………………. 18 Qualify and Quantify Library for Target Enrichment …………………………………………………………… 21 B. SureSelect Target Enrichment …………………………………………………………………………………. 23 Hybridize TELL-SeqTM Library to the Exome Capture Panel …………………………………………………… 23 Prepare Streptavidin-coated Magnetic Beads……………………………………………………………………. 25 Capture the Hybridized DNA using Streptavidin-coated Beads …………………………………………….. 26 Amplify Captured Library ……………………………………………………………………………………………….. 27 Clean Up Captured Library ……………………………………………………………………………………………… 28 Qualify and Quantify Captured Library for Sequencing……………………………………………………….. 29
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1. Introduction
This protocol explains how to prepare target enriched indexed paired-end TELL-SeqTM libraries by hybridization capture using a combination of TELL-SeqTM Library Prep Kit and Agilent® SureSelect Target Enrichment System. TELL-SeqTM Library Prep Kit system is also compatible with other Target Enrichment systems such as IDT and Twist Biosciences. The TELL-SeqTM library prep kit uses an innovative Transposase Enzyme Linked Long-read Sequencing (TELL-SeqTM) technology to prepare a paired-end library to generate barcode linked reads from an Illumina® sequencing system. Agilent® SureSelect Target Enrichment System allows for the enrichment of targeted regions using highly specific capture probes. Each Target Enrichment System has a unique panel of probes that allow for specific regions to be captured that is compatible with TELL-SeqTM libraries.
Genomic DNA Input Recommendations
5ng Genomic DNA is required for this protocol. High molecular weight (HMW) DNA is critical for successful TELL-SeqTM sequencing.
· For human genome, minimum sample DNA size should be greater than 40Kb. · HMW DNA ranging from 100Kb to 300Kb are optimal material for best human phasing
application. · Avoid breaking the HMW DNA during handling. Remove low molecular weight DNA (identified as
a smear less than 10Kb on a gel) in the sample if they present a significant portion in the DNA sample. Use a fluorometric-based method to quantify input DNA. If you use the Qubit dsDNA BR Assay Kit or HS Kit, use at least 2 L of each DNA sample for a measurement. Avoid methods that only measure total nucleic acid concentration, such as NanoDrop or other UV absorbance methods. For accurate measurement of HMW DNA concentration, dilute the concentrated DNA to the working concentration (0.4ng/l to 1ng/l) in a Tris buffer (pH 7.5-8.0) several hours to a day before the concentration measurement and library preparation. Genomic DNA should be stored in a Tris buffer with pH ranging from 7.5 – 8.0 or a low TE buffer (10mM Tris-HCl, 0.1 mM EDTA, pH 8.0). For assessing the purity of a DNA sample, the ratio of absorbance measurement at 260 nm to absorbance at 280 nm can be used. This protocol is optimized for DNA with absorbance ratio values of 1.82.0. If there is excessive RNA in the DNA sample, it should be removed with a RNase treatment.
Patent pending.
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2. Kit Contents

TELL-SeqTM Library Prep Kit, Standard Size (2 Boxes) Box 1 of 2: TELL-SeqTM Library Reagent Box 1 V1 (PN 100035)

NOTE: Do not freeze and thaw Box 1 reagents for more than 6 times.

Component Name

Cap Color

Volume (L)

Storage Temperature

5× Reaction Buffer

CAP Blue

120

-25°C to -15°C

Barcoding Enzyme

CAP Black

-25°C to -15°C

Cofactor II

CAP Amber

120

-25°C to -15°C

Exonuclease

CAP Yellow

-25°C to -15°C

Stabilizer

CAP Violet

-25°C to -15°C

Suspension Buffer EZ

CAP Natural

180

-25°C to -15°C

Tagging Enzyme

CAP Red

-25°C to -15°C

2× PCR Master Mix

CAP Pink

150

-25°C to -15°C

Enhancer 10× Primer Ia

CAP Green

CAP White

-25°C to -15°C -25°C to -15°C

a For use with 10× Primer II in the TELL-SeqTM Library Multiplex Primer Kit together for library amplification.

Box 2 of 2: TELL-SeqTM Library Reagent Box 2 V1 (PN 100036)

Component Name

Cap Color

Volume (L)

TELL Bead Plexb

CAP Orange

Wash Solution

CAP White

4500

Stop Solutionc

CAP Natural

960

b TELL Bead Plex works well on both Illumina and non-Illumina Sequencing Systems.

Storage Temperature 2°C to 8°C 2°C to 8°C 2°C to 25°C

c Prior to use, if the Stop Solution is not clear or has white precipitates, warm the tube up at 37C. Vortex to dissolve any precipitate. After the first use, store resuspended Stop Solution at room temperature for future use.

CAUTION
TELL-Read pipeline v1.1 or above is required to analyze sequencing data generated from TELL-SeqTM libraries prepared with TELL Bead Plex.

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TELL-SeqTM Library Prep Kit, HT24 (2 Boxes) Box 1 of 2: TELL-SeqTM Library Reagent Box 1 V1, HT24 (PN 100037)
NOTE: Do not freeze and thaw Box 1 reagents for more than 6 times.

Component Name

Cap Color

Volume (L)

Storage Temperature

5× Reaction Buffer

CAP Blue

720

-25°C to -15°C

Barcoding Enzyme

CAP Black

144

-25°C to -15°C

Cofactor II

CAP Amber

720

-25°C to -15°C

Exonuclease

CAP Yellow

-25°C to -15°C

Stabilizer

CAP Violet

-25°C to -15°C

Suspension Buffer EZ

CAP Natural

1080

-25°C to -15°C

Tagging Enzyme

CAP Red

144

-25°C to -15°C

2× PCR Master Mix

CAP Pink

900

-25°C to -15°C

Enhancer 10× Primer Ia

CAP Green

108

CAP White

180

-25°C to -15°C -25°C to -15°C

a For use with 10× Primer II in the TELL-SeqTM Library Multiplex Primer Kit together for library amplification.

