BiteSized Bioinformatics: Flash Introduction to Qiime2
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This document provides an overview of analyzing 16S rRNA gene amplicon sequencing data using Qiime2. It begins with an introduction to metabarcoding and using 16S rRNA for microbial community profiling. It then demonstrates the bioinformatics analysis workflow in Qiime2, including quality control, clustering sequences into OTUs, assigning taxonomy, and generating an OTU table with metadata. The document is intended as a training source to familiarize users with common analysis steps and biases or assumptions in 16S amplicon analysis.
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BiteSized Bioinformatics: Flash Introduction to Qiime2
- 1. BiteSized Bioinformatics Andrea Telatin, Gut Microbial Health Quadram Institute, UK Flash introduction to Qiime2
- 2. Today’s Menu • 16S Amplicons: experiment • 16S Amplicons: analysis • Qiime 2
- 4. Metabarcoding – Sogin et. al 2006 “Gene sequences, most commonly those encoding rRNAs, provide a basis for estimating microbial phylogenetic diversity and generating taxonomic inventories of [...] microbial populations.”
- 5. Questions • Who is in the sample? • What are they doing?
- 6. Questions • Who is in the sample? • What are they doing? metagenomics ≠ amplicon sequencing 16S Amplicons, Metagenomics Metagenomics
- 7. Anatomy of a gene: 16S rDNA
- 8. Biases? • Sampling (replicate or lie) • Cell breaking (spores, cell wall,…) • Amplify (primers? cycles?) • Sequencing • Bioinformatics (assumptions, database for annotation…)
- 9. Biases? • Sampling (replicate or lie) • Cell breaking (spores, cell wall,…) • Amplify (primers? cycles?) • Sequencing • Bioinformatics (assumptions, database for annotation…) That’s why we compare different samples
- 11. Overview • QC • Cluster all the filtered reads Representative sequences • Count frequencies OTU table • Assign taxonomy to each OTU
- 12. Comparison This paper: offers a comparison of methods that can be used as a review of biases and trade offs…
- 13. A training source: USEARCH USEARCH is a monolithic package written by Robert Edgar, an independent bioinformatics researcher. Can be used to see how we manipulate reads to make an annotated OTU table. Binary download: http://drive5.com/
- 14. A training source Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa uniques.fa OTUS.fa OTUS_tax.fahttp://drive5.com/usearch/manual/pipe_examples.html
- 15. @1.control::D00420:186:HF5VMBCX2:1:1103:13149:23543 1:N:0:AGTCAA TACGTAGGGAGCAAGCGTTGTCCGGATTTACTGGGTGTAAAGGGTGCGTAGGCGGCTAGGCAAGTCAGAC + IIIAGGGGGIIIGIIGIGGGGGJJJJJJJJJJJJJJJJJJJJ?JJJJJJJJJJJJJJJJJJJJJJJJJJJ @1.control::D00420:186:HF5VMBCX2:1:2203:5458:59908 1:N:0:AGTCAA TACGTATGGTGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGCAGGCGGTGCGGCAAGTCTGAT + GIIIIGGAGGGIIIIIIGI<GGJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ @1.control::D00420:186:HF5VMBCX2:1:2113:15314:56029 1:N:0:AGTCAA TACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGT + GGIIGIIIIIGIIIIGIIGIIGJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ @1.control::D00420:186:HF5VMBCX2:1:2203:17011:74430 1:N:0:AGTCAA AACGTATGGAGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGTGTAGGTGGCCATGCAAGTCAGAA + IIIIIIIGIIIIIIIIIIIIIIJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ @1.control::D00420:186:HF5VMBCX2:1:1109:15763:18147 1:N:0:AGTCAA TACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGT + A training source Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa filtered.fa uniques.fa OTUS.fa Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa unique.fa OTUS.fa OTUS_tax.fa tracing sample IDs
