The document discusses metabarcoding analyses and taxonomic classification using QIIME2. It covers the QIIME2 workflow for taxonomic classification which includes alignment-based and machine learning approaches. The least common ancestor (LCA) approach is used for taxonomic assignment rather than simple BLAST to account for the lowest common taxon that contains all the taxa identified. Taxonomic barplots can be generated in QIIME2 to visualize classification results.

1. Metabarcoding analyses using
WORKSHOP
June 28th and 29th 2021

2. Taxonomic classification
QIIME2 workflow
https://docs.qiime2.org/2021.4/tutorials/overview/

3. Taxonomic databases
QIIME2 workflow
https://docs.qiime2.org/2021.4/tutorials/overview/
CP013078.2406498.2408039
D_0__Bacteria;
D_1__Proteobacteria;
D_2__Gammaproteobacteria;
D_3__Burkholderiales;
D_4__Alcaligenaceae;
D_5__Bordetella;
D_6__Bordetella pertussis
Reference 7-level taxonomy:
>CP013078.2406498.2408039
AGAGATTGAACTGAAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGGATGCTTTACACATGCAAGT
CGGACGGCAGCACGGGCTTCGGCCTGGTGGCGAGTGGCGAACGGGTGAGTAATGTATCGGAACGTGC
CCAGTAGCGGGGGATAACTACGCGAAAGCGTGGCTAATACCGCATACGCCCTACGGGGGAAAGCGGG
GGACCTTCGGGCCTCGCACTATTGGAGCGGCCGATATCGGATTAGCTAGTTGGTGGGGTAACGGCCTAC
CAAGGCGACGATCCGTAGCTGGTTTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGA
CTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATCCAGCCATCCCGCGTG
TGCGATGAAGGCCTTCGGGTTGTAAAGCACTTTTGGCAGGAAAGAAACGGCACGGGCTAATATCCTGT
Reference 7-level sequence:

4. Taxonomic classifier
QIIME2 workflow
>CP013078.2406498.2408039
AGAGATTGAACTGAAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGG
ATGCTTTACACATGCAAGTCGGACGGCAGCACGGGCTTCGGCCTGGTGGC
GAGTGGCGAACGGGTGAGTAATGTATCGGAACGTGCCCAGTAGCGGGGG
ATAACTACGCGAAAGCGTGGCTAATACCGCATACGCCCTACGGGGGAAAG
CGGGGGACCTTCGGGCCTCGCACTATTGGAGCGGCCGATATCGGATTAGC
TAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATCCGTAGCTGGTTTGA
GAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGG
GAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATCCAGCC
ATCCCGCGTGTGCGATGAAGGCCTTCGGGTTGTAAAGCACTTTTGGCAGG
AAAGAAACGGCACGGGCTAATATCCTGTGCAACTGACGGTACCTGCAGAAT
AAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAA
GCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTCGGAAAG
AAAGATGTGAAATCCCAGGGCTTAACCTTGGAACTGCATTTTTAACTACCG
GGCTAGAGTGTGTCAGAGGGAGGTGGAATTCCGCGTGTAGCAGTGAAAT
GCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAGCCTCCTGGGATA
ACACTGACGCTCATGCACGAAAGTGTGGGGAGCAAACAGGATTAGATACC
CTGGTAGTCCACGCCCTAAACGATGTCAACTAGCTGTTGGGGCCTTCGGG
CCTTGGTAGCGCAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGT
CGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGG
ATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACAT
GTCTGGAATCCCGAAGAGATTTGGGAGTGCTCGCAAGAGAACCGGAACA
CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAG
TCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACGAAAGGGCACTCTA
ATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTC
CTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGGACAG
AGGGTTGCCAACCCGCGAGGGGGAGCCAATCCCAGAAACCCGGTCGTAG
TCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAAT
CGCGGATCAGCATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCG
CCCGTCACACCATGGGAGTGGGTTTTACCAGAAGTAGTTAGCCTAACCGC
AAGGGGGGCGATTACCACGGTAGGATTCATGACTGGGGTGAAGTCGTAA