Box 2 of 2: TELL-SeqTM Library Reagent Box 2 V1, HT24 (PN 100038)

Component Name

Cap Color

Volume

TELL Bead Plexb

CAP Orange

456 L

Wash Solution

CAP Blue

28.5 mL

Stop Solutionc

CAP White

5.76 mL

b TELL Bead Plex works well on both Illumina and non-Illumina Sequencing Systems.

Storage Temperature 2°C to 8°C 2°C to 8°C 2°C to 25°C

PRO TIP: TWO TELL-SeqTM Library Prep Kits, HT24 including both Box 1 and Box 2 can pair with of any TELL-SeqTM Library Multiplex Primer Kits.

CAUTION
TELL-Read pipeline v1.1 or above is required to analyze sequencing data generated from TELL-SeqTM libraries prepared with TELL Bead Plex.
TELL-SeqTM Library Multiplex Primer (1-8) Kit (PN 100003)

Component Name 10× Primer II, T501 10× Primer II, T502 10× Primer II, T503 10× Primer II, T504

Cap Color CAP Blue CAP Black CAP Green CAP Yellow

Volume (L) 15 15 15 15

Storage Temperature -25°C to -15°C -25°C to -15°C -25°C to -15°C -25°C to -15°C

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10× Primer II, T505 10× Primer II, T506

CAP Violet CAP Natural

-25°C to -15°C

10× Primer II, T507

CAP Red

-25°C to -15°C

10× Primer II, T508

CAP Orange

-25°C to -15°C

PRO TIP: One TELL-SeqTM Library Multiplex Primer (1-8) Kit contains enough primers to be used with FOUR TELL-SeqTM WGS Library Prep Kits.

TELL-SeqTM Library Multiplex Primer (9-16) Kit (PN 100009)

Component Name

Cap Color

Volume (L)

Storage Temperature

10× Primer II, T509

CAP Blue

-25°C to -15°C

10× Primer II, T510 10× Primer II, T511

CAP Amber CAP Green

-25°C to -15°C

10× Primer II, T512

CAP Yellow

-25°C to -15°C

10× Primer II, T513 10× Primer II, T514

CAP Violet CAP Orange

-25°C to -15°C

10× Primer II, T515

CAP Red

-25°C to -15°C

10× Primer II, T516

CAP Natural

-25°C to -15°C

PRO TIP: ONE TELL-SeqTM Library Multiplex Primer (9-16) Kit contains enough primers to be used with FOUR TELL-SeqTM WGS Library Prep Kits, Standard Size.

TELL-SeqTM Library Multiplex Primer (17-24) Kit (PN 100010)

Component Name

Cap Color

Volume (L)

Storage Temperature

10× Primer II, T517

CAP Amber

-25°C to -15°C

10× Primer II, T518

CAP Blue

-25°C to -15°C

10× Primer II, T519

CAP Yellow

-25°C to -15°C

10× Primer II, T520

CAP Green

-25°C to -15°C

10× Primer II, T521

CAP Black

-25°C to -15°C

10× Primer II, T522

CAP Violet

-25°C to -15°C

10× Primer II, T523

CAP Orange

-25°C to -15°C

10× Primer II, T524

CAP Red

-25°C to -15°C

PRO TIP: ONE TELL-SeqTM Library Multiplex Primer (17-24) Kit contains enough primers to be used with FOUR TELL-SeqTM WGS Library Prep Kits, Standard Size.

TELL-SeqTM Illumina® Sequencing Primer Kit (PN 100004)

Component Name
Read 1 Primer Read 2 Primer Index 1 Primer Index 2 Primer

Cap Color
CAP Black CAP White CAP Red CAP Yellow

Concentration
100M 100M 100M 100M

Volume (L)
100 100 100 100

Storage Temperature -25°C to -15°C -25°C to -15°C -25°C to -15°C -25°C to -15°C

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TELL-SeqTM Illumina® Sequencing Primer Kit, HT (PN 100013)

Component Name
Read 1 Primer Read 2 Primer Index 1 Primer Index 2 Primer

Cap Color
CAP Black CAP White CAP Red CAP Yellow

Concentration
100M 100M 100M 100M

Volume (L)
600 600 600 600

Storage Temperature -25°C to -15°C -25°C to -15°C -25°C to -15°C -25°C to -15°C

TELL-SeqTM Target Blocker (PN 100019)

Component Name TELL-SeqTM Target Blocker

Cap Color Volume (L)

CAP White

Storage Temperature
-25°C to -15°C

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3. Consumables and Equipment (not provided)

Consumables

Consumable

Supplier

0.2 mL PCR tube or strip tube 20 L pipette tip (standard and wide orifice) 200 L pipette tip (standard and wide orifice) Ethanol 200 proof (absolute) for molecular biology (500 mL) Nuclease-free water AMPure XP Agilent Bioanalyzer High Sensitivity DNA Analysis Kit* TapeStation High Sensitivity D5000 ScreenTape Assay*
Qubit dsDNA HS Assay Kit
Qubit Assay Tubes
Dynabeads MyOne Streptavidin T1
Agilent SureSelect XT HS Capture Library Human All Exon 7 Agilent SureSelect XT HS Target Enrichment Kit, ILM Hyb Module (Post PCR), 16 Rxn TE buffer, pH 8.0

General lab supplier General lab supplier General lab supplier Sigma-Aldrich, # E7023 General lab supplier Beckman, # A63880 Agilent, # 5067-4626 Agilent, # 5067-5592, #5067-5593 Thermo Fisher Scientific, # Q32851 or Q32854 Thermo Fisher Scientific, # Q32856 Thermo Fisher Scientific, # 65601, 65602 or 65603 Agilent, # G9704N, G9705N or G9706N
Agilent, #G9916B
General lab supplier

*Depends on which system is available in the user facility.
Equipment
Equipment

Supplier

Thermo Cycler Magnetic stand for 0.2 mL PCR tubes Tube Rotator Incubator (for 35C) Vortexer Microcentrifuge Agilent Bioanalyzer* Agilent TapeStation*
Qubit® Fluorometer 3.0
Ice Bucket SpeedVac Vacuum Concentrators (Optional)
*Depends on which system is available in the user facility.