- 16. >Filt1 TACGTAGGGAGCAAGCGTTGTCCGGATTTACTGGGTGTAAAGGGTGCGTAGGCGGCTAGGCAAGTCAGACGTGAAAACCA TGGGCTCAACCTGTGGACTGCGGTTGAAACTGTTTAGCTTGAGTGAAGTAGAGGTAGGCGGAATTCCCGGTGTAGCGGTG AAATGCGTAGAGATCGGGAGGAACACCAGTGGCGAAGGCGGCCTACTGGGCTTTAACTGACGCTGAGGCACGAAAGCATG GGTAGCAAACAGGATTAGAAACCCGCGTAGTCC >Filt2 TACGTATGGTGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGCAGGCGGTGCGGCAAGTCTGATGTGAAAGCCC GGGGCTCAACCCCGGTACTGCATTGGAAACTGTCGTACTAGAGTGTCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTG AAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGATAACTGACGCTGAGGCTCGAAAGCGTG GGGAGCAAACAGGATTAGAAACCCTGGTAGTCC >Filt3 TACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCC ATCGCTTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTG GAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTG GGGAGCGAACAGGATTAGAAACCCCCGTAGTCC >Filt4 AACGTATGGAGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGTGTAGGTGGCCATGCAAGTCAGAAGTGAAAATCC GGGGCTCAACCCCGGAACTGCTTTTGAAACTGTAAGGCTAGAGTGCAGGAGGGGTGAGTGGAATTCCTAGTGTAGCGGTG AAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGACTGTAACTGACACTGAGGCTCGAAAGCGTG A training source Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa filtered.fa uniques.fa OTUS.fa Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa unique.fa OTUS.fa OTUS_tax.fa
- 17. >Uniq1;size=6103; TACGTATGGTGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGCAGGCGGTGCGGCAAGTCTGATGTGAAAGCCC GGGGCTCAACCCCGGTACTGCATTGGAAACTGTCGTACTAGAGTGTCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTG AAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGATAACTGACGCTGAGGCTCGAAAGCGTG GGGAGCAAACAGGATTAGAAACCCCCGTAGTCC >Uniq2;size=5788; TACGTATGGTGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGCAGGCGGTGCGGCAAGTCTGATGTGAAAGCCC GGGGCTCAACCCCGGTACTGCATTGGAAACTGTCGTACTAGAGTGTCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTG AAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGATAACTGACGCTGAGGCTCGAAAGCGTG GGGAGCAAACAGGATTAGATACCCCCGTAGTCC >Uniq3;size=5082; TACGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGATGGATGTTTAAGTCAGTTGTGAAAGTTT GCGGCTCAACCGTAAAATTGCAGTTGATACTGGATATCTTGAGTGCAGTTGAGGCAGGCGGAATTCGTGGTGTAGCGGTG AAATGCTTAGATATCACGAAGAACTCCGATTGCGAAGGCAGCCTGCTAAGCTGCAACTGACATTGAGGCTCGAAAGTGTG GGTATCAAACAGGATTAGAAACCCCCGTAGTCC >Uniq4;size=3438; TACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCC CGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTG AAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTG A training source Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa filtered.fa uniques.fa OTUS.fa Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa unique.fa OTUS.fa OTUS_tax.fa propagating “metadata”
- 18. >Otu1 TACGTATGGTGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGCAGGCGGTGCGGCAAGTCTGATGTGAAAGCCC GGGGCTCAACCCCGGTACTGCATTGGAAACTGTCGTACTAGAGTGTCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTG AAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGATAACTGACGCTGAGGCTCGAAAGCGTG GGGAGCAAACAGGATTAGAAACCCCCGTAGTCC >Otu2 TACGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGATGGATGTTTAAGTCAGTTGTGAAAGTTT GCGGCTCAACCGTAAAATTGCAGTTGATACTGGATATCTTGAGTGCAGTTGAGGCAGGCGGAATTCGTGGTGTAGCGGTG AAATGCTTAGATATCACGAAGAACTCCGATTGCGAAGGCAGCCTGCTAAGCTGCAACTGACATTGAGGCTCGAAAGTGTG GGTATCAAACAGGATTAGAAACCCCCGTAGTCC >Otu3 TACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCC CGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTG AAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTG GGGAGCAAACAGGATTAGAAACCCCCGTAGTCC >Otu4 TACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCC ATCGCTTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTG A training source Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa filtered.fa uniques.fa OTUS.fa Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa unique.fa OTUS.fa OTUS_tax.fa
- 19. >Otu8;tax=d:Bacteria(1.0000),p:"Bacteroidetes"(0.9991),c:"Bacteroidia"(0.9961),o:"Bacteroidale (0.9952),f:Bacteroidaceae(0.9910),g:Bacteroides(0.9910); TACGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCGGATGCTTAAGTCAGTTGTGAAAGTTT GCGGCTCAACCGTAAAATTGCAGTTGATACTGGGTGTCTTGAGTACAGTAGAGGCAGGCGGAATTCGTGGTGTAGCGGTG AAATGCTTAGATATCACGAAGAACTCCGATTGCGAAGGCAGCTTGCTGGACTGTAACTGACGCTGATGCTCGAAAGTGTG GGTATCAAACAGGATTAGAAACCCCCGTAGTCC >Otu2;tax=d:Bacteria(1.0000),p:"Bacteroidetes"(0.9994),c:"Bacteroidia"(0.9939),o:"Bacteroidale (0.9939),f:Bacteroidaceae(0.9915),g:Bacteroides(0.9915); TACGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGATGGATGTTTAAGTCAGTTGTGAAAGTTT GCGGCTCAACCGTAAAATTGCAGTTGATACTGGATATCTTGAGTGCAGTTGAGGCAGGCGGAATTCGTGGTGTAGCGGTG AAATGCTTAGATATCACGAAGAACTCCGATTGCGAAGGCAGCCTGCTAAGCTGCAACTGACATTGAGGCTCGAAAGTGTG GGTATCAAACAGGATTAGAAACCCCCGTAGTCC >Otu1;tax=d:Bacteria(1.0000),p:Firmicutes(0.9991),c:Clostridia(0.9889),o:Clostridiales(0.9889) :Lachnospiraceae(0.9573),g:Roseburia(0.5720); TACGTATGGTGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGCAGGCGGTGCGGCAAGTCTGATGTGAAAGCCC GGGGCTCAACCCCGGTACTGCATTGGAAACTGTCGTACTAGAGTGTCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTG AAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGATAACTGACGCTGAGGCTCGAAAGCGTG GGGAGCAAACAGGATTAGAAACCCCCGTAGTCC A training source Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa filtered.fa uniques.fa OTUS.fa Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa unique.fa OTUS.fa OTUS_tax.fa we must rely on “metadata”
