Resources

Grant Writing

Facilities and Equipment - The Advanced Genomic Technologies Core of the Brain & Behavior Institute (BBI-AGTC) at University of Maryland

The Advanced Genomic Technologies Core of the Brain & Behavior Institute (BBI-AGTC) is a full-service sequencing facility serving the University of Maryland community and external clients. Situated in the Bioscience Research Building at the heart of the life science community on campus, the mission of the BBI-AGTC is to make state-of-the-art genomic technologies accessible to researchers at a reasonable cost. Services and technologies offered include DNA and RNA qualitative and quantitative analysis, high-throughput sequencing (up to 800M reads per run), whole genome library preparation and sequencing, RNA library preparation - including stranded RNA prep with rRNA depletion, and single-cell genomic/expression profiling. Equipment includes a sequencer (Illumina NextSeq 1000), a single cell controller (10X Genomics Chromium), an automated liquid handling station (Eppendorf epMotion 5075tc), a real-time PCR machine (Roche LightCycler 96), an Agilent TapeStation 4150, and a high performance ultrasonicator for DNA shearing (Covaris SureSelect S2).

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Acknowledging the BBI-AGTC

The existence of core facilities depends in part on proper acknowledgement in publications. Please acknowledge the help of the Brain & Behavior Institute - Advanced Genomic Technologies Core (BBI-AGTC) in your reports and publications.

Evidence of our contribution to your scientific output is a key metric for the management of the core, as it allows us to 1) assess our scientific impact, 2) justify institutional investment, and 3) eventually pursue external grant funding with the ultimate goal of ensuring a continuous operation and providing our essential services to the scientific community.

Please use the following text in all publications:

"Part of this work was conducted at the Brain & Behavior Institute - Advanced Genomic Technologies Core (BBI-AGTC), which is supported by the BBI and the University of Maryland, College Park."

It would be also appreciated if you sent a copy of the accepted manuscript or published paper to bbi-agtcumd.edu. This would facilitate our task when we prepare our yearly institutional report.

Enrichment Workshops

January 26, 2022

This workshop was held on January 26, 2022, 12:00-1:00 pm on Zoom. The presentations are posted below and accessible to those affiliated with UMCP. Unfortunately, we ran out of time and did not want to rush through the second presentation which will be presented in full at a later date.

  • DNA/RNA quality and quantity evaluation for NGS workflows - April Hussey (Core Manager)
    • Presentation slides with additional notes (portrait): PDF
    • Presentation slides only (landscape): PDF
    • Recording: MP4
  • Considerations for DNA-seq and RNA-seq read length and coverage - Najib El-Sayed (Core Director)
May 5, 2021

This workshop was held on May 5, 2021 on Zoom. The presentation is posted below and accessible to those affiliated with UMCP.

  • Biology at True Resolution: Resolving Biology with single-cell genomics, multi-omics, and spatial transcriptomics - Bradley Toms, M.S. (10x Science & Technology Advisor)
    • Presentation slides (landscape): PDF
    • Recording: MP4
April 21, 2021

This workshop was held on April 21, 2021 on Zoom. The presentation is posted below and accessible to those affiliated with UMCP.

  • NextSeq1000, Enabled Aapplications, and Informatics @ BBI-AGTC - Dan Gheba, Tara Kesteloot, Bao Ho (Illumina Team)
    • Presentation slides (landscape): PDF
    • Recording: MP4

Evaluating Nucleic Acid Quality for NGS

Library preparation methods are sensitive to any contaminants present in the sample and the quality of the input material directly affects the quality of the library. There are many places in the lysis, precipitation, and purification process where contaminants can be introduced or incompletely removed from the nucleic acid sample. It is the responsibility of the customer to understand and troubleshoot the properties of their biological samples and the chosen nucleic acid extraction/purification method. Spectrophotometric and fluorometric assay output must be appropriately evaluated as it relates to the species represented and the extraction method used. Samples should be stored at the appropriate temperature and in nuclease-free water or low TE buffer (10 mM Tris-HCl, pH 8.0-8.5, 0.1 mM EDTA) only in sterile, nuclease-free tubes. Samples destined for NGS must not contain >1 mM EDTA or any of the contaminants described below.

