The Genomics Core offers sequencing on an Illumina MiSeq desktop sequencer for targeted sequencing, small-genome RNA sequencing, and ChIP-seq. The MiSeq is capable of generating long reads (up to 600 bp reads), up to 25 million reads per run, and is the smallest and fastest sequencing instrument on the Illumina platform.
The MiSeq is not capable of producing the amount of coverage needed for an RNA-seq experiment for large genomes (i.e. Human/Mouse). Projects requiring more in-depth sequencing will be outsourced to Psomagen or Yale Center for Genome Analysis.
For additional information regarding the capabilities of the MiSeq, please visit Illumina's website.
Designing Your Next Generation Sequencing Run
Type of Run - Single Read (SR) or Paired End (PE)
Single end reads produce reads from one end of a fragment to the other end. Paired-end reads produce bidirectional reads: one read from the 5' to 3' direction and one read from the 3' to 5' direction. Single end reads are faster and cheaper.
MiSeq Reagent Kit V2 (50, 300, 500 Cycles)
|Read Length||Total Time||Output||Quality Score (>Q30)|
|2 x 25 bp||~5.5 hours||750-610 Mb||>90%|
|2 x 150 bp||~24 hours||4.5-5.1 Gb||>80%|
|2 x 250 bp||~39 hours||7.5-8.5 Gb||>75%|
Depth of Coverage (DNA)
Coverage refers to the average number of times a single base is read during a sequencing run. The more frequently a base is sequenced, the more reliable a base is called, resulting in better quality data.
Depth of Coverage (RNA)
Determining the depth of coverage for an RNA-Seq experiment is difficult because different transcripts are expressed at different levels. A useful metric for RNA-Seq is determining the total number of mapped reads. It is important to distinguish between the total number of reads and mapped reads; not all reads will map to your reference genome, therefore, the number of usable reads (or mapped reads) will be less than the number of total reads. The number of mapped reads depend on the library type, sample quality, and how complete your reference genome is. Please refer to the ENCODE Consortium Guidelines for RNA-Seq standards.
Samples are subject to biological variation and replicates are statistically important for identifying those differences. While removing replicates may reduce your sequencing cost, it is important to note that there are many factors that may cause a sequencing run or sample to fail. If you don't have sufficient replicates, you may have to repeat your sequencing run. The EBC recommends at least 4 biological replicates for every experiment.
Both DNA and RNA samples may be submitted for Illumina library construction. Guidelines for submitting library-worthy samples to the Genomics Core can be found on our Sample Submission page.
We offer library preparation services using commercially available kits from Illumina. Please visit Illumina's website to view complete kit descriptions and select which best fits your experimental design. If you are unsure of which kit to select, please let us know and we will be happy to work with you to determine the kit best suited for your project.
Construction of high quality libraries is critical to successful sequencing runs, specifically in terms of the number of reads generated and the validity of the sequences obtained. While you are welcome to submit user prepared libraries, the Genomics Core recommends that starting material be submitted to the core and library construction is performed by core personnel.
Multiplexing on the Illumina platform is accomplished by indexing (barcode tagging) of samples. This allows for sequencing of multiple libraries in a single flow cell. This is an advantageous strategy when the read output per lane is much greater than required for a single library (ex: ChIP or small RNA applications). Illumina offers TruSeq kits that enable multiplexed sequencing of up to 96 libraries.
PCR-free library preparation is an additional option to consider. PCR-free library construction has been shown to reduce bias and increase fidelity in some cases. The only limitation is that a greater amount of starting material (approximately 5-fold) is required.