Facile, high quality sequencing of bacterial genomes from small amounts of DNA.

Momchilo Vuyisich,Patrick Chain,Karen Davenport,Ayesha Arefin,Kim Mcmurry,Beverly Parson-Quintana,Matthew Scholz,Shihai Feng,Jennifer Price,Cheryl Gleasner

doi:10.1155/2014/434575

Momchilo Vuyisich, Patrick Chain + Show 8 more

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https://doi.org/10.1155/2014/434575

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Abstract

Sequencing bacterial genomes has traditionally required large amounts of genomic DNA (~1 μg). There have been few studies to determine the effects of the input DNA amount or library preparation method on the quality of sequencing data. Several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input DNA. In this study, we evaluated the NEBNext Ultra library preparation reagents for sequencing bacterial genomes. We have evaluated the utility of NEBNext Ultra for resequencing and de novo assembly of four bacterial genomes and compared its performance with the TruSeq library preparation kit. The NEBNext Ultra reagents enable high quality resequencing and de novo assembly of a variety of bacterial genomes when using 100 ng of input genomic DNA. For the two most challenging genomes (Burkholderia spp.), which have the highest GC content and are the longest, we also show that the quality of both resequencing and de novo assembly is not decreased when only 10 ng of input genomic DNA is used.

Highlights

The rapid improvement in quality, quantity, and cost of generation sequencing (NGS) has resulted in commensurate improvements in analysis techniques
The commoditization of bacterial genome sequencing has led to more complex applications: clinical and agricultural diagnostics [1,2,3,4], outbreak detection and monitoring [5,6,7], human health studies [8, 9], biocatalysis [10, 11], environmental studies [12], and many others [13, 14]
Our findings indicate that low DNA input amounts are sufficient to generate high quality sequencing data that can be used for genome resequencing or de novo assembly

Summary

Introduction

The rapid improvement in quality, quantity, and cost of generation sequencing (NGS) has resulted in commensurate improvements in analysis techniques. The availability of kits for library preparation, rapid and high content sequencing, and mature data analysis pipelines for genome resequencing and assembly had drastically reduced costs and improved reliability of these results. Existing library preparation methods have several reported limitations These include high variability of evenness and completeness of genome coverage as a function of %GC content, input DNA quantities, and sequencing technology [15,16,17,18,19]. These impact the amount of sequencing data required and the quality of genome assembly and analysis. Our findings indicate that low DNA input amounts are sufficient to generate high quality sequencing data that can be used for genome resequencing or de novo assembly (if combined with long fragment data)

Materials and Methods

Results and Discussion

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