There are three aspects of nucleic acid quality that must be evaluated collectively:

Metrics Evaluation Method
1. Purity UV absorbance spectrophotometric (NanoDrop)
2. Quality RIN, % DV200, DIN, fragment size fluorometric (TapeStation, BioAnalyzer), gel electrophoresis
3. Quantity concentration, total amount fluorometric (Qubit)

Total RNA

1. Purity 2. Quality
  • To prevent degradation, store samples in nuclease-free ultrapure water or low TE buffer (10 mM Tris-HCl, pH 7.0-8.0, 0.1 mM EDTA) at -80°C
  • Acceptable RIN values depend on the selected library preparation method and vice versa. See Submissions for more information
  • For total RNA samples derived from mixed eukaryote/prokaryote samples, such as those modeling host-pathogen interactions, the rRNA of the predominant species can be evaluated and considered representative of the overall quality
  • TapeStation RIN values may not be reliable for mixed species or plant total RNA samples presenting with bacterial/chloroplast/mitochondrial and cytosolic rRNA as the only analysis options are "eukaryote" (18S, 28S) and "prokaryote" (16S, 23S). Thus visual evaluation of the electropherogram is necessary
3. Quantity
  • Fluorescence-based detection utilizing RNA-specific dyes is more accurate than spectrophotometric measurements, as the presence of DNA or other contaminants can result in overestimation of the sample amount
  • Perform a DNase treatment since gDNA can carry over from the interphase of organic extractions or from overloading the silica matrix of solid phase RNA purification methods
  • Minimum input amounts and maximum input volumes depend on the selected library preparation method. See Submissions for more information

Genomic DNA

1. Purity 2. Quality
  • To prevent fragmentation, store samples in nuclease-free ultrapure water or low TE buffer (10 mM Tris-HCl, pH 7.0-8.0, 0.1 mM EDTA) at RT or +4°C
  • gDNA should be largely intact and of high average molecular weight
  • Highly fragmented DNA will negatively impact library quality
  • Environmental or microbiome DNA samples intended for taxonomic or metagenomic analyses should be evaluated in terms of the highest quality that is reasonably achievable
  • The AGTC does not offer a cost-effective means to evaluate gDNA integrity so is is the customer's responsibility to do this prior to sample submission.
3. Quantity
  • Fluorescence-based detection utilizing DNA-specific dyes is more accurate than spectrophotometric measurements, as the presence of RNA or other contaminants can result in overestimation of the sample amount
  • Minimum input amounts and maximum input volumes depend on the selected library preparation method. See Submissions for more information

Microvolume Spectrophotometer (NanoDrop) Notes

  • Use the same solution the nucleic acid is resuspended/eluted in (usually nuclease-free water or low TE buffer) to blank the spectrophotometer
  • Absorbance ratios are not reliable for samples with concentrations <20 ng/μL and are still relatively variable (with a tendency to be too high) at values up to 50 ng/µL
  • Acidic solutions will under-represent the A260/A280 ratio by 0.2-0.3, while a basic solution will over-represent the ratio by 0.2-0.3
  • Absorbance ratios can be misleading if multiple contaminants are involved or if the pH is off so visual inspection of the absorbance plot is necessary
  • Common contaminants and absorbance wavelengths:
    • carbohydrates: ~230 nm
    • EDTA: ~230 nm
    • guanidine HCl (DNA): ~230 nm
    • guanidine isothiocyanate (RNA): ~260 nm
    • phenol: ~230 nm and ~270 nm
    • protein: ~280 nm
    • ethanol & isopropanol: indistinguishable, causes a uniform increase in signal

Additional References