Applied Biosystems General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier Agilent Agilent Thermo Fisher Scientific, # Q33216, Q33217 or Q33218 General lab supplier Thermo Fisher Scientific

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4. TELL-SeqTM Human Exome Capture Workflow
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5. Protocol

A. TELL-SeqTM WGS Library Prep

The following protocol describes a modified TELL-SeqTM whole genome sequencing library preparation procedure with TELL-SeqTM Library Prep kit using human DNA samples. Different genomic DNA sample types can also be substituted following the same protocol. TELL-SeqTM libraries are compatible with other Target Enrichment systems, but Agilent SureSelectXT HS Target Enrichment System using SureSelect Human All Exon V8 capture probes is used as an example in protocol. For use in other Target Enrichment Systems please follow protocol for TELL-SeqTM Library Prep (pg. 10-22) to generate appropriate libraries. TELL-SeqTM libraries can be used as DNA Input following the protocol of each specific Target Enrichment System along with TELL-SeqTM Target Blockers being used in addition to specified blockers.
Barcode DNA

Consumables

Input genomic DNA (User)

Genome Size Large (Human)

Input Amount 5 ng

Reaction Vol (L) Preps/Kit

150

NOTE:
1. Genomic DNA should be stored and diluted in a Tris buffer with pH ranging from 7.5 to 8.0 or a low TE buffer (10mM Tris-HCl, 0.1 mM EDTA, pH 8.0).
5× Reaction Buffer (Kit Box 1, CAP Blue) Cofactor II (Kit Box 1, CAP Amber) Barcoding Enzyme (Kit Box 1, CAP Black) TELL Bead or TELL Bead Plex (Kit Box 2, CAP Orange) Suspension Buffer EZ (Kit Box 1, CAP Natural) Nuclease-free water (User) 0.2 mL PCR tube or strip tube (User) 20 L and 200 L wide orifice pipette tips (User)

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II. Preparation 1. Prepare the following consumables:

Item 5× Reaction Buffer CAP
Cofactor II CAP
Barcoding Enzyme CAP TELL Bead Plex CAP Suspension Buffer EZ CAP Nuclease-free water

Storage -25°C to -15°C
-25°C to -15°C
-25°C to -15°C 2°C to 8°C
-25°C to -15°C Room Temperature

Instruction
Thaw at room temperature. Vortex to mix, then centrifuge briefly. Keep on ice. Vortex to mix, then centrifuge briefly. Keep at room temperature in the dark. Close the tube cap tightly after each use. Flick the tube 4 to 5 times to mix. Centrifuge briefly. Keep on ice. Centrifuge briefly. Keep on ice. Close the tube cap tightly after each use to avoid any evaporation.
Thaw at room temperature. Vortex to mix, then centrifuge briefly. Keep at room temperature.
Keep at room temperature.

2. Set up a tube rotator in a 35C incubator (see Step 7 of the Procedure section).
CAUTION Use wide orifice pipette tips to transfer and mix high molecular weight genomic DNA to avoid breaking the DNA. If wide orifice pipette tips are not available, cut 2mm-3mm off a standard pipette tip top with a clean razor blade before use.
III. Procedure 1. Vortex TELL Bead Plex vigorously for at least 30 seconds. Pulse spin (centrifuge for no more than 1 second) to bring down the bead solution present on the lid or sides of the tube. Right before use, pipet the TELL Bead Plex with a 200 L tip up and down 5 times to make sure all the beads are resuspended properly. 2. In a 0.2 mL PCR tube, assemble each reaction in the following order.

Reagent
5× Reaction Buffer CAP Nuclease-free water Cofactor II CAP TELL Bead Plex CAP (0.5M barcodes/L)

Volume per reaction (L)
Large Genome (150 L)
30 20 Z (Z is the DNA vol)
30 19

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3. Mix well by pipetting up and down for 10 times or vortexing vigorously for 5 seconds and pulse spin to bring the solution down to the bottom. Add Barcoding Enzyme.

Reagent

Volume per reaction (L) Large Genome

Barcoding Enzyme CAP

6 L

4. Mix well by pipetting up and down for 8 times. Avoid introducing air bubbles when pipetting by keeping the pipette tip at the bottom of the solution in the tube.
5. Use a wide orifice pipette tip, add following reagent.

Reagent
Sample genomic DNA Suspension Buffer EZ CAP

Volume per reaction (L)
Large Genome
Z ( 15) 45

NOTE: Suspension Buffer EZ is highly viscous. Use caution and pipette slowly to ensure that correct volume is delivered.

6. Set pipette volume at 110 L. Use a wide orifice pipette tip, gently mix the solution by slowly pipetting up and down 6-8 times. Avoid introducing many air bubbles when pipetting by keeping the pipette tip at the bottom of the solution in the tube.
7. Place the sample tube on a tube rotator in a 35°C incubator and rotate slowly (10-15 rpm) for 30 minutes.

Note: Proper tube rotation is critical to preserve HMW DNA properties and to facilitate the correct barcoding process. Recommended mixing systems are shown below (left side). Mixing systems that do not rotate or that generate vigorous shaking are incompatible with preservation of HMW DNA properties and TELL-Seq; some of these systems are also shown below (right side).
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Stabilize DNA

I. Consumables Stabilizer (Kit Box 1, CAP Violet)

II. Preparation 1. Prepare the following consumables:

Item Stabilizer CAP

Storage -25°C to -15°C

Instruction
Flick the tube 4 to 5 times to mix. Centrifuge briefly. Keep on ice.

III. Procedure 1. Retrieve the sample tube from the 35°C incubator. 2. Add Stabilizer into the tube.

Reagent

Volume per reaction (L) Large Genome

Stabilizer CAP

3. Set pipette volume at 110 L. Use a wide orifice pipette tip, gently mix the solution by slowly pipetting up and down 6-8 times. Avoid creating many bubbles.
4. Place the sample tube back on the tube rotator in the 35°C incubator and rotate it slowly (10-15 rpm) for 30 minutes.

