Illumina Next-generation Sequencing

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Service Manager: Aaron Becker
Service Email: next-gen@umn.edu
Phone: 612-624-6269

HiSeq 2500/2000:

llumina’s HiSeq 2500 configuration allows the use of Illumina's two lane low cells and rapid SBS chemistry. The primary benefits of the 2500 configuration are the ability to cluster on instrument, generate longer reads (2 x 150 bp) with higher sequencing quality in later chemistry cycles with reduced run times. Note that while individual flow cell run times may be reduced, we forecast a limited reduction in project turnaround times using this platform due to lower lane yield and multiplexing capacity. Researchers may choose full lanes or half lanes. Read More

llumina’s HiSeq 2000 is the largest next-generation high-throughput sequencing system. This very flexible system allows single read or paired end reads with lengths of 50 or 100 base pairs. This is the preferred platform for RNA-seq applications and is capable of handling a variety of whole genome applications as mentioned previously. The HiSeq 2000 can be operated in single or dual flow cell mode and its independent operable flow cells allow applications requiring different read lengths to run simultaneously. It also boasts the ability to multiplex several samples per lane for increased sample throughput. Researchers may choose full lanes or half lanes.  Read More

MiSeq:

The Illumina MiSeq is a single-lane cousin to the HiSeq 2000. The MiSeq permits very rapid runs, and has a form factor (one lane per run) suited to pilot runs, low-coverage experiments and some metagenomic applications. It is capable of generating more than 6 Gb of data for each 2 x 250 base pair run. The MiSeq also has the ability to multiplex several samples per run. Currently, the system allows single read with a length of 50 or paired end reads with lengths of 150 or 250 base pairs.   Read More

 cBot

Fully automated clonal cluster generation for Illumina sequencing.

Read More

More information

Illumina instrumentation and applications used in our laboratory, visit www.illumina.com

Visit Minnesota Supercomputing Institute/Galaxy for more information regarding analysis.

Additional information regarding:

The ENCODE Project: ENCyclopedia Of DNA Elements

SEQanswers the next generation sequencing community Forum

Service Options

Illumina HiSeq 2500, HiSeq 2000, MiSeq

Illumina DNA Sequencing

Service Description: UMGC 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 (PicoGreen assay). For a sample to pass QC, mass must be 300 ng.

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 Sequencing

Service Description: UMGC 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.

Roche GS FLX (454) Sequencer

Roche GS FLX Sequencer utilizes Pyrosequencing to produce millions of long reads with exceptional accuracy in a single run. The process involves generation of a single-stranded template DNA library, emulsion-based clonal amplification of the library and data generation via sequencing-by-synthesis.

This service is provided by the UMGC for University of Minnesota researchers only. The UMGC outsources this service to the University of Illinois at Urbana-Champaign. Please inquire within for pricing and further details.

***The UMGC also provides full service Illumina Nextera, Nextera mate pair, RNA-Seq stranded, small RNA, ChiP-Seq as well as Illumina Exome and Agilent SureSelect library creation and sequencing. Please inquire within for more information.

Service Rates

Rates include three general cost categories: 1) QC, 2) Library Preparation, & 3) Sequencing. Our current internal rates are listed for each category.

ILLUMINA SEQUENCING – Quantification:  Internal Rates

 
DNA Quantification Service Options Standard Scale High Scale
PicoGreen. any # samples
$3.37
-
Agilent Capillary Electrophoresis.  any # samples
$8.57
-
RNA Quantification Service Options Standard Scale High Scale
RiboGreen. any # samples
$3.40
-
Agilent Capillary Electrophoresis. any # samples
$6.40
-

Additional Library Quantification, Size Selection, and

Pooling Service Options

Standard Scale High Scale
Library QC. For libraries prepared outside of the UMGC. any # samples
$50.88
-
Caliper XT. Size Selection. DNA. Minimum 4 samples. 4+ samples
$54.40 per sample
-
Library Pooling. 96-well plate. 1-2 plates
$91.38 per plate
3-4 plates
$67.14 per plate
ILLUMINA SEQUENCING – LIBRARY CREATION: Internal Rates 

