Something old, something new: Nextera and CopyControl technologies

With the advent of next-generation sequencing techniques, genomic cloning has taken on a new role in de novo and reference-genome sequencing. Previously, Kitzman et al.¹ generated massive amounts of shotgun sequence data from a human library by sequencing 120 pools of CopyControl™ fosmid clones using Nextera™ DNA library preparation technology. This method generated sequencing data of sufficient depth to assemble a complete haplotype-resolved genome for the individual being studied.

However, a recent study by Naka et al.² used a combination of direct genomic next-generation sequencing and CopyControl fosmid cloning to generate and confirm the de novo sequence of the bacterium Vibrio anguillarum strain 775. While deep sequencing generated 32X coverage of the bacterial genome, there were gaps in the sequence data that required confirmation, or contig “gap-filling.” The CopyControl fosmid library allowed the generation of sequence data confirming not only the taxonomy of the new organism, but also demonstrating the synergistic approach of using deep sequencing techniques together with classical Sanger-based sequencing.

1. Kitzman, J. et al. (2010). Haplotype-resolved genome sequencing of a Gujarati Indian individual Nature Biotechnology, 29 (1), 59-63 DOI: 10.1038/nbt.1740
2. Naka, H. et al. (2011). Complete Genome Sequence of the Marine Fish Pathogen Vibrio anguillarum Harboring the pJM1 Virulence Plasmid and Genomic Comparison with Other Virulent Strains of V. anguillarum and V. ordalii Infection and Immunity, 79 (7), 2889-2900 DOI: 10.1128/IAI.05138-11

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Nextera technology featured at ASM 111th General Meeting

Less is more--Evaluation of a low-input, transposase-mediated protocol for rapid generation of high-throughput sequence libraries

Epicentre’s Nextera™ library preparation kits have taken the metagenomics field by storm. Researchers are taking advantage of the rapid, simple Nextera library preparation to determine species, as well as specific enzyme activities, present in a given environmental sample. In a poster at the 111th General Meeting of the American Society for Microbiology (ASM) in New Orleans, Marine et al. used the Nextera DNA Sample Prep Kit (Roche 454-Compatible) to dissect a mock sample in a cultivation-independent bacteriophage preparation. The library, consisting of five mycobacteriophages and four cyanophages, had a relatively broad range of GC content (35.3%-64.7%) and read abundance (28.1%-0.3%).

The results showed that the Nextera library preparation did not compromise the relative abundance of each of the phages in the preparation, and scarce members of the population remained detectable in the presence of more populous library members. The assembled genomes of four of the phages from the Nextera-generated sequence data covered >99% of the genomes at near-perfect identity. GC content was found to affect the coverage of the genomes, though the researchers attributed part of the GC bias to amplification bias in the emulsion PCR prior to sequencing.

The researchers concluded that the Nextera technology is a good alternative to older library preparation methods, and generated quality sequence data from a variety of origins from small quantities (50 ng) of input DNA.