Tagment DNA
IV. Consumables Tagging Enzyme (Kit Box 1, CAP Red) Exonuclease (Kit Box 1, CAP Yellow)
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V. Preparation 1. Prepare the following consumables:

Item Tagging Enzyme CAP Exonuclease CAP

Storage -25°C to -15°C -25°C to -15°C

Instruction
Flick the tube 4 to 5 times to mix. Centrifuge briefly. Keep on ice.
Flick the tube 4 to 5 times to mix. Centrifuge briefly. Keep on ice.

2. Use the same tube rotator in the 35C incubator.

VI. Procedure 1. Retrieve the sample tube from the 35°C incubator. 2. Add Tagging Enzyme and Exonuclease into the tube.

Reagent

Volume per reaction (L) Large Genome

Tagging Enzyme CAP

Exonuclease CAP

3. Set pipette volume at 110 L. Use a wide orifice pipette tip, gently mix the solution by slowly pipetting up and down for 8 times. For this step, the mixing needs to be very thorough. Avoid creating many bubbles.
4. Place the sample tube back on the tube rotator in the 35°C incubator and rotate it slowly for 10 minutes. When necessary, different amount of Tagging Enzyme can be used to adjust the library size.
NOTE: If a longer insert library is preferred, less amount of Tagging Enzyme can be used in the reaction. On the other hand, if a shorter insert library is preferred, up to 6µL Tagging Enzymes can be used in the reaction.

5. Proceed to next step immediately after the incubation.

Wash Beads
I. Consumables Stop Solution (Kit Box 2, CAP Natural or stored at room temperature after the first use) Wash Solution (Kit Box 2, CAP White) 0.2 mL PCR tube or strip tube (User)

II. Preparation 1. Prepare the following consumables:

Item

Storage Instruction

15 100032-USG V5.0

Stop Solution CAP Wash Solution CAP

2°C to 25°C 2°C to 8°C

Check for any precipitates. If present, incubate the buffer at 37°C for 10 minutes, and vortex until they dissolve. Store at room temperature for future use.
Bring to room temperature.

2. Set up a thermo cycler with the following program: · Preheat lid option to 100°C · 63°C forever
III. Procedure

1. Place the sample tube on a magnetic stand for 1 minute or until the solution is clear. 2. While the tube is on the magnetic stand, aspirate and discard the supernatant without
disturbing beads. 3. Remove the tube from the magnetic stand. Add 120 L Wash Solution to the sample tube.
Pipet to resuspend the beads. If necessary, pulse spin to bring the solution down. 4. Place the sample tube back on the magnetic stand for 1 minute or until the solution is clear. 5. While the tube is on the magnetic stand, aspirate and discard the supernatant without
disturbing beads. 6. Remove the tube from the magnetic stand. Add 80 L of Stop Solution to the tube. 7. Pipet several times to resuspend the beads. If necessary, pulse spin to bring the solution
down. 8. Incubate the tube at room temperature for 5 minutes. 9. Place the sample tube back on the magnetic stand for 1 minute or until the solution is clear. 10. While the tube is on the magnetic stand, aspirate and discard the supernatant without
disturbing beads. 11. Remove the tube from the magnetic stand. Add 120 L Wash Solution to the PCR tube. Pipet
to resuspend the beads. 12. Transfer all the bead solution into a new 0.2ml PCR tube. 13. Incubate the tube at 63C on the PCR thermocycler for 3 minutes. 14. Place the new sample tube on the magnetic stand at room temperature for 1 minute or until
the solution is clear. 15. While the tube is on the magnetic stand, aspirate and discard the supernatant without
disturbing beads. 16. Remove the tube from the magnetic stand. Add 120 L Wash Solution to the PCR tube. Pipet
to resuspend the beads. If necessary, pulse spin to bring the solution down. 17. Incubate the tube at 63C on the PCR thermocycler for 3 minutes. 18. Place the sample tube on the magnetic stand at room temperature for 1 minute or until the
solution is clear. 19. While the tube is on the magnetic stand, aspirate and discard the supernatant without
disturbing beads. Use a P20 pipette to remove any remaining supernatant. 20. Remove the tube from the magnetic stand. Resuspend the beads in 20 L of Wash Solution.

16 100032-USG V5.0

NOTE: This is a SAFE STOPPING POINT. The washed beads can be stored at 2°C to 8°C for two weeks.

Amplify Library

Consumables

2× PCR Master Mix (Kit Box 1, CAP Pink)

10× Primer I (Kit Box 1, CAP White)

10× Primer II, T50# (Multiplex Primer Kit)

Nuclease-free water (User)

0.2 mL PCR tube or strip tube (User)

II. Preparation 1. Prepare the following consumables:

Item 2× PCR Master Mix CAP 10× Primer I CAP 10× Primer II, T50# Enhancer CAP Green Nuclease-free water

Storage -25°C to -15°C -25°C to -15°C -25°C to -15°C -25°C to -15°C Room Temperature

Instruction
Thaw at room temperature. Flick the tube 4 to 5 times to mix, then centrifuge briefly. Keep on ice. Thaw at room temperature. Vortex to mix, then centrifuge briefly. Keep on ice. Thaw at room temperature. Vortex to mix, then centrifuge briefly. Keep on ice. Thaw at room temperature. Vortex to mix, then centrifuge briefly. Keep at room temperature.
Keep at room temperature.

2. Set up Library Amplification Program (LAP) on a thermo cycler as following: · 63°C 2 minutes · 72°C 2 minutes · 98°C 30 seconds · [98°C 15 seconds, 63°C 20 seconds, 72°C 30 seconds] x Cycle Number · 72°C 3 minutes · 4°C forever
NOTE:
Cycle number is flexible based on downstream applications and requirement. Recommend starting with 13 cycles. Higher cycle number will generate more TELL-Seq library as an input for Hybridization and Capture, but higher duplication rates for the final sequencing analysis. Lower cycle number may decrease duplication rate, but lower DNA inputs may result in decreased capture efficiency and reduced library complexity. Please refer to the end of Qualify and Quantify Library for Hybridization and Capture for further considerations when determining appropriate cycle number.