Please inquire for availability of additional library preparation methods.
DNA Library Preparation Service Options Standard Scale
TruSeq Nano. any # samples
$86.29
Nextera. XT.  any # samples
$79.40
Nextera. Standard.  any # samples
$149.09
Nextera. Mate-pair.  any # samples
$360.84
PCR free. any # samples
$166.25
ChiP_Seq. any # samples
$161.06
RNA Library Preparation Service Options Standard Scale
RNA. any # samples
$141.25
Small RNA. any # samples
$227.85
Stranded mRNA. 1-12 samples
$262.61
Stranded. With RiboZero rRNA reduction. 1-12 samples
$354.48
Agilent Custom Capture Service Options Standard Scale
Agilent SureSelect Target Enrichment. Custom capture. Excludes SureSelect baits.

any # samples
Inquire

ILLUMINA SEQUENCING – Sequencing Runs:  Internal Rates

HiSeq 2500 Sequencing Service Options Unit Standard Scale 
HiSeq 2500. High-output Mode. Single-read. 50 cycles. per lane* any # lanes
$1,166.80
HiSeq 2500. High-output Mode. Paired-end. 50 cycles. per lane*  any # lanes
$1,905.65
HiSeq 2500. High-output Mode. Paired-end. 125 cycles. per lane*  any # lanes
$2,761.75
HiSeq 2500. Rapid Mode. Single-read. 50 cycles. per lane*  any # lanes
$1,218.95
HiSeq 2500. Rapid Mode. Single-read. 100 cycles. per lane*  any # lanes
$1,535.33
HiSeq 2500. Rapid Mode. Paired-end. 50 cycles. per lane*  any # lanes
$1,631.71
HiSeq 2500. Rapid Mode. Paired-end. 100 cycles. per lane* any # lanes
$2,099.56
HiSeq 2500. Rapid Mode. Paired-end. 150 cycles. per lane* any # lanes
$2,650.05
HiSeq 2500. Rapid Mode. Paired-end. 250 cycles. per lane* any # lanes
$3,972.55
HiSeq 2000 Sequencing Service Options Unit Standard Scale
HiSeq 2000. Single-read. 50 cycles. per lane* any # lanes
$1,078.39
HiSeq 2000. Single-read. 100 cycles. per lane* any # lanes
$1,358.48
HiSeq 2000. Paired-end. 50 cycles. per lane* any # lanes
$1,754.31
HiSeq 2000. Paired-end. 100 cycles. per lane* any # lanes
$2,033.27
HiSeq 2000. Paired-end. 116 cycles. Dual Indexed Run. per lane any # lanes
$2,317.20
MiSeq Sequencing Options Unit Standard Scale
MiSeq. Single-read. 50 cycles. V2. per lane any # lanes
$846.76
MiSeq. Paired-end. 150 cycles. Nano Kit V2. per lane any # lanes
$904.62
MiSeq. Paired-end. 250 cycles. Nano Kit V2. per lane any # lanes
$1,035.75
MiSeq. Paired-end. 150 cycles. Micro Kit V2. per lane any # lanes
$1,229.31
MiSeq. Paired-end. 150 cycles. V2. per lane any # lanes
$1,418.94
MiSeq. Paired-end. 250 cycles. V2. per lane any # lanes
$1,549.70
MiSeq. Paired-end. 75 cycles. V3. per lane any # lanes
$1,257.05
MiSeq. Paired-end. 300 cycles. V3. per lane any # lanes
$2,004.25

*Clients may choose to purchase half lanes or full lanes.

External Rates

Please consult with UMGC staff to obtain a quote. Contact Aaron Becker at next-gen@umn.edu.

Submission Forms & Guidelines

How to Order

Researchers can download the appropriate submission form for submitting samples or submitting created libraries. Once the form has been completed, please submit the submission by emailing next-gen@umn.edu.

DNA Sample Submission Guidelines (Illumina TruSeq Nano)
  1. 300 ng of DNA diluted in 25 ul nuclease free H2O is requested.

