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Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Published on: April 4, 2018

Library preparation and data analysis packages for rapid genome sequencing.

Kyle R Pomraning¹, Kristina M Smith, Erin L Bredeweg

¹Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing (CGRB), Oregon State University, Corvallis, OR, USA.

Methods in Molecular Biology (Clifton, N.J.)

|October 16, 2012

Summary

This summary is machine-generated.

This study presents an updated protocol for creating Illumina sequencing libraries for high-throughput sequencing (HTS). The method enables cost-effective, in-house library preparation and multiplexing for comparative genomics research.

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Area of Science:

Genomics
Molecular Biology
Bioinformatics

Background:

High-throughput sequencing (HTS) is crucial for comparative genetics and genomics.
Decentralized sequencing is increasingly common in non-specialized labs.
Standardized protocols are needed for accessible HTS library preparation.

Purpose of the Study:

To provide an updated, accessible protocol for constructing single- and paired-end Illumina sequencing libraries.
To enable cost-effective, in-house library preparation for diverse research settings.
To facilitate multiplexing through the generation of barcoded libraries independent of commercial kits.

Main Methods:

Detailed protocol for preparing Illumina sequencing libraries from purified genomic DNA.
Method for generating barcoded/indexed libraries for multiplexed sequencing.
Discussion of various approaches for analyzing sequencing data.

Main Results:

An updated, robust protocol for Illumina library construction is described.
The protocol supports both single- and paired-end sequencing.
A method for independent library barcoding enabling multiplexing is presented.

Conclusions:

The protocol empowers laboratories to perform HTS library preparation in-house.
Multiplexing via barcoded libraries enhances throughput and reduces costs.
End-users should stay informed about evolving alignment and counting algorithms for data analysis.