Rapid, high-throughput library preparation for next-generation sequencing

Abstract

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

Summarizing Specific Profiles in Illumina Sequencing from Whole-Genome Amplified DNA

“…Advancement in library preparation also permits their creation from just a few nanograms of DNA. 5 Nevertheless, obtaining even nanograms of starting material can be challenging in certain applications. One solution is to pool many samples to obtain sufficient DNA to construct one library.…”

Ultra-low-input, tagmentation-based whole-genome bisulfite sequencing

“…Second, an oligonucleotide replacement scheme (Supplemental Fig. S1B; Grunenwald et al 2011;Gertz et al 2012) was utilized to ensure that each strand would have adaptors covalently attached to both ends of the molecule. Specifically, this entails initial transposition with a single adaptor in which the double-stranded transposase recognition sequence is truncated to 16 bp (Tm = 36°C), thereby facilitating its post-incorporation removal by denaturation.…”

Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

“…It is worth noting that Nextera technology employs in vitro transposition to prepare sequencer-ready libraries and requires no heat treatment. However, the GC-bias of a Nextera library has been found to be comparable to that observed with a physical shearing method 11 , 30 , probably owing to insertion site bias or amplification-generated bias.…”