In this issue
GenoFest 2017: Clinical Applications
University of Minnesota Genomics Center (UMGC)
The UMGC is pleased to announce our annual Open House and Festival of Genomics — aka "GenoFest" This year, we highlight clinical applications in collaboration with Lab Medicine & Pathology along with our newest Illumina Sequencer, NovaSeq
GenoFest will be held in the Cancer & Cardiovascular Research Building on Thursday, November 2, 2017.
We understand that sequencing wait times can cause bottlenecks, especially for pilot projects. UMGC has two options for researchers who need to complete small runs quickly, at a lower cost. For an additional fee, we also offer expedited runs on our Illumina MiSeq and HiSeq sequencers.
The UMGC offers a shared MiSeq service called Stowaway Sequencing (Stowaway-Seq). Stowaway-Seq runs every three weeks and only offers one option (2x300 bp paired-end). The MiSeq flow cell is split into 8 “lanes,” which users may purchase lanes on a first-come, first-served basis.
The service is intended to provide access to Illumina NGS for those users who need low-depth sequencing coverage. The service is most useful for targeted amplicon sequencing or small pilot projects to assess custom applications. It is not suitable for whole genome sequencing, RNA-Seq or other high coverage applications. The UMGC can barcode amplicon libraries for users (preferred). Clients who opt to make their own custom libraries must discuss indexing options with UMGC staff in advance of submission to avoid potential conflicts with other stowaways.
Please see the UMGC Sequencer Home for additional details and run schedule. Or, contact Aaron Becker if you would like to discuss the Stowaway-Seq service.
Illumina has lowered prices for their MiSeq Micro and Nano v2 kits. Paired-end Micro and Nano runs are a great option for studies that do not require significant read depth or yield. Typically, this includes amplicon sequencing or a small number of bacterial genomes. Micro kits (4 M reads per run) are only available in 2x150 bp configuration, while Nano kits (1 M reads) are available in 2x150 bp and 2x250 bp options.
Pricing for UMN researchers
Nano 2x150 bp PE - $574.26
Nano 2x250 bp PE - $642.49
Micro 2x150 bp PE - $757.62
We are now offering expedited project sequencing for MiSeq and HiSeq runs for an additional fee (TBD). Expedited projects are run on either MiSeq 2x250 bp or 2x300 bp runs or HiSeq 2500 Rapid runs (multiple run configurations available). Expedited runs are not available on the HiSeq 2500 High Output instrument (”Franklin”). Turnaround time for library preparation and sequencing is guaranteed to be two weeks from time of samples passing UMGC QC steps. Users will be asked to review sample QC and provide approval for moving projects to next steps.The UMGC reserves the right to limit sample numbers and expedited instrument time to ensure that standard operations are not adversely affected. We expect that most expedited requests of a reasonable size and scope will be accommodated. Please contact the UMGC the NGS team if you would like to discuss our expedited service.
In the Fall of 2016, the UMGC acquired a Pacific Biosciences Sequel instrument with funds from the Office of the Vice President for Research. This has enabled us to offer true long-read sequencing to our clients: we are consistently seeing average read lengths above 10 kb. Updates to the instrument are ongoing and are expected to continue to increase average polymerase read lengths, enabled lower sample inputs, and improve sequencing metrics and analysis. In addition, the Sequel already provides an approximately four- to six-fold increase in output per SMRT cell compared to the older RSII instrument, with high-quality libraries providing 3-4 Gb of data per cell.
Pacific Bioscience sequencing can be especially useful in de novo genome assembly due to its lack of GC-bias and ability to span repetitive regions. Our standard library prep offers a 20 kb size-selected library from
IsoSeq is a non-quantitative RNA library prep/sequencing methodology for acquiring full-length mRNA information for analysis of isoforms or
Amplicons that are too large (>600bp) for sequencing on the Illumina platform can be sequenced using PacBio. Similar to our high-throughput indexing service for Illumina, client's custom primers are modified to include an additional universal tail sequence. In a subsequent round of PCR, UMGC will add 16-bp indices for multiplexing on the Sequel. Amplicons as large as 10 kb can be multiplexed together using this method. Up to 96 amplicons can be barcoded separately and multiplexed on a single SMRT cell. Expect around 150,000 reads per SMRTCell using this method after demultiplexing.