17 100032-USG V5.0

Genome Size Large

Vol of Beads Used (B) for PCR 20 L

PCR Volume 75 L

Cycle Number 12-14

III. Procedure 1. Vortex beads vigorously for 10 seconds to resuspend the beads. Pulse spin to bring solution down. Using a 20 L tip, pipet the beads up and down 5 times to make sure all the beads are resuspended properly. Immediately transfer entire bead solution amount of to a new PCR tube. 2. Place the PCR tube on a magnetic stand for 1 minute or until the solution is clear. 3. While the tube is on the magnetic stand, remove 20 L supernatant without disturbing beads. Remove the PCR tube from the magnet. 4. Add following reagents to the PCR tube.

Reagent
Nuclease-free water 2× PCR Master Mix CAP 10× Primer I CAP 10× Primer II, T50# Enhancer CAP Green

Volume per reaction (L)
Large Genome (75 L) 16 L 37.5 L 7.5 L 7.5 L 4.5 L

5. Mix well by vortexing or pipetting. Pulse spin to bring solution down. 6. Place the tube on the thermal cycler and run the LAP program (see above) with proper
number of cycles. 7. After PCR amplification, use 2 L PCR product for quality check on a Bioanalyzer or a
TapeStation. See Qualify and Quantify Library section for instruction.
PRO TIP: If QC check shows the library yield is relatively low, put the tube with remaining PCR product back to the thermocycler and amplify for another one or two extra cycles before moving to Clean Up Library section.

NOTE: This is a SAFE STOPPING POINT. The PCR product can be stored at -25°C to -15°C for one month.

Clean Up Library
I. Consumables AMPure XP (User) 18 100032-USG V5.0

Ethanol 200 proof (absolute) for molecular biology (User) Nuclease-free water (User) 0.2 mL PCR tube or strip tube (User)

II. Preparation 1. Prepare the following consumables:

Item Fresh 75% (v/v) ethanol
AMPure XP
Nuclease-free water TE buffer, pH 8.0

Storage Room Temperature
2°C to 8°C
Room Temperature Room Temperature

Instruction
Require 400 L per sample. Mix 1.5 mL Ethanol (200 proof) with 0.5 mL Nuclease-free water. Vortex to mix and keep at room temperature. Bring it to room temperature for at least 20 minutes and vortex vigorously to resuspend the beads before use. Keep at room temperature.
Keep at room temperature.

III. Procedure 1. Briefly centrifuge the sample PCR tube to bring all solution down. 2. Place the tube on the magnetic stand for 1 minute or until the solution is clear. 3. While the tube is on the magnetic stand, transfer the supernatant to a new 0.2 mL PCR tube without disturbing beads. 4. Measure the volume of transferred supernatant (PCR product) with a pipette. 5. Add following reagents into the PCR product to a total volume of 100 L.

Reagent PCR product Nuclease-free water

Volume per reaction 75 L
To final 100 L total

6. Vortex vigorously to resuspend the AMPure XP solution and add 78 L AMPure XP into the 100 L PCR product.
7. Mix by pipetting up and down 10 times. 8. Incubate at room temperature for 5 minutes. 9. Place the tube on the magnetic stand for 1 minute or until the solution is clear. 10. Aspirate and discard the supernatant without disturbing AMPure beads. 11. While keeping the tube on the magnetic stand, add 200 L freshly prepared 75% ethanol
into the tube. Let it sit for 30 seconds. 12. Aspirate and discard the supernatant without disturbing beads. 13. Repeat steps 11-12 one more time, keeping the tube on the magnetic stand for the whole
time. 14. Keep the tube on the magnetic stand with cap open and allow the tube to dry for 1-2
minutes to evaporate traces of ethanol. DON’T over dry the beads. 15. Remove the tube from the magnetic stand and add 20 L nuclease-free water to the beads. 16. Pipette or vortex to resuspend the beads. Let it sit for 5 minutes. 17. Put the tube on the magnetic stand for 1 minute or until the solution is clear.
19 100032-USG V5.0

18. Recover 18 L of the supernatant to a new tube. Be careful not to disturb the beads. 19. The supernatant contains the TELL-SeqTM library.
NOTE: This is a SAFE STOPPING POINT. The purified TELL-Seq library can be stored at -25°C to -15°C for a month.
20 100032-USG V5.0

Qualify and Quantify Library for Target Enrichment

Consumables

Agilent High Sensitivity DNA Kit or TapeStation High Sensitivity D5000 ScreenTape Assay

(User) Qubit dsDNA HS Assay Kit (User) TE buffer, pH 8.0 (User)

NOTE:
Standard qPCR library quantitation assay for Illumina system works for TELL-Seq library, but it is not required.

II. Preparation 1. Prepare the necessary consumables as required by Bioanalyzer or TapeStation and Qubit.
III. Procedure 1. Use 1 L of library for Agilent High Sensitivity DNA Kit or 2 L of library for TapeStation High Sensitivity D5000 ScreenTape Assay. 2. Check the saved uncleaned PCR product from the Amplify Library section at the same time. Uncleaned PCR product may have a high level of primer dimer and adapter dimer. It requires a two-fold dilution with nuclease-free water before loading onto a Bioanalyzer chip or TapeStation tape to avoid interfering with lower marker signal. 3. A good-sized library should have most library fragments under 1000 bp (Figure 1).

