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Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

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Published on: October 27, 2011

Preparing Mate-Paired Illumina Libraries Using Cre Recombinase.

Ze Peng¹, Nandita Nath², Zhiying Zhao²

¹United States Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA. zpeng@lbl.gov.

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

|August 18, 2017

Summary

We developed CLIP-PE, a new method using Cre-LoxP recombination and inverse PCR to create large DNA libraries. This approach improves genome assembly and structural variant discovery using next-generation sequencing.

Keywords:

Cre-LoxP recombination Illumina sequencing Mate-paired libraries

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Area of Science:

Genomics
Molecular Biology
Bioinformatics

Background:

Large insert mate pair reads are crucial for de novo genome assembly and structural variant detection.
Existing methods face challenges in efficiently capturing and analyzing large DNA fragments.

Purpose of the Study:

To introduce Cre-LoxP inverse PCR paired end (CLIP-PE) methodology for enhanced genome assembly.
To leverage Cre-LoxP recombination and inverse PCR for improved analysis of large DNA fragments.

Main Methods:

Utilized the Cre-LoxP recombination system for efficient circularization of large DNA fragments.
Employed inverse PCR to enrich for DNA fragments containing both ends.
Incorporated restriction enzymes to create recognizable junction sites between ligated ends.

Main Results:

Successfully generated CLIP-PE libraries with jumping pairs up to 22 kb.
Demonstrated significant improvements in genome assembly quality using CLIP-PE libraries.
Validated the effectiveness of CLIP-PE for structural variant discovery.

Conclusions:

CLIP-PE offers a robust method for creating large DNA libraries for genomic studies.
The methodology enhances the accuracy and contiguity of genome assemblies.
CLIP-PE is adaptable to current and future next-generation sequencing platforms.