(SW8) Microbiome and Metagenomics Benchtop to Bioinformatics; Technical approaches to Sequencing, DNA Extractions, and Data Analysis

Workshop Sponsors:

GIGA
Illumina
Zymo Research

 

Sunday, April 22

8:00 am – 4:00 pm

Microbiome sequencing has become an increasingly important activity for NGS core facilities due to interest in host-associated microbial communities in human health. Historically, there have been relatively few core NGS facilities performing library preparation and sequencing of amplicons, shotgun metagenome sequencing, and shotgun meta-transcriptome sequencing. Likewise, experience in analysis of these data has often been limiting in standard bioinformatics core facilities. To address these historical limitations, we are providing a workshop following library preparation, library Q/C and balancing of libraries in pools, sequencing, primary bioinformatics analysis, statistical analyses and visualization of data for both 16S rRNA gene amplicon sequencing and shotgun metagenome sequencing. A series of talks from members of the DNA Services (DNAS) Facility and the Core for Research Informatics (CRI) within the Research Resources Center (RRC) at the University of Illinois at Chicago (UIC) will be followed by a question-and-answer panel discussion, and if time permits, a brief slideshow highlighting innovative presentations and analyses of microbiome data. The primary focus of the workshop will be on DNA-based analysis, but consideration of direct RNA sequencing and RNA metatranscriptome sequencing will also be provided. In addition, the session will highlight the ease of conducting NGS amplicon sequencing protocols for genes other than the 16S rRNA gene.

Organizer

Stefan J. Green, DNA Services (DNAS) Facility, Research Resources Center (RRC), University of Illinois at Chicago (UIC) and Scott Tighe, Advanced Genomics Lab,  University of Vermont

Speakers

Stefan J. Green, DNAS, RRC, UIC
Kevin Kunstman, DNAS, RRC, UIC
Mark Maienschein-Cline, Core for Research Informatics (CRI), RRC, UIC
George Chlipala, CRI, RRC, UIC
Scott Tighe, UVM

Workshop Goals

The goals of the workshop include: (a) Introduction to the field of microbiome sequencing, (b) Helping core facility staff determine which sequencing approach will meet their customers’ needs (e.g. targeted amplicon sequencing or shotgun metagenome sequencing), and also determine what nucleic acid extraction strategy should be used. (c) Demonstrating an incredibly easy protocol that will allow core facilities to offer immediately microbiome sequencing to investigators in their own institutions, (d) Understanding methods to recover high quality DNA and RNA, (e) Understanding what extraction methods to use for specific sample types, (f) Discussing the applications of the Oxford Nanopore and PacBio for 16s and shotgun sequencing applications, (g) Providing core facility staff an overview of bioinformatics pipelines for analysis of amplicon and shotgun metagenome data that can be implemented at their home institution, (h) Providing bioinformatics core facility staff with an overview of the types of statistics and visualization strategies that are employed to analyze microbial communities, and (i) Provide an open forum for attendees to ask questions of workshop speakers.

Target Audience

Core directors, technicians, graduate and undergraduate students involved (or getting involved) with studies in metagenomics, microbiome, DNA/RNA extractions and other microbial analysis. We have seen that there are relatively few facilities that like to perform full workflow sequencing of amplicons for microbiome studies, and this workshop will empower them to perform these assays in-house. Scientists would be from the field of genomics/metagenomics.

Abstract

Microbiome sequencing has become an increasingly important activity for NGS core facilities due to interest in host-associated microbial communities in human health. Historically, there have been relatively few core NGS facilities offering high performance DNA extractions, library preparation and sequencing of amplicons, shotgun metagenomes, and shotgun meta-transcriptomes. Likewise, experience in analysis of these data has often been limiting in standard bioinformatics core facilities. To address these historical limitations, we are providing a full day workshop including Sample recovery, DNA/RNA extraction techniques, library preparation, library Q/C and balancing of libraries in pools, sequencing, primary bioinformatics analysis, statistical analyses and visualization of data for both 16S rRNA gene amplicon sequencing and shotgun metagenome sequencing. A series of talks from members of the DNA Services (DNAS) Facility and the Core for Research Informatics (CRI) within the Research Resources Center (RRC) at the University of Illinois at Chicago (UIC) will be followed by a question-and-answer panel discussion, and if time permits, a brief slideshow highlighting innovative presentations and analyses of microbiome data. The primary focus of the workshop will be on DNA-based analysis, but consideration of direct RNA sequencing and RNA meta-transcriptome sequencing will also be provided. In addition, the session will highlight the ease of conducting NGS amplicon sequencing protocols for genes other than the 16S rRNA gene.

DRAFT AGENDA [7-8 hr total]

  1. Introduction (15 min; SJG)

  2. Basics of PCR-based microbiome sequencing (45 min; SJG)

  3. 16S rRNA Gene Amplicon Bioinformatics Using QIIME (60 min; GC)

  4. Basics of shotgun metagenome sequencing (30 min; TBD)

  5. Bioinformatics of shotgun metagenome sequence data (60 min; TBD)

  6. Techniques in DNA and RNA Extractions for Soils, Air, Swabs and Fecal pellets (2hrs)

  7. Giga Sponsors (2) -1hr

  8. Round table discussion (30 min; All speakers)

Moderately-Detailed Agenda

  1. Introduction (SJG, 15 min)

    1. Pros and cons of 16S rRNA gene amplicon sequencing and shotgun metagenomics
    2. Decision tree for helping customers decide on sequencing approach
       
  2. Basics of PCR-based microbiome sequencing [Book chapters available as reference] (SJG, 45 min)

    1. One-step PCR approach
    2. Two-step PCR approach
    3. One-step PCR + Ligation approach
    4. Primer selection and primer design
    5. Amplicon Q/C
    6. Pooling
    7. Size selection (if necessary)
    8. Sequencing issues related to unique features of amplicons

      1. Primers for initiating sequencing reaction
      2. phiX spike-in
      3. Read lengths
    9. Raw data output examples
    10. Long read sequencing options
    11. Managing complex sample pools
       
  3. 16S rRNA Gene Amplicon Bioinformatics Using QIIME (GC, 60 min)

    1. Basic Q/C of raw sequence data
    2. Merging, trimming, primer removal
    3. Chimera detection and removal
    4. Clustering (OTU97 / sub-OTU)
    5. Annotation
    6. Rarefaction
    7. Ordination
    8. Statistical tests

      1. ANOSIM
      2. Group-significance testing
      3. Alpha diversity measures
      4. LEFSe
    9. Network / correlation analyses
    10. Examples of useful figures
       
  4. Basics of shotgun metagenome sequencing (TBD, 30 min)

    1. Selecting library preparation protocols
    2. Size selection?
    3. Sequencing Q/C
    4. Sequencing platform choices
    5. Long read sequencing options (including Pacbio, Nanopore, Hi-C, etc)
       
  5. Techniques for DNA/RNA extractions
  1.   Contrasting new and old methods
  2. Kits and reagents for preservation of gDNA length
  3. Methods for improved recovery
  4. Approaches to RNA
  5. Quality assessment of nucleic acids
     
  1. Bioinformatics of shotgun metagenome sequence data (TBD, 60 min)

    1. Basic Q/C of raw sequence data
    2. Quality trimming and adapter removal
    3. When to merge?
    4. High-throughput annotation

      1. Taxonomy only: OneCodex, Kraken, Diamond, etc.
      2. Functional gene annotation
    5. Statistical Analyses
    6. Pathway analyses
    7. Examples of useful figures
       
  2. Round Table Discussion (30 min)

Platinum Sponsors of the ABRF