5. Taxonomic classifier
QIIME2 workflow
>CP013078.2406498.2408039
AGAGATTGAACTGAAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGG
ATGCTTTACACATGCAAGTCGGACGGCAGCACGGGCTTCGGCCTGGTGGC
GAGTGGCGAACGGGTGAGTAATGTATCGGAACGTGCCCAGTAGCGGGGG
ATAACTACGCGAAAGCGTGGCTAATACCGCATACGCCCTACGGGGGAAAG
CGGGGGACCTTCGGGCCTCGCACTATTGGAGCGGCCGATATCGGATTAGC
TAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATCCGTAGCTGGTTTGA
GAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGG
GAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATCCAGCC
ATCCCGCGTGTGCGATGAAGGCCTTCGGGTTGTAAAGCACTTTTGGCAGG
AAAGAAACGGCACGGGCTAATATCCTGTGCAACTGACGGTACCTGCAGAAT
AAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAA
GCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTCGGAAAG
AAAGATGTGAAATCCCAGGGCTTAACCTTGGAACTGCATTTTTAACTACCG
GGCTAGAGTGTGTCAGAGGGAGGTGGAATTCCGCGTGTAGCAGTGAAAT
GCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAGCCTCCTGGGATA
ACACTGACGCTCATGCACGAAAGTGTGGGGAGCAAACAGGATTAGATACC
CTGGTAGTCCACGCCCTAAACGATGTCAACTAGCTGTTGGGGCCTTCGGG
CCTTGGTAGCGCAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGT
CGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGG
ATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACAT
GTCTGGAATCCCGAAGAGATTTGGGAGTGCTCGCAAGAGAACCGGAACA
CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAG
TCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACGAAAGGGCACTCTA
ATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTC
CTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGGACAG
AGGGTTGCCAACCCGCGAGGGGGAGCCAATCCCAGAAACCCGGTCGTAG
TCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAAT
CGCGGATCAGCATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCG
CCCGTCACACCATGGGAGTGGGTTTTACCAGAAGTAGTTAGCCTAACCGC
AAGGGGGGCGATTACCACGGTAGGATTCATGACTGGGGTGAAGTCGTAA

6. Taxonomic classifier
QIIME2 workflow
>CP013078.2406498.2408039
AGAGATTGAACTGAAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGG
ATGCTTTACACATGCAAGTCGGACGGCAGCACGGGCTTCGGCCTGGTGGC
GAGTGGCGAACGGGTGAGTAATGTATCGGAACGTGCCCAGTAGCGGGGG
ATAACTACGCGAAAGCGTGGCTAATACCGCATACGCCCTACGGGGGAAAG
CGGGGGACCTTCGGGCCTCGCACTATTGGAGCGGCCGATATCGGATTAGC
TAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATCCGTAGCTGGTTTGA
GAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGG
GAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATCCAGCC
ATCCCGCGTGTGCGATGAAGGCCTTCGGGTTGTAAAGCACTTTTGGCAGG
AAAGAAACGGCACGGGCTAATATCCTGTGCAACTGACGGTACCTGCAGAAT
AAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAA
GCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTCGGAAAG
AAAGATGTGAAATCCCAGGGCTTAACCTTGGAACTGCATTTTTAACTACCG
GGCTAGAGTGTGTCAGAGGGAGGTGGAATTCCGCGTGTAGCAGTGAAAT
GCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAGCCTCCTGGGATA
ACACTGACGCTCATGCACGAAAGTGTGGGGAGCAAACAGGATTAGATACC
CTGGTAGTCCACGCCCTAAACGATGTCAACTAGCTGTTGGGGCCTTCGGG
CCTTGGTAGCGCAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGT
CGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGG
ATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACAT
GTCTGGAATCCCGAAGAGATTTGGGAGTGCTCGCAAGAGAACCGGAACA
CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAG
TCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACGAAAGGGCACTCTA
ATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTC
CTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGGACAG
AGGGTTGCCAACCCGCGAGGGGGAGCCAATCCCAGAAACCCGGTCGTAG
TCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAAT
CGCGGATCAGCATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCG
CCCGTCACACCATGGGAGTGGGTTTTACCAGAAGTAGTTAGCCTAACCGC
AAGGGGGGCGATTACCACGGTAGGATTCATGACTGGGGTGAAGTCGTAA

7. Taxonomic classification
QIIME2 workflow
https://docs.qiime2.org/2021.4/tutorials/overview/
Alignment based
Naive-Bayes machine learning

8. Least common ancestor approach (LCA)
Why not use a simple BLAST?
* ASV
* * *
Best BLAST G G or H D or E or F or G or H
LCA G C A

9. Taxonomic barplots
QIIME2 workflow
https://docs.qiime2.org/2021.4/tutorials/overview/