- 20. A training source Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa filtered.fa uniques.fa OTUS.fa Sample_1.fastq Sample_2.fastq Sample_3.fastq merged.fq filtered.fa unique.fa OTUS.fa OTUS_tax.fa OTU Table example #OTU S1 S2 S3 S4 S5 Otu1 14303 347 17513 474 14954 Otu2 10636 1312 7312 1486 11395 Otu4 3229 2662 7050 3612 3292 Otu3 382 9054 467 10925 400 Otu5 6523 1001 8289 1446 6546 Otu9 679 5963 1058 8398 682 Otu23 5025 413 6982 830 4976 Otu6 1213 6380 570 10908 1191 Otu7 2172 922 3836 1202 2278 Otu11 46 2386 70 2458 116 Otu8 3911 2136 3706 1997 4082
- 21. Qiime 2 the first things to know
- 22. Qiime is… … an open-source bioinformatics pipeline for performing microbiome analysis from raw DNA sequencing data. • It’s a suite of wrappers built around third party tools • The wrapping is consistent and makes easier to perform the whole analysis • …but hiding technicalities can lead to misinterpretations
- 23. Metadata file (was Mapping File) A major problem in bioinformatics workflow is access to metadata
- 24. Qiime2 is… • a complete rewrite of Qiime 1 • addresses the most common problems users found (is this good?) • introduce the new concept of artifacts (input/output packages with metadata) • improves the workflow • It’s the recommended version: http://4ngs.com/go/hV
- 25. Qiime2 artifacts qza qzv Qiime2 archive It’s the output format of all Qiime2 programs. It’s a ZIP files with both data and metadata. Qiime2 visualization It’s the output format for plots/charts and tables that the user could desire to inspect. It’s an HTML document (web page) embedded in a “ZIP with metadata”, like qza. http://view.qiime2.org
- 26. Qiime2 command: first example qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path ./reads/ --source-format CasavaOneEightSingleLanePerSampleDirFmt --output-path ./imported_reads.qza Sample01_S10_L001_R1_001.fastq Sample01_S10_L001_R2_001.fastq Sample02_S11_L001_R1_001.fastq Sample02_S11_L001_R2_001.fastq reads imported_reads.qza
- 27. Qiime2 command: first example imported_reads.qza $ unzip -t demux-paired-end.qza Archive: demux-paired-end.qza testing: e2150a41-6d7c-4e13-99ee-36f57ab1f2fb/VERSION OK testing: e2150a41-6d7c-4e13-99ee-36f57ab1f2fb/metadata.yaml OK testing: e2150a41-6d7c-4e13-99ee-36f57ab1f2fb/provenance/VERSION OK testing: e2150a41-6d7c-4e13-99ee-36f57ab1f2fb/provenance/metadata.yaml OK testing: e2150a41-6d7c-4e13-99ee-36f57ab1f2fb/provenance/citations.bib OK testing: e2150a41-6d7c-4e13-99ee-36f57ab1f2fb/provenance/action/action.yaml OK testing: e2150a41-6d7c-4e13-99ee-36f57ab1f2fb/data/64_S64_L001_R2_001.fastq.gz OK testing: e2150a41-6d7c-4e13-99ee-36f57ab1f2fb/data/34_S34_L001_R1_001.fastq.gz OK testing: e2150a41-6d7c-4e13-99ee-36f57ab1f2fb/data/60_S60_L001_R2_001.fastq.gz OK testing: e2150a41-6d7c-4e13-99ee-36f57ab1f2fb/data/19_S19_L001_R1_001.fastq.gz OK
- 30. Installation https://docs.qiime2.org/2018.6/install/ Natively with Miniconda wget https://data.qiime2.org/distro/core/qiime2-2018.6-py35-linux-conda.yml conda env create -n qiime2-2018.6 --file qiime2-2018.6-py35-linux-conda.yml rm qiime2-2018.6-py35-linux-conda.yml Docker docker pull qiime2/core:2018.6 docker run -t -i -v $(pwd):/data qiime2/core:2018.6 qiime
- 31. The “Moving Pictures” tutorial https://docs.qiime2.org/2018.6/tutorials/moving-pictures/
- 33. Alpha diversity Sample’s richness of species
- 34. Beta diversity Measures the change or difference in composition across samples A nice example of the importance of metadata for meaningful outputs!
- 35. Statistical tests For each OTU, can we measure a significant difference in abundance CTRL vs TREATED?