The CGRB began offering Illumina MiSeq high throughput DNA sequencing service in April 2013.  Cluster generation, sequencing, and analysis are all done on a single instrument.  The sequencing process takes place on a flow cell with 1 channel. Multiple samples can be run at once by using indices for each sample. The advantage of the MiSeq over the HiSeq 3000 are the read lengths and turnaround time.  2x300bp reads are supported on the MiSeq and takes ~3 days to run.  With v.3 kits the MiSeq can produce >25 million reads or 15GB per run.  With v.2 kits the MiSeq can produce >15 million reads or 7.5 GB per run with standard flow cells.  There is also the option of using micro and nano flow cells which produce up to 4 million and 1 million reads per run (1.2Gb & 500Mb).  Actual output can vary depending on cluster density.

Information about the HTS mailing list and library prep kits that can be used with the MiSeq are found on the HiSeq 3000 information page.  Libraries prepped for the HiSeq 3000 can be run on the MiSeq and vice versa (with one exception of libraries prepped with the older pre-TruSeq genomic single end prep kits).  The Nextera™ XT library prep kit is made for small genomes, plasmids, and amplicons, ideal for the MiSeq.  The Core Facilities offers Nextera XT library prep as an optional service.  Additionally, for those who wish to sequence amplicons, there are protocols available using a 2-step PCR approach for creating libraries. 

Important: Low diversity (at each cycle of sequencing, most bases are the same; base distribution not balanced) libraries can be problematic when run on the MiSeq instrument.  Therefore, a control must be spiked into the sample by the Core Facilities prior to sequencing.  For highly diverse libraries, it is recommended to spike in PhiX at 1-5%, but for low diversity libraries (amplicons, RAD-seq, etc.) it is recommended to spike in at least 10% and lower the cluster density.  Failure to follow these guidelines can result in a failed sequencing run, in which the CGRB Core Facilities cannot be held responsible.  It is most important to have high diversity during the first several cycles of sequencing, as this is when the critical calculations for the entire run are made (cluster id, matrix & phasing, & image registration).  There are additional options to correct for low diversity.  These include increasing the number of different libraries per run, reducing cluster density, shearing long amplicons, and careful library design if using the 2-step PCR approach for amplicon library prep.  Diversity can be introduced during primer design. 

Contact Mark Dasenko for more information.

Ilumina MiSeq

 

Please note that as of July 2014, Illumina only supports 2x300bp reads on the MiSeq