Sanger sequencing is the process of generating 600- to 800-base single reads of a DNA sequence using fluorescent dye-labeled chain terminators and capillary electrophoresis. This sequencing method allows researchers to identify mutations in select genes that may cause disease or help diagnose and treat disease.
- Consultations. A consultation is optional for Sanger sequencing. The Molecular Biology Core has many years of experience with troubleshooting Sanger sequencing issues. Assistance is available free of charge by contacting the core.
- Results. Sanger results, which are delivered as chromatogram files to the DNASEQPICKUP server, can be viewed and analyzed using various software programs available online or through the Mayo Clinic Research Computing Facility or Molecular Biology Core.
Next-generation sequencing is the process of sequencing DNA or RNA by producing millions of sequence reads in a massively parallel manner. Also known as deep sequencing, next-generation sequencing allows for the sequencing of the entire genome, exome or transcriptome. This makes it possible to identify complex genetic differences that may cause disease or help diagnose and treat disease.
- Consultations. A free-of-charge consultation is required before submitting a service request for next-generation sequencing. The consultation will include discussion of experimental design, sample requirements, submission procedures, costs and data analysis requirements.
- Results. Next-generation sequencing produces much larger data sets than does Sanger sequencing. Results are delivered as FASTQ or BAM files to the next-generation sequencing data delivery server. While investigators can perform their own downstream analysis, they are encouraged to work with the Mayo Clinic Bioinformatics Core due to the size and complexity of the data. The Molecular Biology Core and Bioinformatics Core have integrated work flows to take advantage of the various analytical pipelines developed by the Bioinformatics Core.
- Applications. Applications of next-generation sequencing include:
- De novo sequencing. De novo sequencing is the process of sequencing unknown genomes or transcriptomes. It may require a combination of several applications, including Sanger sequencing and short- and long-read next-generation sequencing.
- Whole-genome sequencing. Whole-genome sequencing is the process of sequencing genomes that have a known reference sequence to identify differences and changes in an individual's genomic makeup.
- Mate pair sequencing. Mate pair sequencing involves sequencing the ends of long fragments (2 to 5 kilobases) of DNA to identify large structural rearrangements or indels.
- Targeted resequencing. Targeted resequencing, which includes whole-exome capture, custom capture and amplicon sequencing, refers to the process of sequencing a subset of a known genome in an effort to identify single-nucleotide polymorphisms (SNPs).
- ChIP sequencing (ChIP-seq). ChIP-seq is sequencing in combination with chromatin immunoprecipitation to identify protein-DNA interactions that can affect gene expression.
- Methyl sequencing (methyl-seq). Methyl-seq is sequencing in combination with bisulfite conversion to identify methylation patterns of genomic regions that can affect gene expression.
- Whole-transcriptome sequencing. Whole-transcriptome sequencing, also known as RNA sequencing (RNA-seq), is the process of sequencing expressed genes in the genome. It can also identify splice variants, gene fusions and mutations.
- MicroRNA sequencing (miRNA-seq). MiRNA sequencing targets the various miRNAs expressed in the cell that can control RNA translation by translational repression, target degradation and gene silencing.