NextGen Library Construction
Next-generation sequencing (NGS) technology utilizes a massively parallel approach to quantitatively assess copy number and sequence changes in DNA or RNA samples. It is rapidly becoming a preferred method for integrated approaches that simultaneously measure transcript copy numbers and sequence-based alterations.
In collaboration with the Sequencing Core at Mayo Clinic, the Gene Expression Core (GEC) provides services for NGS by producing the libraries that are required for NGS analysis. Using Illumina TruSeq technology, mRNA and miRNA libraries are generated from high-quality total RNA in preparation for analysis on the Illumina HiSeq 2000.
Illumina TruSeq mRNA library construction. The Illumina TruSeq mRNA procedure creates cDNA libraries from total RNA in preparation for NGS analysis.
RNA sample quality is assessed, followed by polyA selection and fragmentation of 100 nanograms to 1 microgram of starting total RNA. Subsequently, the fragments undergo reverse transcription with random primers and second-strand synthesis to generate double-stranded cDNA. Ends are repaired and adenylated, followed by adaptor and index ligation.
Finally, the products are denatured and PCR-enriched to generate the final genomic library. The libraries undergo quality control for quantitative and qualitative thresholds. The GEC then provides the samples to the Sequencing Core to be run on the Illumina HiSeq 2000.
Illumina TruSeq miRNA library construction. The Illumina TruSeq miRNA procedure generates small RNA libraries in preparation for NGS analysis. Adapters are ligated onto the 3' and 5' ends of each miRNA, and then the miRNA products are PCR-enriched and purified. Five hundred nanograms to 1 microgram of total RNA is used as input for this assay.
As is done for the TruSeq mRNA assay, products are then provided to the Sequencing Core to be run on the Illumina HiSeq 2000.
The cost for each project is dependent on experimental design, vendor costs and time required for completion. This information is provided in each project proposal, which must be approved by the principal investigator prior to project initiation.
For more information or to initiate a project, contact us at 507-255-1086 or by email.