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Training options & Documents

• Training Requests & Suggestions

• Links: Software, qPCR, Technical info, & Training

• Equipment & Protocol Doc's

• Self-paced online Training

• In-house Training

• Vendor Training

Training Requests & Suggestions

Please submit training requests and topic suggestions through the Core's email address (Genomics Core). For training, please indicate how many people from your lab would like to attend and provide some tentative dates/times you would be available. For topic suggestions, please include your rationale for the topic suggestion and your best guess as to how popular training on that topic would be to others at LSU.

Links:  Software, qPCR, Technical info, & Training

  • Software for DNA Analysis
  • Quantitative and Real-time PCR
  • Technical Information, Apps, & Training

  • Documents:  Equipment & Protocols

  • All Documents (Equipment & Protocols)
  • Ion Torrent S5 (NGS)
  • ABI 3130xl (DNA Sequencing/Fragment Analysis)
  • ABI QuantStudio·6 (qPCR)
  • Diagenode Bioruptor NGS
  • Agilent Bioanalyzer
  • E-gel (DNA Size-selection)
  • BluePippin (DNA Size Selection for NGS)
  • SpeedVac SPD1010
  • Typhoon 8600 Variable Mode Imager
  • Eppendorf Centrifuge 5810R
  • Victor·3 Multilabel Counter
  • Qubit 4.0 (DNA/RNA/Protein quantitation; RNA Integrity)
  • Nanodrop (Nucleic Acid & Protein quantitation)
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    Self-paced online Training?

    There are many training opportunities through self-paced online webinars. For instance, some of the course offerings at ThermoFisher Instrument and Applications Training are free, recorded sessions. In particular, you should consider viewing materials at CE Sequencing & Fragment Analysis and at Real-Time PCR. As of February 2019, the following free online sessions were available:

  • CE Sequencing & Fragment Analysis:
  • – GeneMapper Software Theory & Software Demos (2 hrs total).
    – CE Sanger Analysis Modules on Thermo Fisher (TF) Cloud.
  • qPCR Instrument Specific Software Training:
  • – qPCR Software Video Training Series.
  • qPCR Software and Application training:
  • – Introduction to TF Cloud.
    – Genotyping Data Analysis Using GT App on TF Connect.
    – Introduction to High-Resolution Melt (HRM).
    – High Resolution Melt Analysis Using HRM App on TF Connect.
    – Gene Expression Data Analysis Using RQ App on TF Connect.
    – Real-Time PCR Video Training Series (22-part series covering qPCR fundamentals, including an in-depth look at gene expression).
    – Set up experiment using Design & Analysis App on TF Cloud.
    – Standard Curve Analysis Using SC App on TF Connect.
    – Presence Absence Test Analysis using PA App on TF Connect.
    – Gene Expression Data Analysis Using Expression Suite Software.
  • qPCR Training Series (~30-60 min each):
  • – Module 1: Fundamentals of Real-Time PCR.
    – Module 2: Real-Time PCR Assay Design Choices.
    – Module 3: Normalization.
    – Module 4: Multiplexing.
    – Module 5: Real-Time PCR Reagent Choices.
    – Module 6: Theory of Real-Time Quantitative PCR Analysis.

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    In-house Training

    The Genomics Core can help labs with training personnel in various molecular biology techniques and software. If your training needs are not listed here, please make a suggestion as described above.

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    Vendor Training

    The Genomics Core periodically arranges for vendors to provide local training opportunities... either on its own or at the behest of clients. Training sessions can be virtual or in-person. If there is sufficient interest, vendors are sometimes even willing to put on a workshop rather than just presentations. Listed below are some of the training opportunities available through ThermoFisher.

  • qPCR:
  • 3130xl Applications:
  • NGS by Ion Torrent:
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    In-House Training

    Project Development

    It can be daunting to flesh out the "path" for achieving the aims of a project, especially for beginning graduate students. For instance, there are often many potential molecular biology approaches, which vary in the level of effort, time and funding required as well as the information they can deliver. Feel free to use the Genomics Core as a sounding board to help you identify what approaches are most suitable for your project's needs in the context of your available resources.

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    Topics may include the following:
  • Overview of DNA sequencing issues.
  • Template preparation protocols.
  • DNA sequencing reaction/cleanup protocols.
  • Master-mix calculations.
  • Master-mix preparation.
  • Reaction set-up, including PCR machine.
  • Online submission, including Excel sheet preparation.
  • Reaction cleanup.
  • Reaction resuspension.
  • SequenceAnalysis software (basecalling)
  • Sequence Scanner (review data quality).
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    Sequence Scanner software

    Most researchers skip the critical step of QC'ing their Sanger-Sequencing data, often simply trusting the sequence text file without even examining the electrophoretic trace; this practice can lead to erroneous conclusions and lost time/money. Discover how to use Sequence Scanner (freeware!) to properly and quickly QC Sanger-Sequencing data. Of particular importance, this software enables users to examine the raw data behind the pretty electropherogram... and detect when that pretty data might not be especially reliable. For an example, please visit Why Analyze Data with Sequence Scanner? in our Science Aid Center.

