Inexpensive multiplexed library preparation for megabase-sized genomes.

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Whole-genome sequencing is crucial in modern biology.
• High sample preparation costs, especially for small genomes, limit its application.
• Existing protocols are expensive and time-consuming.

Purpose of the Study:

• To develop a cost-effective and rapid protocol for preparing multiplexed genomic libraries.
• To reduce the cost of library preparation for Illumina sequencing.
• To enable large-scale sequencing of small genomes and other applications.

Main Methods:

• Optimized Nextera tagmentation reaction in small volumes.
• Replaced expensive reagents with cheaper alternatives.
• Streamlined protocol, omitting unnecessary steps.

Main Results:

• Achieved a library preparation cost of $8 per sample, ~6 times cheaper than Nextera XT.
• Completed library preparation for 96 samples in under 5 hours.
• Enabled pooling of hundreds of samples on a single HiSeq lane using custom barcodes.

Conclusions:

• The developed protocol significantly reduces costs and time for genomic library preparation.
• This method is highly applicable for re-sequencing microbial/viral genomes, evolution experiments, and environmental samples.
• The protocol supports diverse sequencing applications, including RNA sequencing and large amplicon sequencing.