4. Library can be stored at -25°C to -15°C. 5. Make a 10-fold diluted TELL-SeqTM library sample: dilute 2 L of TELL-SeqTM library with 18 L
of nuclease-free water. Use 4 L diluted library to check the concentration with the Qubit dsDNA HS Assay Kit.
21 100032-USG V5.0

6. Use the concentration (ng/L) and volume to calculate total mass of each TELL-SeqTM Library going into the SureSelectXT HS Target Enrichment System process. 500 ng -1,000 ng TELLSeqTM library per sample is recommended for optimal results, but library inputs as low as 250 ng per sample are possible though performance can be negatively impacted. NOTE: There are volume constraints for the Hybridization and Capture protocol. 12 L is the max volume allowed for DNA input. Careful consideration should be used to ensure that the DNA library volume falls within this range. Ideally use all available TELL-Seq library material after QC into the Hybridization and Capture reaction.
7. (Optional) TELL-SeqTM libraries can be concentrated using SpeedVac Vacuum Concentrator. If a SpeedVac will be used for concentrating library, TELL-SeqTM library after AMPure XP cleanup can be eluted from XP beads with at least 30 L nuclease-free water for better recovery. After QC, the clean TELL-SeqTM library can be concentrated to desired volume for Hybridization and Capture. NOTE: Other Target Enrichment Systems can be used like IDT and Twist Biosciences using TELLSeqTM libraries created. Follow specified Target Enrichment protocols using TELL-SeqTM libraries as DNA Input. Use 5ul of TELL-SeqTM TargetSeq Blocker in addition to detailed blockers in each protocol.
22 100032-USG V5.0

B. SureSelect Target Enrichment
The following protocol is a modification of the Hybridization and Capture portion SureSelect XT HS Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library Protocol (Agilent, G9702-90000). Modifications allow for compatibility of TELL-SeqTM WGS Library Prep with Agilent SureSelect XT HS Target Enrichment System and All Exon V8 Capture Probes. Agilent reagents for just Hybridization and Capture can be purchased separately in 16-reaction format (Agilent Part Number G9916B). If using other Target Enrichment Systems follow the specified protocols for each system and substituting TELL-SeqTM libraries as DNA Input. 5ul of TELL-SeqTM Target Blockers is also needed in addition to detailed blockers in each Target Enrichment protocol

Hybridize TELL-SeqTM Library to the Exome Capture Panel

I. Consumables SureSelect XT HS and XT Low Input Blocker Mix, 16 Rxn (Agilent, SureSelect XT HS Target
Enrichment Kit, ILM Hyb Module, Box 2 (Post PCR), CAP Blue) SureSelect RNase Block, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb
Module, Box 2 (Post PCR), CAP Violet) SureSelect Fast Hybridization Buffer, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit
ILM Hyb Module, Box 2 (Post PCR), Bottle) TELL-SeqTM Target Blocker (UST, TELL-SeqTM Target Blocker Box, CAP White) SSel XT HS and XT Low Input Human All Exon V7 (Agilent, SSel XT HS and XT Low Input
Human All Exon V7, CAP Red) Nuclease-free water (User) 0.2 mL PCR tube or strip tube (User) TELL-SeqTM Library (User)

Input Amount Reaction Vol (L)

500 -1,000 ng

Up to 12 L

II. Preparation 1. Prepare the following consumables:

Item

Storage Instruction

SureSelect XT HS and XT Low Input Blocker Mix CAP SureSelect RNase Block CAP
SureSelect Fast Hybridization Buffer

-25°C to -15°C -25°C to -15°C -25°C to -15°C

Thaw on ice. Vortex to mix, then centrifuge briefly. Keep on ice. Thaw on ice. Vortex to mix, then centrifuge briefly. Keep on ice.
Thaw and keep at room temperature

TELL-SeqTM TargetSeq Blocker CAP
TELL-SeqTM DNA Library SSel XT HS and XT Low Input Human All Exon V8 CAP

-25°C to -15°C -25°C to -15°C -85°C to -75°C

Thaw at room temperature. Vortex to mix, then centrifuge briefly. Keep on ice.
Thaw and keep at ice Thaw on ice. Vortex to mix, then centrifuge briefly. Keep on ice.

23 100032-USG V5.0

2. Set up the Hybridization Program (HP) on a thermo cycler (with the heated lid ON) with the program below. Start the program, then immediately press the Pause button, allowing the heated lid to reach temperature while you set up the reactions.

Segment Number 1 2 3
4 5

Number of Cycles 1 1 1
60 1

Temperature
95°C 65°C 65°C 65°C 37°C 65°C

Time
5 minutes 10 minutes 1 minute 1 minute 3 seconds
Hold

III. Procedure 1. Place 5001000 ng of each prepared TELL-SeqTM library sample into the strip tube wells and then bring the final volume in each well to 12 µl using nuclease-free water if needed. Ideally make the total amplified TELL-SeqTM library DNA, within the 5001000 ng range and use all for the hybridization reaction. 2. To each TELL-SeqTM library sample well, add 5 µl of SureSelect XT HS and XT Low Input Blocker Mix and add 5 µl of TELL-SeqTM Target Blocker. Cap the wells then vortex at high speed for 5 seconds. Spin the strip tube briefly to collect the liquid release any bubbles. 3. Transfer the tubes to the thermal cycler and press the Play button to resume the HP program set up.
NOTE:
The thermal cycler must be paused during Segment 3 (see HP) to allow additional reagents to be added to the Hybridization wells, as described in step 6. During Segments 1 and 2 of the thermal cycling program, begin preparing the additional reagents as described in step 4 and step 5. If needed, you can finish these preparation steps after pausing the thermal cycler in Segment 3.

4. Prepare a 25% solution of SureSelect RNase Block (containing 1 volume of RNase Block with 3 volume of water), according to the table below. Prepare the amount required for the number of hybridization reactions in the run, plus excess.

Volume per reaction (L)

Reagent

Volume for 1 Volume for 8 reactions Volume for 24 reactions

Reaction

(includes excess)

SureSelect RNase Block CAP

0.5 L

4.5 L

12.5 L

Nuclease-free water

1.5 L

13.5 L

37.5 L

5. Prepare the Capture Library Hybridization Mix in the following order.

Reagent

Volume per reaction (L)

24 100032-USG V5.0

Volume for 1 Reaction

25% RNase Block solution

2 L

SSel XT HS and XT Low Input Human All Exon V7 CAP

5 L

SureSelect Fast Hybridization Buffer

6 L

Volume for 8 reactions (includes
excess) 18 L
45 L
54 L

Volume for 24 reactions (includes
excess) 50 L
125 L
150 L

Combine the listed reagents at room temperature. Mix well by vortexing at high speed for 5 seconds then spin down briefly. Proceed immediately to step 6. 6. Once the thermal cycler starts Segment 3 of the HP (1 minute at 65°C), press the Pause button. With the cycler paused, and while keeping the DNA + Blocker samples in the cycler, transfer 13 µl of the room- temperature Capture Library Hybridization Mix from step 6 to each sample well. Mix well by pipetting up and down slowly 8 to 10 times. The hybridization reaction wells now contain approximately 35 µl 7. Make sure that all wells are completely sealed. Vortex briefly, then spin strip tube briefly to remove any bubbles from the bottom of the wells. Immediately return the strip tube to the thermal cycler. 8. Press the Play button to resume the thermal cycling program to allow hybridization of the prepared DNA samples to the Capture Library.