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    qPCR software (QuantStudio·6)

    Mechanics of starting instrument & computer, setting up the run, creating & saving templates for future runs, when to use Melt Curves, performing basic QC using on-instrument software, & exporting data.

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    DNA Quantitation Methods

    Pros & cons of quantifying DNA using Bioanalyzer chips, Gel electrophoresis, Nanodrop, Qubit, or qPCR. Primary emphasis is on applications to NGS work with respect to library construction.

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    DNA Size-Selection Methods

    Pros & cons of size-selecting DNA using the Bluepippin, Columns, E-gels, or standard agarose gel electrophoresis. Primary emphasis is on applications to NGS work with respect to library construction.

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    PCR Basics

    Proper technique for setting up reactions (e.g., methods to prevent pipetting errors), typical cycling parameters (including what's really needed vs. historically used), and gel electrophoresis of the finished reactions. Additional topics could include best practices for DNA/Primer Resuspension, PCR Cycling Parameters, and PCR/qPCR Plastics Considerations.

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    Restriction Digest Basics

    Basics of single-digests and double-digests, using the NEB Catalog & Technical Reference as a guide.

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    Online GC Submissions & Biosci-Booked (Genomics)

    Covers various topics, such as the difference between a 'Standard' & a 'Biosci-Booked (Genomics)' Login ID; the mechanics of applying for a Login ID & submitting or amending online requests (including filling out the Excel templates); options for DNA Sanger-Sequencing (including electrophoresis-only vs. full-service); & available services and supplies.

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    Vendor Training

    Fundamentals of Real-Time PCR

    Module 1 (ThermoFisher): Overview of real-time PCR instrument choices and how real-time PCR differs from endpoint PCR. How can you use real-time PCR in your research? What is the difference between TaqMan® chemistry and SYBR® Green chemistry?

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    qPCR Assay Design Choices

    Module 2 (ThermoFisher): What should you consider when planning your gene expression assays? What is the impact of multitranscript assays and assay specificity? How do you design custom assays?

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    qPCR Normalization

    Module 3 (ThermoFisher): What is normalization and why is it important? What are the types of normalizers and what are their functions? How does multigene normalization work?

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    qPCR Multiplexing

    Module 4 (ThermoFisher): What is multiplexing, and when and how do you use it?

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    qPCR Reagent Choices

    Module 5 (ThermoFisher): Covers aspects of real-time PCR chemistries, such as hot start, UNG, passive reference dye, Fast vs. standard master mixes and reverse transcription.

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    qPCR Theory of Analysis

    Module 6 (ThermoFisher): Perform dynamic range testing with standard curves, quantify your data using the ΔΔCt method, and gain a better understanding of absolute vs. relative quantification.

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    Protein Thermal Shift

    Protein Thermal Shift (PTS) is a technique to detect changes in protein stability using a real-time PCR instrument. PTS is used to identify optimal buffers for protein storage or crystallization, and to measure the impact of molecules interacting with the protein, such as ligands, nucleic acids or other proteins.

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    3130xl Special Purpose Assays

    Some potential topics are listed below; please visit Sequencing Applications for some additional possibities.
  • De novo sequencing.
  • Targeted DNA sequencing.
  • Next-generation sequencing validation.
  • CRISPR validation.
  • Microbial sequencing.
  • Mitochondrial sequencing.
  • Gene expression analysis.
  • Methylation analysis.
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    3130xl GeneMapper software

    Provides DNA sizing and quality allele calls for microsatellite and SNP genotyping analysis. A typical training sessionwould include Fragment Analysis Fundamentals, covering experimental design, optimizing PCR, optimizing capillary electrophoresis, selecting the appropriate size standard, and troubleshooting analysis issues. For additional potential topics, please see the chapters in the DNA Fragment Analysis by Capillary Electrophoresis User Guide.

    The full version of GeneMapper is available at no charge on the Client Computer workstation in the Genomics Core, and the freeware version, PeakScanner, can be accessed at Thermo Fisher Cloud. For a full list of TF Cloud apps, see Connect Data Analysis Apps.

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    3130xl Minor Variant Finder Software

    Designed for accurate detection and reporting of minor variants in Sanger Sequencing traces; available at no charge on the Client Computer workstation in the Genomics Core. Minor Variant Finder removes background noise by comparing test samples with a normal control sample, producing a list of candidate variants and cleaned electropherograms for users to review and confirm candidate variants on both forward and reverse strands. Within the region of strand overlap for test and control .ab1 files, the software can identify 5% minor variants with a sensitivity ≥95% & specificity ≥99%.

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    3130xl SeqScape Software

    Resequencing package designed for mutation detection & analysis, SNP discovery and validation, pathogen sub-typing, allele identification, and sequence confirmation. SeqScape is available at no charge on the Client Computer workstation in the Genomics Core.

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    3130xl Cloud analysis

    ThermoFisher: Applied Biosystems Sanger Analysis Modules are cloud-based secondary data analysis tools that bring together multiple data sets in one place, making it easier to view, store, and analyze Sanger sequencing data. These free modules can be accessed at Connect Data Analysis Apps.

