SW3: Next-Generation Sequencing of Microbial Communities: Strategies for Project Design, Execution and QIIME Analysis

Session Organizer: Chris L. Wright, Assistant Director, DNA Sequencing
W.M. Keck Center for Comparative and Functional Genomics
​University of Illinois at Urbana-Champaign

Saturday, March 28th, 2015

 

  • Giga Sponsor:     

 

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    Objective:  This workshop will study many of the current strategies being applied to microbial community analysis in regards to project design, sequencing execution, and analysis utilizing QIIME.  QIIME (Quantitative Insights into Microbial Ecology) is an open-source software package that is used for comparison and analysis of microbial communities. The workshop will use the latest version of QIIME (1.9.0), scheduled for release in December 2014.

     

    The target attendees are Core Director and Supervisors who consult with customers on project setup and design, as well as bioinformaticians who want to become more familiar with the sequencing side of these projects or who have no or limited experience with QIIME.  The larger target group includes researchers setting up microbial community analysis projects, students analyzing these projects, and Core technicians looking to gain more understanding into this rapidly growing field.

     

    Structure:

  • Pre-Meeting Primers:  Three pre-meeting training sessions (Primers) will occur during the 3 weeks prior to the workshop.  While not mandatory, it is essential that attendees understand the material presented in these self-paced primers to take full advantage of the applications discussed during the workshop.  Attendees will be given readings and/or assignments that should take ~2 hours to complete.  The last Primer will also contain basic iPython instruction.  Each assignment will be followed up by a 1 -hour conference call or on-line meeting where organizers will be available to answer basic questions on the material.
     
  • Workshop Day:  The workshop will focus on applications of the information learned in the Primers, followed by iPython-guided tutorial of QIIME analysis from fastq file to biome to diversity analysis. 
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  • Important Registration and Primer Class Information:

    • Registration for this workshop is limited to 40 attendees only—register early to ensure your seat!
    • Registration will close on March 20th, 2015. Because this workshop relies on information learned during the 3 pre-meeting training Primers, attendees are strongly encouraged to register before March 7th, 2015.
    • Primer material will be provided on March 7th, March 14th, and March 21st with Q&A sessions for the weekly material held on the following Thursday from 11 am -12 pm (Central Time). FAQs will be updated and available to all attendees.

       

      Workshop topics will include:        

       

      1. Overview of single- and dual-indexing amplicon preparation methods
      2. Overview of Fluidigm and Nextera amplicon setup
      3. Project Design: How to apply the above (one shoe does not fit all)
      4. Illumina MiSeq setup and run parameters for optimal amplicon sequencing
      5. Processing data from the instrument:  generation of demultiplexed or bulk files, single- or dual- index processing, multiple primers used, trimming primer sequences, etc.
      6. Review of iPython and the command line
      7. Getting started in QIIME: I have data files, now what??  
      8. Mapping files, sequence QC and demultiplexing in QIIME
      9. From sequences to a biom table
      10. Diversity analysis (alpha, beta, and taxonomy plots)
      11. Finding interesting OTUs and other analysis tools
      12. Putting it all together: Discussion and wrap-up
         

Schedule at a glance (food and refreshments provided during breaks and lunch):

Part 1: Amplicon design and applications

8:00 - 8:10:

Welcome and Introductions

8:10 – 8:40:

FLUIDIGM Presentation—overview of FLUIDIGM setup and applications. — Andrew O’Guin. Field Applications Specialist, Fluidigm, Inc.

8:40 – 9:10:

ILLUMINA Presentation-- Overview of single- and dual-indexing methods, Nextera amplicon set-up, MiSeq setup and run parameters and BaseSpace applications. — Gary Nunn, PhD. Staff Field Applications Scientist, Illumina, Inc.

9:10 – 9:30:

Project Design: How to apply the above (one shoe does not fit all). — Chris Wright and Marie Adams

  • Project size, when to do it yourself, samples per project/per run, single interrogation or multiple primer sets, which variable region or gene-specific target to use, etc.
  • Processing data from the instrument: demultiplexed or bulk files, single- or dual- index, multiple primers used, trimming primer sequences, and other considerations.

9:30 – 9:45:

Questions and answers

Part 2: QIIME analysis: QIIME Masters Adam Robbins-Pianka and Justine Debelius

9:45 – 10:00:

Review of iPython and the command line.

10:00 – 10:30:

Break

10:30 – 11:00:

Getting started in QIIME: I have data files, now what??

11:00 - 12:00:

Mapping files, sequence QC and demultiplexing in QIIME

12:00 – 1:00:

Lunch

1:00 – 2:00:

From sequences to a biom table

2:00 – 2:30:

Break

2:30 – 3:30:

Diversity analysis (alpha, beta, and taxonomy plots)

3:30 – 4:00:

Finding interesting OTUs and other analysis tools

4:00 - 4:30:

Putting it all together: Discussion and wrap-up

Other information:
Attendees will be expected to bring their own laptops. No special software is needed other than the ability to connect to the Internet. We will utilize an iPython notebook through Google Chrome to facilitate the QIIME analysis.