HTG Sample Preparation

Samples submitted for Illumina Sequencing services will be analyzed by appropriate quality control measures (NanoDrop, Qubit, Bioanalyzer, gel electrophoresis) to access the quantity and quality of the sample.

Libraries

Samples that pass quality control steps will be used for generating an Illumina sequencing library using the appropriate sample prep kit. If the quality of a sample is suspect or if there is insufficient sample to construct a sequencing library, a member of the core facility will contact the investigator.

Investigators have the option of constructing the sequencing library within their own laboratory. A fraction (15 ul) of the library can then be submitted to the Microarray and Genomic Analysis Core Facility for sequencing. Following the construction of a sequencing library, an aliquot of the library will be run on an Agilent Bioanalzyer chip to validate the quality and size range of the library.

The library will also be qualified by qPCR to establish the molarity of cluster-forming molecules in the library. This step enables us to load an appropriate quantity of the library on a flowcell as a means to optimize cluster density during the sequencing run. We are able to further optimize cluster denisty if additional lanes of the same sample are run on future sequencing runs. Sequencing libraries will then be run on either the Illumina Genome Analyzer IIx or the HiSeq2000.

The Illumina HiSeq 2000 and the MiSeq uses real time analysis software in addition to analysis pipeline software to call bases from the image files, generate reads, and deconvolute index tags on the sequencing libraries.

Sequence Reads

Sequence reads will be made available to the investigator for download from GNomEx. Sequence reads can be mapped to a to a genome build of choice by contacting the Cancer Bioinformatics (CBI) Shared Resource. See pipeline user guide. Additional bioinformatic analysis can be requested from CBI. Tools for data analysis that were developed by CBI may be found at USeq.