NextGen DNA Sequencing Resources
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Core Resources
Authorship credit should be given if members of the UF ICBR NextGen DNA Sequencing staff have provided services or expertise that is critical to a manuscript. Although other circumstances may warrant authorship, authors are those that meet the items below:
- Agrees to be accountable for accuracy and/or integrity of the work and expertise. (In all our work the person executing the work takes the fullest degree of responsibility for accuracy and integrity.)
- Provides substantial contributions to the experimental design, acquisition, and analysis/interpretation of the work.
- Provides input on drafting of the work (often figures) and approves of the work to be submitted/published. (If a member of the facility has made substantial contributions it is expected that s/he will be given the opportunity to review drafts of manuscripts for accuracy/fidelity and should indicate agreement before a draft is moved forward to publication.)
If a member of UF ICBR NextGen DNA Sequencing has contributed work to a manuscript, but does not meet the criteria for authorship, the corresponding author should acknowledge the UF ICBR NextGen DNA Sequencing staff member who provided the data and their contributions should be specified.
In an effort to make acknowledgments easier than ever, UF | ICBR now offers Research Resource Identifiers (RRIDs)– persistent and unique identifiers for referencing a research resource. Copy and paste the appropriate core RRID (below) for citation. Proper acknowledgment of UF | ICBR in publications and awards validates our role in driving research forward and provides a platform to improve our services and instrumentation.
| NextGen DNA Sequencing (NS) | RRID:SCR_019152 |
The NextGen DNA Sequencing core at the Interdisciplinary Center for Biotechnology Research (ICBR) provides researchers with quality, massively parallel, high-throughput sequencing data using the most current instrumentation, at a reasonable cost. We currently support the most popular platforms: Illumina NovaSeq X Plus, Illumina MiSeq, Illumina iSeq, PacBio SEQUEL IIe, ONT PromethION, and ONT MinION Mk1c. Together, these instruments cover a broad range of powerful applications, thus allowing biologists to investigate and obtain answers to questions that were not attainable until recently. Our free consultation services aim to help researchers navigate through the complex matrix of experimental options represented by the available sequencing technologies. Some of these important considerations include: read length, error rate, predominant type of error, data output/run, speed, cost, etc. The ever-broadening range of applications include (but are not limited to) de novo sequencing of whole genomes, targeted sequencing, transcriptome sequencing (RNA-seq), chromatin immunoprecipitation (ChIP) sequencing, methylation analysis, and metagenomics.
Sample Requirements
For DNA:
- Purity: Abs 260/280= 1.8-2.0 and Abs 260/230> 1.5 as determined in the NanoDrop or equivalent.
- Integrity: Fragments should be > 20 kb
For RNA:
- Purity: Abs 260/280= 1.8-2.0 and Abs 260/230> 1.5 as determined in the NanoDrop or equivalent.
- RNA Integrity Number (RIN) must be 7or higher
| Comparison of minimum sample for library construction between Oxford NanoPore (ONT) and PacBio (PB) | |||
| Application | NanoPore | PacBio | Comments |
| Large-insert genomic DNA without barcoding | Smaller amounts if library is to be sequenced in 1-2 runs (1-5 µg) | Larger amounts. However, the library yield is typically good for many runs (2-20 µg) | Sample input mass for library prep varies depending on the desired insert size. ONT has no upper limits for input genomic fragment size. Ligation and loading are much faster for ONT |
| Large-insert genomic DNA with barcoding | Similar amounts if library is to be sequenced in 2-3 runs (1-5 µg/sample) | Similar amounts. However, the library yield is typically good for many runs (1-10.0 µg/sample) | Sample input mass for library prep varies depending on the desired insert size. PB has an upper limit of 15-20 kb for proper demultiplexing. No size limit for ONT. Ligation and loading are much faster for ONT |
| RNAseq, cDNA with Amp | 50 ng total RNA or 1-2 ng mRNA | 300 ng | Initial steps of protocols are very similar. However, the adaptor ligation and loading steps are much faster for ONT |
| Direct RNAseq | 500 ng poly-A plus RNA (mRNA) | N/A | Sequencing output is ~1/10th in number of reads versus cDNA with Amp |
Getting Started
When planning your NGS project, please consider the following checklist.
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Core personnel can:
- Inform you on available instrumentation and sample prep services.
- Help you navigate through the many library construction and sequencing choice to optimize your results, based on your research goals.
To discuss a Next Generation Sequencing project contact ICBR-NextGen@ad.ufl.edu or call 352-273-8050 or generate a request for consultation in iLab.
To request NGS services:
- Create an account in our CrossLab system portal. To request an iLab account follow direction on our website (https://biotech.ufl.edu/ilab/)
- Once you have an account, use your credentials to log into iLab>UF Core>NextGenSeq>Request Services>fill out the form.
- In iLab, you will need to fill out the service-specific form (e.g., General Services, NovaSeq X Plus sequencing, PacBio Sequel IIe Sequencing, etc.). You will not be allowed to complete your submission unless you add information to required fields (e.g., payment information). Always click “Save” and the bottom of the form to complete iLab submission and get a project ID. Print your iLab form to submit with your samples.
Prepare your sample/libraries for shipping or delivery to our facility:
- Transfer your samples into 1.5 ml microcentrifuge tubes in concentration and volume as indicated in our “Sample requirements”
- Label all samples clearly. Please simplify sample names
- Place samples in boxes of appropriate size for large number of samples or inside 50 ml conical tubes for small number of samples.
- Prepare to submit any QC documentation you may have on your sample(s): Gel pictures, TapeStation traces, table with barcoding information, etc.
- Pack your sample(s) on ice for DNA or frozen for RNA. Include your iLab service request form and bring your samples to the NGS lab (room 178, CGRC, 2033 Mowry Road).
If shipping, please use this address:
Attention: ICBR-NGS
2033 Mowry Road
Room 178, CGRC bldg.
Gainesville, FL 32610
- Once your sample(s) has been received in the core lab, a project manager will contact you and let you know if any other information about your sample(s) is needed.
- The project manager will also be able to inform you of approximate turn around time.
Once all requested services have been completed, the project manager will notify you and let you know how to access your data. - For sequencing projects, you need to make arrangements for receipt and long-term storage of large volumes of data. ICBR is unable to store sequencing data for longer than 6 months from the original date of data delivery. ICBR will no longer support data delivery in hard drives, starting in the Fall 2019.
- Illumina sequencing data can be accessed through BaseSpace (FASTQ). The ICBR Bioinformatics core will also contact you for delivery all raw data via Globus or HiperGator.
- PacBio sequencing data are also delivered digitally by our Bioinformatics core via Globus or HiperGator.