-
Notifications
You must be signed in to change notification settings - Fork 24
TOPIC: Amplicons
- Lesson 1: Introduction to Amplicons
- Lesson 2: A longer discussion of Amplicons
- Lesson 3a: Analyzing amplicon data with DADA2 in Qiime2
- Lesson 3b: RStudio + DADA2 using DECIPHER for taxonomic classification
- Lesson 4: Preparing Output from Amplicon Pipelines for Analysis
- Lesson 5: Ordination - Linked with Lesson 8 in Topic R
Date posted: 11 April 2020
Author(s): Dr. Liz Suter and Dr. Sarah Hu
Instructor(s): Dr. Liz Suter and Dr. Sarah Hu
- What are amplicons?
- What are the different pipelines for processing amplicon data?
- What are the available methods for producing count tables?
Date posted: 15 April 2020
Author(s): Dr. Liz Suter, Dr. Ben Tully, Dr. Mike Lee, Dr. Ella Sieradzki, Dr. Chris Trivedi, Dr. Sarah Hu
Instructor(s): Dr. Liz Suter, Dr. Ben Tully, Dr. Mike Lee, Dr. Ella Sieradzki, Dr. Chris Trivedi, Dr. Sarah Hu
An informal discussion among instructors about topics such as:
- What are your preferred workflows for analyzing amplicons?
- What are your opinions on certain caveats associated with amplicon analyses?
Discussion of particular questions can be found at the following times in the lesson:
-
How important is it to use ASVs over OTUs?
- Callahan et al. 2017- values of AVS over OTUs
- Summary of that paper’s main points at Happy Belly Bioinformatics here
- Is it still acceptable to use a 454 dataset?
- Are -omics approaches replacing amplicon approaches?
-
Should we delete singletons?
- Edgar 2017: Evidence that 97% clustering falsely inflates singletons
-
Have there been any recent advances for getting enough DNA from low biomass samples?
- Some kit options for 50ng input and 1ng input
- It's important to account for contamination when working with low biomass! A blog post from Rose Kantor's website.
- What kind of server do you use for running your amplicon pipeline?
- What does your typical amplicon workflow look like?
- Is there a difference between calling DADA2 in Qiime2 vs calling it on its own?
- Which interface do you use for running your code?
- How do you manage and store your data?
- How do you treat your count tables to account for the way that high throughput sequencing ‘collects’ data (ie. as relative abundances rather than absolute counts)?
Prerequirements:
Date posted: 25 May 2020
Author(s): Dr. Liz Suter
Instructor(s): Dr. Liz Suter
- Example analysis of 16S rRNA amplicon data using DADA2 implemented in Qiime2
Prerequirements:
- Miniconda in order to use cutadapt from the command line
Date posted: 7 June 2020
Author(s): Dr. Christopher Trivedi
Instructor(s): Dr. Christopher Trivedi
- DADA2 pipeline in RStudio using DECIPHER and idTaxa for taxonomy assignment
Date posted: 22 June 2020
Author(s): Dr. Liz Suter
Instructor(s): Dr. Liz Suter
- Importing files into R
- Removing contamination
- Checking sequencing depth with rarefaction curves
- Removing singletons
- Normalization
- Exporting formatted files from R
Date posted: 26 June 2020
Author(s): Dr. Liz Suter
Instructor(s): Dr. Liz Suter
- R crossover tutorial, see Topic R Lesson 8 for more
- Using the output from Qiime2 analysis in Lesson 4
- Import ASV table into phyloseq
- Explore functionality of phyloseq: making tree, re-rooting tree, bar plot of taxa
- Ordinations with phyloseq: PCoA, weighted UniFrac PCoA, NMDS
Primary tools/programs used:
Date posted: 7 July 2020
Author(s): Dr. Sarah Hu
Instructor(s): Dr. Sarah Hu
Content | Video presentation - Part 1 | Video presentation - Part 2
- QIIME2, mothur, DADA2, phyloseq, and vegan documentation
- Tutorials
- Riffomonas from Pat Schloss's group
- Mike Lee's workflow using DADA2 and phyloseq in R
- Vegan tutorial
- GUI options
Home -- Topics -- Unix -- R -- Amplicons -- Metagenomics -- Functional Annotation -- Transcriptomics -- Networks -- Python -- Reproducibility Challenge -- BVCN Conference 2021 -- Infrastructure Used -- Jupyter -- MyBinder -- Get Involved!