UTMB’s Next Generation Sequencing (NGS) Core utilizes an Illumina NextSeq 550 sequencing system to perform massively parallel sequencing for genetic analysis. The NGS Core offers support in library construction from various template sources; RNA (total, poly A+, ncRNA and miRNA), chromatin immunoprecipitated DNA (ChIP-Seq) and DNA (genomic exome and amplicon-derived). Library complexity is assessed using qPCR prior to amplification. Illumina NGS technology uses adapter-ligated template molecules to populate a hollow glass flow cell. Individual target molecules are then amplified to create template clusters. Templates are sequenced using reversible, fluorescent-tagged terminator nucleotides. The NextSeq 550 has the capacity to create over 400 million clusters across the flow cell. With the capability of either single-end or paired-end sequence reads and 50 to 150 base read lengths, the NextSeq 550 can generate up to 120 Gb from a single sequence run. Costs are reduced by indexing (“bar coding”) individual template libraries, allowing multiple libraries to be sequenced in each a single runs.
The Molecular Genomics (MG) Core provides technical support in both the design and performance of real time quantitative PCR (both Taqman and SYBR-based assays), SNP discrimination analysis and Sanger-based DNA sequence analysis. Confirmation of gene expression data is available using quantitative RT-PCR (Q-PCR) analysis (absolute or relative). Both Taqman (Applied Biosystems /Life Technologies) and SYBR Green-based assays are offered. In addition to commercial assays, the MG Core will design a custom assay for any target sequence. RNA samples are quantified using a Nanodrop Spectrophotometer (Nanodrop Technologies) and qualified by analysis on an RNA Nanochip using the Agilent 2100 Bioanalyzer (Agilent Technologies). Assays are analyzed on an ABI Prism 7500 Sequence Detection System (Life Technologies/ Applied Biosystems). Data analysis is performed using the ΔΔ method for relative gene expression analysis.