title: Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA.
abstract: Construction of DNA fragment libraries for next-generation sequencing can prove challenging, especially for samples with low DNA yield. Protocols devised to circumvent the problems associated with low starting quantities of DNA can result in amplification biases that skew the distribution of genomes in metagenomic data. Moreover, sample throughput can be slow, as current library construction techniques are time-consuming. This study evaluated Nextera, a new transposon-based method that is designed for quick production of DNA fragment libraries from a small quantity of DNA. The sequence read distribution across nine phage genomes in a mock viral assemblage met predictions for six of the least-abundant phages; however, the rank order of the most abundant phages differed slightly from predictions. De novo genome assemblies from Nextera libraries provided long contigs spanning over half of the phage genome; in four cases where full-length genome sequences were available for comparison, consensus sequences were found to match over 99% of the genome with near-perfect identity. Analysis of areas of low and high sequence coverage within phage genomes indicated that GC content may influence coverage of sequences from Nextera libraries. Comparisons of phage genomes prepared using both Nextera and a standard 454 FLX Titanium library preparation protocol suggested that the coverage biases according to GC content observed within the Nextera libraries were largely attributable to bias in the Nextera protocol rather than to the 454 sequencing technology. Nevertheless, given suitable sequence coverage, the Nextera protocol produced high-quality data for genomic studies. For metagenomics analyses, effects of GC amplification bias would need to be considered; however, the library preparation standardization that Nextera provides should benefit comparative metagenomic analyses.
authors.affiliation: Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Marine
authors.forename: Shawn W
authors.initials: SW
authors.lastname: Polson
authors.forename: Jacques
authors.initials: J
authors.lastname: Ravel
authors.forename: Graham
authors.initials: G
authors.lastname: Hatfull
authors.forename: Daniel
authors.initials: D
authors.lastname: Russell
authors.forename: Matthew
authors.initials: M
authors.lastname: Sullivan
authors.forename: Fraz
authors.initials: F
authors.lastname: Syed
authors.forename: Michael
authors.initials: M
authors.lastname: Dumas
authors.forename: K Eric
authors.initials: KE
authors.lastname: Wommack
mesh_terms: D001435:Bacteriophages / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D015723:Gene Library; D016679:Genome, Viral; D056186:Metagenomics / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D019895:Transposases / Q000378:metabolism*
chemical_list: D004247:DNA; D019895:Transposases