TruSight Cardio Sequencing Kit Reference Guide (15063774 v01)

TruSight Cardio Sequencing Kit Reference Guide (15063774 v01)

  Chapter 1  Overview

Overview

Introduction

DNA Input Recommendations

Additional Resources

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TruSight Cardio Sequencing Kit Reference Guide

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Introduction

This protocol explains how to prepare up to 48 indexed paired-end libraries followed by enrichment using the TruSight Cardio Sequencing Kit.

This protocol fragments and adds adapter sequences onto template DNA to generate indexed libraries that can be carried through enrichment for targeted resequencing applications.

The TruSight Cardio protocol offers:

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Fast and easy sample preparation

} Prepare up to 12 or 48 enriched libraries in approximately 1.5 days, with approximately 5 hours of hands-on time

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Low DNA input and excellent data quality

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Excellent data quality with low input of 50 ng

} Access precious samples with no affect on performance

} Ability to archive samples for subsequent analysis

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High enrichment rates, low duplicates, and exceptional coverage uniformity

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Efficient use of sequencing

} Reliable variant calling

} Reduced hands-on time with the most cost-effective, high-throughput workflow

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DNA Input Recommendations

Using an enzymatic DNA fragmentation step allows TruSight Cardio library preparation to be more sensitive to DNA input than mechanical fragmentation methods. Accurate quantification of the starting gDNA is essential to enrichment success.

Quantify the starting gDNA using a fluorometric-based method specific for double-stranded

DNA (dsDNA) and run samples in triplicate. Avoid methods that measure total nucleic acid content, such as NanoDrop or other UV absorbance methods. Common contaminants such as ssDNA, RNA, and oligos are not substrates for the TruSight Cardio Sequencing Kit.

} Make sure that the starting DNA does not contain more than 1 mM EDTA and is free of organic contaminants, such as phenol and ethanol.

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DNA samples can contain substances that interfere with the Nextera tagmentation reaction and result in unexpected library insert sizes. An additional sample cleanup and quantification step before library preparation ensures optimal conditions for library preparation.

The TruSight Cardio protocol has been optimized for 50 ng of total gDNA. A higher mass input of gDNA can result in incomplete tagmentation and larger insert sizes, and can affect enrichment performance. Conversely, a lower mass input of gDNA or low quality gDNA in the tagmentation reaction can generate smaller than expected insert sizes, which can be lost during subsequent cleanup steps and result in lower diversity.

To minimize gDNA sample input variability into the tagmentation step, perform a 2-step method of gDNA normalization. After the initial quantification, gDNA samples are normalized to 10 ng/µl. Samples are then quantified using a similar fluorometric-based method and normalized to a final 5 ng/µl.

TruSight Cardio Sequencing Kit Reference Guide

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Additional Resources

Visit the TruSight Cardio Sequencing Kit support page on the Illumina website for documentation, software downloads, training resources, and information about compatible

Illumina products.

Resource

Custom Protocol Selector

TruSight Cardio Sequencing Kit

Protocol Guide (document #

15069812)

TruSight Cardio Sequencing Kit

Checklist (document # 15069811)

Nextera Rapid Capture

Enrichment Low-Plex Pooling

Guidelines Technical Note

Description

http://support.illumina.com/custom-protocol-selector.html

A wizard for generating customized end-to-end documentation that is tailored to the library prep method, run parameters, and analysis method used for the sequencing run.

Provides only protocol instructions.

The protocol guide is intended for experienced users. For lesser experienced users, see the TruSight Cardio Sequencing

Kit Reference Guide.

Provides a checklist of the protocol steps.

The checklist is intended for experienced users. For new or less experienced users, see the TruSight Cardio Sequencing Kit

Reference Guide.

Provides pooling guidelines and dual indexing strategies for

Nextera Rapid Capture Enrichment library preparation.

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Document # 15063774 v01

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