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.

When free transposon ends are used in the reaction, the target DNA is fragmented and the transferred strand of the transposon end oligonucleotide is covalently attached to the 5′ end of the target fragment (Fig. 1a). The size distribution of the fragments can be controlled At present, next-generation sequencing platforms use slightly different technologies for sequencing, such as pyrosequencing, sequencing by synthesis or sequencing by ligation. However, most platforms adhere to a common library preparation procedure, with minor modifications, before a 'run' on the instrument. This procedure includes fragmenting the DNA (sonication, nebulization or shearing), followed by DNA repair and end polishing (blunt end or A overhang) and, finally, platform-specific adaptor ligation. This process typically results in considerable sample loss with limited throughput. To streamline

Epigenomics is increasingly becoming an important field of research, and the ability to detect and quantify DNA methylation accurately is now critical for numerous fields of study, including disease biology and gene expression. The differential reactivities of methylated and nonmethylated cytosines in DNA with sodium bisulfite forms the basis for their identification in the genome by sequencing. Current whole-genome bisulfite sequencing methods require substantial amounts of starting DNA to compensate for the loss due to bisulfite-mediated DNA degradation. To address issues with sample preparation, we have developed the EpiGnome™ Methyl-Seq Kit, which utilizes a novel pre-library bisulfite conversion scheme to prepare whole-genome bisulfite sequencing libraries with no sample loss and with only 50 ng of input genomic DNA.DNA methylation is not evenly distributed in the mammalian genome.In human somatic cells approximately 60%-80% of all CpGs (~1% of total DNA bases) are methylated. Numerous methods are available to investigate methylation patterns, including those that are enrichment based (e.g., as methylated DNA immunoprecipitation sequencing (MeDIP-seq)), restriction enzyme based (e.g., methylation-sensitive restriction enzyme sequencing (MRE-seq)) and bisulfite based (e.g., reduced-representation bisulfite sequencing (RRBS) and wholegenome bisulfite sequencing (WGBS)). Of these, the only method that captures the complete methylome of a given sample is WGBS, as only through this approach-in which unmethylated cytosine residues are converted to uracil-can genome-wide analysis of 5-methylcytosines be achieved. However, a major challenge in WGBS is the degradation of DNA that occurs during bisulfite conversion under the conditions required for complete conversion. Typically, ~90% of input DNA is degraded, something that is especially problematic when only limited starting amounts are available. Additionally, regions that are rich in unmethylated cytosines are more sensitive to strand breaks. As a consequence, a majority of DNA fragments contained in di-tagged NGS DNA libraries treated with bisulfite "post-library construction" can be rendered inactive due to strand breaks in the DNA sequence flanked by the adapter sequences. These mono-tagged templates are then excluded during library enrichment, resulting in incomplete coverage and bias when performing whole-genome bisulfite sequencing.Here, we describe a novel library construction method, called Method overviewWith the EpiGnome™ Methyl-Seq Kit, bisulfite-treated single-stranded DNA (ssDNA) is randomly primed using a polymerase able to read uracil nucleotides to synthesize DNA strands containing a specific sequence tag (Fig. 1). The 3′ ends of the newly synthesized DNA strands are then selectively tagged with a second specific sequence tag using a patented procedure, resulting in di-tagged DNA molecules with known sequence tags at their 5′ and 3′ ends. The di-tagged DNA is enriched in PCR, resulting in double-stranded DNA (dsDNA) with the appropriate sequences...

oligonucleotide is covalently attached to the 5′ end of the target fragment (Fig. 1a). The size distribution of the fragments can be controlled by changing the amounts of transposase and transposon ends (data not shown). Exploiting transposon ends with appended sequences results in DNA libraries that can be used in high-throughput sequencing (Fig. 1b).At present, next-generation sequencing platforms use slightly different technologies for sequencing, such as pyrosequencing, sequencing by

Advances in next-generation sequencing have led to new library preparation methods compatible with multiple sequencing platforms. Current methods (both mechanical and enzymatic) face limitations: multi-step protocols, sample loss, lack of automation and labor costs. With the continued decline of sequencing costs and increase in sample throughput, there is greater demand for more costeffective, streamlined library preparation methods. Here we describe Epicentre's patented Nextera ™ technology, which addresses current issues in sample preparation and provides a simplified procedure amenable to high-throughput workflows. Molecular weight distribution of Nextera-generated librariesThe molecular weight distributions of libraries prepared using Nextera technology can be controlled for the read-length requirements of different sequencers. To show that molecular weight distributions are consistent and reproducible across different sample types, we prepared genomic DNA libraries from phage λ, Escherichia coli and human DNA using both Roche 454-Compatible and Illumina-Compatible Nextera Enzyme Mixes according to the standard protocol (Fig. 1). The reaction conditions were optimized to yield fragment sizes appropriate for the respective sequencers. Each final fragment size includes approximately 100 base pairs (bp) of adaptor sequence; therefore, the actual genomic DNA sequenced is smaller than the apparent molecular weight of the library. It should also be noted that not all sample types will result in similar molecular weight distributions, as many factors (for example, complexity of the sample type and quantification method) can affect the molecular weight distribution. Deep sequencing of Nextera Roche 454-compatible librariesLibraries were prepared from multiple sample types and then sequenced to validate Nextera technology for Roche 454. E. coli, plasmid, cosmid and soy genomic DNA was fragmented and 5′ end-tagged with Nextera Enzyme Mix (Roche-Compatible). Roche

No abstract

No abstract

No abstract