Next Generation Sequencing (NGS) and single cell experiments require careful planning and can be discussed with the facility and bioinformatics cores prior to commencement. While some equipment is available for use by internal users the sequencers are fee-for-service only.
NGS requires a sample to be converted to a sequencing ‘library’ before it can be sequenced. Sample libraries consist of fragmented sample DNA with the necessary sequencing adapters at both ends. These libraries can be mixed and sequenced together provided each has a unique barcode incorporated during preparation. The number of libraries which can be pooled together, and the choice of sequencer, depends on how much ‘coverage’ is required for each sample. The sequencing read length and strategy chosen (eg. 100 bp paired-end reads) will depend on the experimental aims.
The Fluidigm system has a number of applications including qRT-PCR, digital PCR and genotyping. The facility has a C1 module which enables isolation of single-cells for further analysis. The main advantages of the Fluidigm technology are the relatively low use of sample and reagents, minimal pipetting steps and high reproducibility.
The standard qRT-PCR machines (Biorad CFX and ABI Quantstudio 7’s) are available to SAHMRI staff and can be booked using the internal PPMS system.
The first step in the process is to discuss the project with the facility and obtain a quote. Where bioinformatics support is required other staff may be involved as necessary. To proceed with a job please return the quote indicating a purchase order number (external SAHMRI clients) or cost code (internal SAHMRI staff) for the charges.