Category Archives: laboratory

Coming soon: Illumina MiSeq i100

Posted on by | Leave a reply

The CQLS was awarded a 2025 Research Equipment Reserve Fund (RERF) to purchase a new Illumina MiSeq i100.

The CQLS has run an Illumina MiSeq for 12 years as the center of a highly successful low-range sequencing service. Primarily used for microbiome, environmental, and amplicon samples, the MiSeq offers a lower volume of Illumina high-throughput sequencing compared to the CQLS’ Illumina NextSeq. Illumina is transitioning the original MiSeq to end-of-life service. By upgrading to the new MiSeq i100, the CQLS will replicate our current sequencing abilities while drastically reducing instrument maintenance costs for the CQLS and consumable costs for OSU researchers.

The Illumina MiSeq i100 is a next-generation sequencer that sequences low-volume flow cells, 5 Million sequences in 300 or 600 base-pair lengths or 25 Million sequences in 100, 300, or 600 base-pair lengths. The MiSeq i100 is quicker and cheaper to run than other short-read sequencers.

Key features include:

  • Cost efficiency: Cost-effective consumables enable more affordable sequencing
  • Speed: Dramatic reduction in run times: as fast as four hours, with same-day results (4× faster than MiSeq)
  • Room-temperature shipping and storage for reagents: Allowing for greater flexibility to sequence on demand without the need to thaw reagents
  • Simplicity: Simpler, streamlined operations for various levels of sequencing experience

The new MiSeq i100 is upgraded from the original MiSeq in several ways.  Flow cells are lower-cost and higher volume so researchers will be able to obtain more sequence at a lower price. The small volume sequencing (1M) of the original MiSeq is no longer offered but researchers will be able to sequence higher volume runs at a lower or similar price.  Running the instrument is also more streamlined: run times are 4x faster; many reagents and consumables are now stored in room temperature; the instrument is simpler to maintain resulting in a lower yearly service contract price.

MiSeq i100 overview:

https://www.illumina.com/systems/sequencing-platforms/miseq-i100.html

MiSeq i100 specification:

https://www.illumina.com/systems/sequencing-platforms/miseq-i100/specifications.html

MiSeq i100 research applications:

https://www.illumina.com/systems/sequencing-platforms/miseq-i100/applications.html

Leica Stellaris 5 Confocal Microscope

Posted on by

Anne-Marie Girard, CQLS

CQLS has installed a new Leica Stellaris 5 Confocal Microscope System which replaces an older confocal system. With this type of microscope one can obtain 3D sectioning of fluorescently labeled cells, or tissues for clearer, sharper images of specimens. People have used confocal systems to examine structures within living or fixed cells and to examine the dynamics of cellular processes.

3D rendering of veins in maize leaves. Yellow: Pin1a-YFP in cell membrane, Red: DR5-RFP in endoplasmic reticulum. Image courtesy of Camila Medina.

A confocal system has the capacity to image in Z and time to better visualize location in 3D than widefield fluorescence microscope by using a pinhole to eliminate out of focus light. The system has a white light laser (WLL) with tunable excitations from 485 nm up to 685 nm in addition to a 405 nm laser and sensitive HyD S detectors with a detection range from 410 to 850 nm.  Additionally, the Stellaris system also has TauSense, a set of tools based on fluorescence lifetime information with potential to eliminate autofluorescence, and LIGHTNING which expands the extraction of image details for both classical imaging range and beyond the diffraction limit (120nm).

We will be offering free training and imaging time during this fiscal year to those people who have a project ready for imaging and in order to help with grant writing for future imaging projects. Contact Anne-Marie Girard to discuss potential projects or for more information about the system or its capacities.

BluePippin DNA Size Selection

Posted on by

Elizabeth Zepeda and Katie Carter

Our BluePippin instrument is used for DNA size selection. Several genomic applications benefit from collecting only DNA fragments within a specified size range from a pool of DNA. This service is often used before Illumina sequencing to remove undesired PCR peaks or before long read sequencing to increase the proportion of fragments greater than a given length in a library.

Using pulsed-field electrophoresis on pre-cast gel cassettes, a DNA sample is separated and fragments within the target range are eluted into buffer. Each cassette can run up to 5 samples at once. Up to 5 ug of DNA can be loaded into each well.

The following cassettes are available at CQLS:

  • 3% agarose, 100-250bp
  • 2% agarose, 100-600bp
  • 1.5% agarose, 250bp – 1.5kb
  • 0.75% agarose, 1-50kb

The desired size range must lie within the total size range capability of the cassette. For example, between 200-350 bp on a 2% agarose cassette would be an acceptable range.

The expected collection yield of target sizes is approximately 50-80% based on product validation studies. Yield can vary greatly depending on the desired range and input fragment sizes.

Before and after size selection of Illumina sequencing library to remove undesired peak at approx. 300 bp

Contact Katie Carter for inquiries about this service.

Source: sagescience.com

Interview: Pacific Biosciences (PacBio) Sequel Service

Posted on by

Aaron Trippe discusses the changes and challenges of working with the PacBio Sequel since 2016. He discusses improvements in the technology since 2016 and has advice for user who would like to utilize this service.

Aaron Trippe, our long-time PacBio technician, stands next to the CGRB’s Pacific Biosciences Sequel.

Q1: How long have you been running the PacBio sequencing service at the CGRB?

The CGRB was one of the early adopters of the Sequel, the second phase of long read genomic sequencing technology from Pacific Biosciences.  It arrived here on campus in August of 2016.  Since then the technology has made significant improvements to the user-interface, and has tremendously increased read lengths and output. 

Q2: You started up the PacBio sequencing service at the CGRB. What has been the most challenging aspect about developing this service?

Aside from the continually changing and evolving technology, one of the most challenging aspects of the service is getting everything you feed the machine to produce optimal results.  One of the advantages of the technology is that you are sequencing native DNA, but that also makes it challenging when working with an organism that traditionally is difficult to work with and considered problematic.  Finding ways to produce super clean and high molecular weight DNA from just about everything is probably the largest hurdle to working with the technology as a service provider.  The keys to success are definitely within the sample quality.  Having pure, high molecular weight DNA is essential to take advantage of the long read aspect of the technology, and is directly correlated to the quality of the sequencing output.

Q3: What type(s) of project(s) would you recommend to use PacBio’s long read technology?

The technology is great for just about any sequencing application.  With the long reads, you have access to regions of DNA that were not previously accessible due to repetitive regions in genomic DNA.  There is enough output to multiplex several microbial genomes on a single SMRT Cell.  Complete sequences of multiplexed amplicons using Circular Consensus Sequencing for high fidelity reads of shorter inserts. With the read lengths exceeding that of RNA transcripts, Isoform sequencing using the Iso-seq application is also available for obtaining complete transcripts.

Q4: Favorite or most interesting project you’ve worked on?

Since managing the PacBio Sequel, I’ve gotten to work with plants, animals (vertebrates/invertebrates), fungi, bacteria, and insects for the local scientific community, and beyond.  I can’t say that I have had a favorite organism, and they have all been interesting projects, but overcoming challenges with successful results always feels rewarding.

For more information please visit the CGRB website: https://cgrb.oregonstate.edu/core/pacbio

Note: We wish Aaron the best as he purses a new opportunity and are grateful he was able to develop a successful PacBio Service at the CGRB! For future sequencing inquires please contact Katie Carter.

Close up of a PacBio SMRT cell.