    *We recommend researchers check genomic DNA by running approximately 50 ng of the sample on a 1% agarose gel. Intact genomic DNA should appear as a high molecular weight (> 10,000 bp) and with no lower molecular weight smear. If possible, please provide a picture of this gel to the core when submitting your sample.
     
  2. Samples are quantified by the UMGC using fluorimetry (PicoGreen assay). To pass QC, a sample must quantitate >100 ng for 350 bp insert or >200 ng for 500 bp insert.
     
  3. Include a hard copy of the Sequencing Request Form with plate submission and make sure to label the plate with the corresponding ID that matches the request form (Available through UMGC Online Ordering). Also, please follow instructions on form carefully to ensure samples are accepted. Indicate all sequencing parameters required for your experiment on the form. If you are unsure of what to request, please contact next-gen@umn.edu.
     
  4. Send Request Form electronically to next-gen@umn.edu
     
  5. Important: Submit samples in a nuclease free fully skirted PCR plate sealed with foil. UMGC can provide this for a small charge.
     
  6. Please give advance notice of submission date and time so staff can be prepared to receive samples. If shipping samples from outside the University of Minnesota, ship via express shipping carrier on dry ice to the address below:

University of Minnesota Genomics Center
1475 Gortner Ave.
28 Snyder Hall
St. Paul, MN 55108

RNA Sample Submission Guidelines (Illumina TruSeq)
  1. 2-3 ug of total RNA is preferable with a minimum of 1 ug allowable (Please contact UMGC staff if you are unable to obtain this amount). Isolation of total RNA with Qiagen RNeasy is the preferred method. Other equivalent methods are acceptable. If Trizol is used, a subsequent purification with RNeasy is recommended.

    a. Samples are quantified by the UMGC using fluorimetry (RiboGreen assay) and RNA integrity assessed using capillary electrophoresis (e.g., Agilent BioAnalyzer 2100 or Caliper GX), generating an RNA Integrity Number (RIN). For a sample to pass QC, mass must be ≥ 1 ug, with a RIN > 8. 
     
  2. Concentration should be in the range of 50-200 ng/ul. Samples normalized to 100 ng/ul are suggested, but not required. Please dilute samples with nuclease free water.
     
  3. Include a hard copy of the Sequencing Request Form with plate submission and make sure to label the plate with the corresponding ID that matches the request form (Available through UMGC Online Ordering). Also, please follow instructions on form carefully to ensure samples are accepted. Indicate all sequencing parameters required for your experiment on the form. If you are unsure of what to request, please contact Aaron Becker (next-gen@umn.edu).
     
  4. Send Request Form electronically to Aaron Becker (next-gen@umn.edu). 
     
  5. Important: Submit samples in a nuclease free fully skirted PCR plate sealed with foil. (UMGC can provide this for a small charge if you do not have any.)
     
  6. Please give advance notice of submission date and time so staff can be prepared to receive samples. If shipping samples from outside the University of Minnesota, ship via express shipping carrier on dry ice to the address below:
      

University of Minnesota Genomics Center
1475 Gortner Ave.
28 Snyder Hall
St. Paul, MN 55108

Client-made libraries

Please consult with UMGC staff before submitting client-made libraries by emailing next-gen@umn.edu.

Client-made libraries will have the following services performed on them before proceeding with sequencing.

  1. PicoGreen (concentration of library)
  2. DNA Agilent High Sensitivity chip (size of library)
  3. KAPA QC (functionality of library)  

We recommend that pooling be done at UMGC as PicoGreen concentrations are used for balancing libraries. Clients may choose to pool themselves.

Shipping instructions

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. Please give advance notice of submission date and time so staff can be prepared to receive samples. If shipping samples from outside the University of Minnesota, ship via express shipping carrier on dry ice to the address below:

University of Minnesota Genomics Center
1475 Gortner Ave.
28 Snyder Hall
St. Paul, MN 55108

Standards, Guidelines and Best Practices for RNA-Seq

Standards, Guidelines and Best Practices for RNA-Seq:

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