Next-Generation Sequencing

Illumina HiSeq 2000 and Illumina MiSeq:

Illumina DNA Sequencing –

Service Description: BMGC provides full service DNA Sequencing which includes sample QC, library creation and sequencing to your preferred read depth.

QC: Please submit DNA in a 96-well plate. Samples are quantified using flourimetry (Pico Green assay). For a sample to pass QC, mass must be ≥ 1ug.

Library Creation: Steps include DNA shearing (Covaris acoustic shearing), fragment purification and end polishing, and ligation to indexed (barcoded) adaptors. The Library is then size selected (if desired), size distribution validated using capillary electrophoresis, and quantified using fluorimetry (PicoGreen) and via Q‐PCR.

Sequencing: Indexed libraries are then normalized, pooled (depending on number of reads requested), clustered on a flow cell, and loaded onto the instrument for sequencing.

Illumina RNA-Seq –

Service Description: BMGC provides full service RNA-Seq which includes sample QC, library creation, and sequencing to your preferred read.

Samples: Please submit total RNA in a 96- well plate. The preferred RNA extraction method is via QIAGEN RNeasy kit. If you are working with plant or fungi, we recommend that you use the QIAGEN RNeasy Plant mini kit. Organic RNA extraction methods (such as phenol or Trizol) are discouraged. Organic carryover can inhibit the enzymatic reactions used in Illumina library preparation and can increase the risk of failure of library generation.

QC: Samples are quantified using fluorimetry (RiboGreen assay) and RNA integrity assessed using capillary electrophoresis (e.g., Agilent BioAnalyzer 2100), generating an RNA Integrity Number (RIN). For a sample to pass QC, mass must be ≥ 1 ug, with a RIN > 8.

Library Creation: Steps include oligo-dT purification of polyadenylated RNA, and reverse trascription to create cDNA. The cDNA is fragmented, blunt-ended, and ligated to indexed (barcoded) adaptors. The library is size selected (in the case of paired-end RNA-Seq), size distribution validated using capillary electrophoresis, and quantified using fluorimetry (PicoGreen) and via Q-PCR.

Sequencing: Indexed libraries are then normalized, pooled (depending on number of reads requested), clustered on a flow cell, and loaded onto the instrument for sequencing.

***The BMGC also provides full service Illumina ChiP_Seq, Illumina Mate Pair, Illumina Small RNA, Illumina Exome and Agilent SureSelect library creation and sequencing. Please inquire within for more information.

Roche GS FLX (454 Sequencing)

Please inquire within for more information.

Shipping instructions:

Shipping: Samples should be frozen and shipped on dry ice in a 96-well plate. We recommend using plate tape to seal the wells. Place the plate inside of a plastic bag prior to placing on dry ice. Samples should be shipped via an express (next day) shipping carrier to:

BMGC-Sequencing and Analysis Facility

Room 20 Snyder Hall

1475 Gortner Ave.

St. Paul, MN 55108