Prepare Streptavidin-coated Magnetic Beads

I. Consumables Dynabeads MyOne Streptavidin T1 (User) SureSelect Binding Buffer (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module,
Box 1 (Post PCR), CAP)

II. Preparation 1. Prepare the following consumables:

Item

Storage

Dynabeads MyOne Streptavidin T1

2°C to 8°C

SureSelect Binding Buffer, CAP

Room Temperature

Instruction
Centrifuge vigorously. Keep at room temperature.
Keep at room temperature.

III. Procedure 1. Vigorously resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads on a vortex mixer. The magnetic beads settle during storage. 2. For each hybridization sample, add 50 µl of the resuspended beads to wells of a fresh PCR plate or a strip tube. 3. Wash the beads by adding 200 µl of SureSelect Binding Buffer. Mix by pipetting up and down 20 times or cap the wells and vortex at high speed for 510 seconds. 4. Put the plate or strip tube into a magnetic separator device.

25 100032-USG V5.0

5. Wait at least 5 minutes or until the solution is clear, then remove and discard the supernatant.
6. Repeat Steps 3-5 two more times for a total of 3 washes. 7. Resuspend the beads in 200 µl of SureSelect Binding Buffer.

Capture the Hybridized DNA using Streptavidin-coated Beads

I. Consumables SureSelect Wash Buffer 1, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR), CAP) SureSelect Wash CAP Buffer 2, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR), CAP)
II. Preparation 1. Prepare the following consumables:

Item SureSelect Wash Buffer 1, CAP SureSelect Wash Buffer 2, CAP

Storage Room Temperature Room Temperature

Instruction Keep at room temperature. Heat 200 µl aliquots at 70°C. See Step 4

III. Procedure 1. After the hybridization step is complete and the thermal cycler reaches the 65°C hold step, transfer the samples to room temperature. 2. Immediately transfer the entire volume (approximately 30 µl) of each hybridization mixture to wells containing 200 µl of washed streptavidin beads using a multichannel pipette. Pipette up and down 58 times to mix. 3. Incubate the capture strip tube on a 96- well plate mixer, mixing vigorously (at 14001800 rpm) or rotator, for 30 minutes at room temperature. Make sure the samples are properly mixing in the wells. 4. Proceed to next step immediately after the incubation. During the 30-minute incubation for capture, prewarm SureSelect Wash Buffer 2 at 70°C as described below. Place 200 µl aliquots of Wash Buffer 2 in wells of a fresh 96- well plate or strip tubes. Aliquot 6 wells of buffer for each DNA sample in the run. Cap the wells and then incubate in the thermal cycler, with heated lid ON, held at 70°C until used in 5. When the 30-minute incubation period initiated in Step 3 is complete, spin the samples briefly to collect the liquid. 6. Place the strip tube in a magnetic separator to collect the beads. Wait until the solution is clear, then remove and discard the supernatant. 7. Resuspend the beads in 200 µl of SureSelect Wash Buffer 1. Mix by pipetting up and down 1520 times, until beads are fully resuspended. 8. Place the strip tube in the magnetic separator. Wait for the solution to clear (approximately 1 minute), then remove and discard the supernatant. 9. Remove the strip tubes from the magnetic separator and transfer to a rack at room temperature. Resuspend the beads in 200 µl of 70°C prewarmed Wash Buffer 2. Pipette up
26 100032-USG V5.0

and down 1520 times, until beads are fully resuspended. Seal the wells with fresh caps and then vortex at high speed for 8 seconds. Spin the plate or strip tube briefly to collect the liquid without pelleting the beads. 10. Incubate the samples for 5 minutes at 70°C on the thermal cycler with the heated lid on. 11. Place the strip tube in the magnetic separator at room temperature. Wait 1 minute for the solution to clear, then remove and discard the supernatant. 12. Repeat Steps 9-11 five more times, for a total of 6 washes. 13. After verifying that all wash buffer has been removed, add 25 µl of nuclease-free water to each sample well. Pipette up and down 8 times to resuspend the beads. Samples can be stored on ice before amplification
Amplify Captured Library

I. Consumables 5× Herculase II Reaction Buffer (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR), CAP Clear) Herculase II Fusion DNA Polymerase (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR), CAP Red) 100 mM dNTP Mix, (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR), CAP Green) SureSelect Post-Capture Primer Mix (Agilent, SureSelect XT HS Target Enrichment Kit, ILM
Hyb Module Box 2 (Post PCR), CAP Clear)

II. Preparation 1. Prepare the following consumables:

Item

Storage Instruction

5× Herculase II Reaction Buffer CAP Herculase II Fusion DNA Polymerase CAP 100 mM dNTP Mix CAP SureSelect Post-Capture Primer Mix CAP

-25°C to -15°C -25°C to -15°C -25°C to -15°C -25°C to -15°C

Thaw at room temperature. Vortex to mix, then centrifuge briefly. Keep on ice.
Centrifuge briefly. Keep on ice.
Thaw at room temperature. Vortex to mix, then centrifuge briefly. Keep on ice. Thaw at room temperature. Vortex to mix, then centrifuge briefly. Keep on ice.

2. Set up following program on a thermo cycler as following: · 98°C 2 minutes · [98°C 30 seconds, 63°C 30 seconds, 72°C 1 minute] x 9 · 72°C 5 minutes · 4°C forever

IV. Procedure 1. Prepare the appropriate volume of PCR reaction mix. Add following reagents to the PCR tube based on number of reactions.