New services have also been launched to support long-read sequencing. The Agilent TapeStation instrument can perform Genomic DNA sizing for regular quality-control. For applications that are especially sensitive to DNA integrity, we are now using Pulsed-Field Gel Electrophoresis (PFGE) on a case-by-case basis to QC samples for PacBio and 10X Genomics services.
The 10X Genomics’ Chromium single-cell product is a droplet-based technology that enables 3′ mRNA counting of 100-10,000 single cells per cell suspension. Individual cells are lysed then polyadenylated mRNA reverse transcribed/bar-coded within an emulation droplet. The final library is compatible with the Illumina HiSeq 2500 and MiSeq. UMGC provides initial data analysis and visualization of single cell data utilizing Cell Ranger software suite.
|10X Genomics Chromium||C1 Fluidigm|
|Gene Model||3’ gene counting||Whole transcriptome|
|Capture Dynamic Range||100-10,000 cells||1-96 cells|
|Cell Size Restriction||≤ 50 um||5-30 um|
|RNA||polyadenylated mRNA||polyadenylated mRNA|
|Recommended Reads/ Cell||50K||250K|
|Internal Pricing (Capture and Library Prep)||$2,077.60/ cell suspension||$3,568.23/ cell suspension|
Sequencing Run Type
HiSeq High-output PE (26 bp first read, 99 second-read) single-index
MiSeq 75 PE dual-index
Cost Per Lane
Average Total Cost Per Cell
$1.24 (average 3,000 cells/capture
$68.53 (average 70 cells/capture
In order to better understand the robustness of our protocols and to identify failure modes for Sanger Sequencing in the UMGC we carried out a series of experiments testing the effects of DNA template concentration, primer concentration, and sample purity on Sanger sequencing data quality. We determined that the UMGC’s Sanger sequencing processes are robust for a wide range of template and primer concentrations and that the most likely failure modes for submitted samples center around the presence of contaminants. Finally, we compared the contaminant sensitivity profiles of the UMGC processes to two external vendors, ACGT and Genewiz, and found that the effects of template concentration, primer concentration, and contaminants were highly similar across all three providers. These experiments may help to guide researchers in preparing their samples for Sanger sequencing.
Download the Sanger Sequencing White Paper
The UMGC Sequencer Home shows the queue of our Illumina next-generation sequencers. The worksheets track current and queued runs as well as the sample queue for our different sequencers, 2500 High-output, 2500 Rapid, and MiSeq. The exact make-up of queued runs is liable to change depending on the timing of completion of libraries.
Cargill Building Seminar Room 105
1500 Gortner Ave., St. Paul, MN 55108
Thursday, May 25, 2017
NanoString PlexSet is a new, simple, cost-effective multiplex expression assay run on the established nCounter there at UMGC. The PlexSet assay is ideally suited for analysis of high throughput, low to mid-plex research applications. Our well documented “lyse and go” protocols eliminate the need for RNA extraction, purification and amplification and currently saves Big Ag companies tens of thousands in RNA spin columns. Turn-key; custom reagents do not require upfront optimization prior to initiating a new study. Finally, the data analysis and visualization can be done with our free and easy to use nSolver software suite by your own lab in a matter of hours. Some of the applications we can address:
10 AM – 11 AM
Agriculture, animal science and CRISPR applications. Alternatives to qPCR for building large data sets in less time.
11 AM – Noon
3D Biology quantifying DNA, RNA & Protein from a single sample. Applications for CRISPR and a summary of technology seminars from AGBT, AACR and ASCO.
KeyGene and University of Minnesota have entered a strategic licensing agreement which allows the UMGC to offer KeyGene’s proprietary Sequence-Based Genotyping (SBG). SBG technology allows rapid and cost-effective discovery and scoring of genetic variation. KeyGene has established a strong proprietary position on SBG with world-wide IP protection and this collaboration fully enables investigators from industry and academia to advance their research programs through SBG.Download the full press release