  • Quality Check: The Quality Check app automatically checks CE sequence trace quality. It provides a results summary based on quality parameter settings and auto-flags lower quality traces for further inspection. Users can quickly and easily navigate to questionable or borderline data, and analyze, make adjustments, or exclude traces from the study.
  • Variant Analysis: The Variant Analysis app finds variants in samples sequenced on Applied Biosystems genetic analyzers. It reports variants at genomic coordinates and allows users to export variant calls in standard vcf format. It also reports genomic annotations for SNPs and provides links to external databases.
  • Next-Generation Confirmation (NGS): The Next-Generation Confirmation app confirms NGS variants using CE technology. It allows users to easily visualize the variants detected by both NGS and CE platforms and to export confirmed variants in standard vcf format.
  • Peak Scanner: The Peak Scanner app offers peak identification and fragment sizing for application-specific capillary electrophoresis assays.
  • Microsatellite Analysis: The Microsatellite Analysis app is microsatellite genotyping software that allows you to analyze a mixture of DNA fragments, separated by size. This analysis provides a profile of the separation, precisely calculates the sizes of the fragments, and determines the microsatellite alleles present in the sample. Microsatellite analysis is commonly used to study Microsatellite Instability in cancer and Triplet Repeat Expansion in neurodegenerative diseases, and for Species Identification & Characterization and Human Sample Authentication.
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    Ion NGS Microbial Applications

    Targeted or whole genome sequencing (WGS) approaches can be employed effectively on the Ion S5 for Microbial Sequencing.

  • Whole-genome sequencing: Determine the complete sequence of bacterial or viral genomes by constructing de novo libraries. For bacterial species with moderate levels of GC-content, libraries can be constructed with minimal DNA input, using PCR amplification. However, for bacterial species with high GC-contents (e.g., ≥70%), non-amplification techniques should be employed to avoid PCR-bias which can prevent the assembly of large contigs from the data.

  • Targeted sequencing: Analyze specific bacterial genes or perform viral typing with AmpliSeq technology (ultrahigh-multiplex PCR).

  • Metagenomics: Identify bacterial species in mixed samples using the 16S Metagenomics Kit. Using just two primer pools, the kit amplifies seven hyper-variable regions (V2, V3, V4, V6, V7, V8, and V9) of bacterial 16S rRNA; automated analysis is available through Ion Reporter.
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    Ion NGS RNA Applications

    ThermoFisher: The Total RNA-Seq Kit v2 is designed as a complete solution with a common workflow, combining optimized reagents and protocols for discovery of small RNAs and isoforms, coding RNA, noncoding RNA, and alternative splice variants. Strand information is preserved, enabling more accurate determination of the structure & expression level of transcripts, and aiding in the discovery of novel transcription regions from both positive & negative genomic strands.

  • Whole Transcriptomes: Sequence noncoding RNA, splice variants, and alternative transcripts, examining annotated transcripts from any species using polyA-selected RNA and detecting novel transcripts using ribo-depleted total RNA.

  • Small RNAs: Historically, undesirable adapter-dimers were removed from small RNA libraries by gel size-selection; here, cDNA synthesis of the adapter byproduct is inhibited, thus allowing cDNA separation with magnetic bead-based technologies.
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    Ion NGS Targeted Applications

    ThermoFisher: Ion AmpliSeq technology delivers a highly muliplexed, PCR-based workflow allowing researchers to:
    – sequence low quality samples with minimal input DNA or RNA;
    – target one to hundreds of genes in a single run; and,
    – go from sample to results in as little as 24 hours.

  • AmpliSeq panels: for both DNA and RNA, choose from Predesigned ('Ready-to-Use' and Community-designed panels) or 'Made to order' panels. As of 27 April 2020, Ion AmpliSeq Designer includes pre-loaded genomes for Human, Mouse, Chicken, Cow, Pig, Sheep, Chinese hamster, Dog, Maize, Rice, Soybean, and Tomato.

  • Transcriptomes: These RNA panels enable simple high-throughput gene expression studies on mouse or human (covering 20K genes in a single multiplex PCR for library prep). Start with ≥10 ng of total RNA from FFPE, cells, or other sources (no need to polyA-select or ribo-deplete), multiplex up to 8 samples per 540 chip, and enjoy automated analysis.

  • Exomes: The Exome RDY Kit enables simple exome enrichment, generating libraries in <6 hours with <50 minutes of hands-on time. Start with 50 ng gDNA, and cover >97% of the consensus coding sequences with 90% On-Target reads and 90% uniformity. Multiplex two human exomes per 540 chip, and enjoy automated analysis.
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    Ion NGS Library Construction

    ThermoFisher: Covers basic methods for constructing NGS libraries suitable for Ion S5 platform, such as the:
  • Ion AmpliSeq Library Kit;
  • Ion Plus Fragment Library Kit;
  • Ion Total RNA-Seq Kit; and,
  • PCR-based fusion methods.
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