Reagent

Volume per reaction (L)

27 100032-USG V5.0

Volume for 1 Reaction

Nuclease-free water 5× Herculase II Reaction Buffer CAP Herculase II Fusion DNA Polymerase CAP 100 mM dNTP Mix CAP SureSelect Post-Capture Primer Mix CAP

12.5 L 10 L 1 L 0.5 L 1 L

Volume for 8 reactions (includes
excess)
112.5 L 90 L 9 L 4.5 L 9 L

Volume for 24 reactions (includes
excess)
312.5 L 250 L 25 L 12.5 L 25 L

2. Prepare the appropriate volume of PCR reaction mix. Add following reagents to the PCR tube based on number of reactions. Add 25 µl of the PCR reaction mix prepared in Table 29 to each sample well containing 25 µl of bead- bound target- enriched DNA (prepared on page 26 and held on ice).
3. Mix the PCR reactions well by pipetting up and down until the bead suspension is homogeneous. Avoid splashing samples onto well walls; do not spin the samples at this step.
4. Place the tube on the thermal cycler and run the program (see above) with proper number of cycles.
5. When the PCR amplification program is complete, spin the strip tube briefly. Remove the streptavidin- coated beads by placing the plate or strip tube on the magnetic stand at room temperature. Wait 2 minutes for the solution to clear, then remove each supernatant (approximately 50 µl) to wells of a strip tube.

Clean Up Captured Library

I. Consumables AMPure XP (User) Ethanol 200 proof (absolute) for molecular biology (User) Nuclease-free water (User) TE buffer, pH 8.0 (User) 0.2 mL PCR tube or strip tube (User)

II. Preparation 1. Prepare the following consumables:

Item

Storage

Instruction

Fresh 75% (v/v) ethanol AMPure XP Nuclease-free water

Room Temperature 2°C to 8°C Room Temperature

Require 400 L per sample. Mix 1.5 mL Ethanol (200 proof) with 0.5 mL Nuclease-free water. Vortex to mix and keep at room temperature. Bring it to room temperature for at least 20 minutes and vortex vigorously to resuspend the beads before use. Keep at room temperature.

TE buffer, pH 8.0

Room Temperature Keep at room temperature.

III. Procedure 1. Bring solution down with a quick ~1 second spin in the centrifuge.
28 100032-USG V5.0

2. Vortex vigorously to resuspend the AMPure XP solution and add 50 L AMPure XP into each PCR product.
3. Mix by pipetting up and down 10 times. 4. Incubate at room temperature for 5 minutes. 5. Place the tube on the magnetic stand for 1 minute or until the solution is clear. 6. Aspirate and discard the supernatant without disturbing AMPure beads. 7. While keeping the tube on the magnetic stand, add 200 L freshly prepared 75% ethanol
into the tube. Let it sit for 30 seconds. 8. Aspirate and discard the supernatant without disturbing beads. 9. Repeat steps 7-8 one more time, keeping the tube on the magnetic stand for the whole
time. 10. Leave the tube on the magnetic stand with cap open and allow the tube to dry for 1-2
minutes to evaporate traces of ethanol. DON’T over dry the beads. 11. Remove the tube from the magnetic stand and add 25 L TE buffer to the beads. 12. Pipette or vortex to resuspend the beads. Let it sit for 5 minutes. 13. Place the tube on the magnetic stand for 1 minute or until the solution is clear. 14. Recover 23 L of the supernatant to a new tube. Be careful not to disturb the beads. 15. The supernatant contains the captured TELL-SeqTM library.
NOTE:
This is a SAFE STOPPING POINT. The captured TELL-Seq library can be stored at -25°C to -15°C for six months.
Qualify and Quantify Captured Library for Sequencing
I. Consumables Agilent Bioanalyzer High Sensitivity DNA Kit or TapeStation High Sensitivity D5000 ScreenTape Assay (User)
Qubit dsDNA HS Assay Kit (User) TE buffer, pH 8.0 (User)
II. Preparation 1. Prepare the necessary consumables as required by Bioanalyzer or TapeStation and Qubit.
III. Procedure 1. Use 1 L of library for Agilent Bioanalyzer High Sensitivity DNA Kit or 2 L of library for TapeStation High Sensitivity D5000 ScreenTape Assay. 2. To determine the library concentration, set the Region on the Bioanalyzer or TapeStation analysis software from 150 bp to 1000 bp. Record sample Concentration (nM) for this region (see Figure 2). To determine the library size, set the Region from 150 bp to 3000 bp. Record sample Average Size (bp) as Library Size.
29 100032-USG V5.0

CAUTION The concentration reading from the Bioanalyzer (or TapeStation) should be used as a starting point to make necessary dilution or library pooling for sequencing. Verify the concentration of the final diluted sequencing library or library pool with a Qubit dsDNA HS Assay kit (see Step 6).
3. Library can be sequenced immediately or stored at -25°C to -15°C. 4. When sequencing, dilute the library using TE buffer to the concentration recommended by
each Illumina® sequencing system. Make diluted library pool for sequencing if more than one library will be sequenced in the same run. 5. Measure the library concentration with the Qubit dsDNA HS Assay Kit. Use the Average Size value from the Bioanalyzer (or TapeStation) measurement as the library size for conversion of mass concentration into molar concentration (nM). A = Mass Concentration (ng/µL) S = Library Size (bp) Molar Concentration (nM) = (A*1,000,000)/(S*650) Adjust the volume needed in the sequencing preparation if the library concentration measured by Qubit is different from the recommended concentration by more than 10%.
30 100032-USG V5.0

Documents / Resources

UNIVERSAL SEQUENCING 100035 TELL Seq Target Enrichment [pdf] User Guide
100035, 100036, 100037, 100038, 100035 TELL Seq Target Enrichment, 100035, TELL Seq Target Enrichment, Seq Target Enrichment, Target Enrichment

References

User Manual

100035 TELL Seq Target Enrichment

Specifications:

Product Usage Instructions:

1. Introduction

2. Kit Contents

3. Genomic DNA Input Recommendations

4. TELL-SeqTM Human Exome Capture Workflow

5. Protocol

FAQ:

Q: What should I do if the Stop Solution in Box 2 is not clear
or has white precipitates?

Documents / Resources

References

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