Advanced Technologies in Next-Generation Sequencing Platforms and Sample Preparation for Whole Genome, Transcriptome and Metagenomic Sequencing

Next-generation DNA sequencing platforms continue to evolve quickly, along with associated instrumentation and protocols for library preparation. With the range of library types and sequencing options available, correct experimental design is critical for project success. Here, we present the newest developments in the field and how we utilize them across a range of research needs.

The University of Illinois W.M. Keck Center houses 4 Illumina HiSeq 2500, 3 MiSeq, and the Fluidigm Access Array and C1 platforms. As a Beta testing site for NGS companies, we have prepared many TruSeq Synthetic Long Read libraries (formerly known as Moleculo) and applied these long reads across a wide swath of de novo assembly projects. These include plants, animals, insects, and microbial communities. We illustrate how these long-reads fit into a de novo assembly plan to maximize their usage.

In addition, we present a start-to-finish application for 16S rRNA, 18S rRNA, ITS, or other targeted microbial community analysis via amplicon sequencing. Starting from genomic DNA, the Fluidigm Access Array is used to generate pools of up to 384 different metagenomic target amplicons that are ready to load onto the MiSeq platform.  Up to 24 different primer sets can be used within each pool. This approach minimizes PCR errors and cross-contamination while maximizing data from the mixed community being studied.

We present the above strategies and sequencing approaches as the latest developments in a constantly evolving field.