Project Title: Pippin Prep Agarose Gel Based DNA Fragment Isolation System for Next Generation Sequencing Library Construction at the Center for Bioinformatics and Functional Genomics (CBFG)

Project Lead's Name: Andor Kiss

Email: KissAJ@MiamiOH.edu

Phone: (513) 529-4280

Please Choose the Primary Affiliation: CAS

Are There Other Project Team Members?: Yes

Other team member name: Luis Actis

Other team member email address: dengx1@miamioh.edu

Project Details: The project request is for the acquisition of an agarose gel based DNA fragment recovery system for the size selection of specific fragment sizes. Once selected, these fragments are isolated and recovered to be used in construction of next generation sequencing libraries for sequencing on the CBFG's recently acquired Illumina MiSeq Benchtop Sequencer. Although DNA fragment recovery is available via a manual methodology, the efficiency is extremely variable and usually very poor. This has a direct negative impact on the efforts from a many scientific laboratories that use the MiSeq. The usage of a computer controlled agarose matrix based gel purification system, such as the one being requested (https://youtu.be/7MocmyYG-FM) eliminates the cumbersome razor blade based cut & melt approach coupled with and elution ion-exchange chromatography. The Pippen Prep (by Sage Science) is a computer controlled self-contained computer controlled unit that uses precast agarose gel cartridges and a size marker to elute DNA fragments of desired sizes between 90 bp ~ 1500 bp (the typical desired size range for the DNA fragment library insert size). This project/instrument acquisition will complement the recently acquired Illumina MiSeq Benchtop Sequencer, the CybertronPC workstation with CLC Genomics Workbench and the Covaris M220 Ultrasonicator - all of which are required to construct NGS libraries. Usage of the MiSeq has now reached over 70 runs (at about $1000 per run cost → $70,000) and has been utilised in both undergraduate courses at the introductory level (MBI 224 Bacterial Genomics) as well as the senior level classes (BIOL 407/507 Ichthyology; MBI 475/575 Microbial Ecology) - these courses have enrollments of approximately 75 undergraduate students per year. In addition to 13 different active researchers (Actis, Ballish, Berg, Bollman, Dabney-Smith, Dong, Ferguson, Killian, Kiss, Morgan-Kiss, Moore, Shi, and Tomoyasu), we have intense interest from several other PIs that are looking to re-invigorate their research programmes - many are likely to move towards usage very soon. In addition to on-campus usage of the MiSeq and associated instrumentation, we have been performing Illumina MiSeq 16S Earth Microbiome Project metagenomics analysis as part of our turnkey service (https://www.scienceexchange.com/labs/center-for-bioinformatics-functional-genomics). Thus, this instrument would also enhance our revenue stream capacity.

Problem Project Attempts to Solve: The problem is low efficiency recovery of size fractionated DNA for NGS library constructions. Our solution would be the acquisition of a new technology (Pippin Prep) to improve the user experience at a very technically problematic stage of library construction. In particular, several PIs (Berg, Keane, Solomon, Moore, Fisk and Vanni) would benefit from this instrument as it would aid in RAD-Seq protocols. RAD-Seq is the usage of NGS based technologies to evaluate thousands of loci per sample for phylogenetic analyses and evolutionary relationship reconstruction.

Does this project focus on graduate student education or graduate student life?: Yes

If yes, explain: The split between graduate student usage and undergraduate usage is about 25% undergraduate and 75% graduate student usage - although these numbers are a little fuzzy due to projects such as DUOS wherein both graduate students (PhD) & undergraduates work together. Graduate students will benefit by being able to accomplish their graduate work, publish their findings with their PI and contribute directly to new funding. Undergraduates will gain contemporary knowledge about current technologies preparing them for graduate, biomedical or employment.

Does it meet tech fee criteria?: The success of the project will be in the increased usage of the instrumentation and the increased competitiveness of our undergraduates and graduates in terms of their marketable skill sets. In addition, the increased success of our PIs to obtain external funding will be increased. The innovation of our project will be reflected in reduced wastage of time and money in terms of library construction - often our graduate students spend months overcoming bottlenecks in the new approaches required for novel research. Having instrumentation available to them that will permit new avenues of research leads to innovation and fosters creativity.

How will you assess the project?: We will assess the project by recording the instrumentation usage. To date, we've had enormous interest in NGS based technologies with just about every field from microbiology to ecology to cancer biology to plant physiology show interest in using these techniques. With over 70 MiSeq runs in a little over two years, representing about $70,000 spent in reagents, this demonstrates that there is not only interest but a commitment to using the technology and the support instrumentation to make each run a success. One of the most salient measures of success is the excitement and intellectual investment that occurs in undergraduate classes when then are able to perform this NGS and analyse the data in a classroom/laboratory environment. For them, doing something "new" - never been seen before - is a huge motivator and all the instructors involved in these classes routinely have to turn away perspective students from enrolling in their classes.

Have you received tech fee funding in the past?: Yes

What results were achieved?: We've been funded for (1) a high powered computer workstation (hexadeca core AMD Opteron with 128 GiB ECC RAM) and CLC Genomics Software, and (2) a Covaris M220 Ultrasonicator. Both have had a large impact. The computer workstation sees daily use for the last four years. The ultrasconicator has been essentially in fostering the study of epigenetics with the advent of ChIP-Seq NGS studies.

Did you submit a final report?: Yes

What happens to the project in year two?: The CBFG is asking for one-time acquisition cost for the instrument. Our facility has the space and two full time employees that will help maintain and train users on the instrument usuage. Our annual budget - derived from our charge-backs from users performing Sanger Sequencing - will more than cover any incidental costs, or future repair costs. Users will be charged for consumables as they consume them. This model has been in place at the CBFG for the past 12 years and has had the full support of the three past Deans of CAS, including the current Dean.

Hardware: Piggin Prep, $9,900

Total Budget: $10,145