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(kraken-reads)=
# Taxonomic classification of reads
In this section we will focus on the taxonomic classification of shotgun metagenomic reads using two different tools: Kraken 2 and Kaiju. 
We will use the data obtained in the [data retrieval section](../00_data_retrieval.md).

## Approach 1: Kraken 2
Before we can use Kraken 2, we need to build or download a database. We will use the `build-kraken-db` action to fetch the PlusPF database 
from [here](https://benlangmead.github.io/aws-indexes/k2) - this database covers RefSeq sequences for archaea, bacteria, viral, plasmid, 
human, UniVec_Core, protozoa and fungi.
```{code-cell}
mosh annotate build-kraken-db \
    --p-collection pluspf \
    --o-kraken2-db ./cache:kraken2_db \
    --o-bracken-db ./cache:bracken_db \
```

We can now use the `classify-kraken2` command to run Kraken2 using the paired-end reads as a query and the PlusPF database retrieved in the previous step:
```{code-cell}
mosh annotate classify-kraken2 \
    --i-seqs ./cache:reads_filtered \
    --i-db ./cache:kraken2_db \
    --p-threads 72 \
    --p-confidence 0.5 \
    --p-memory-mapping False \
    --p-report-minimizer-data \
    --o-reports ./cache:kraken_reports_reads \
    --o-outputs ./cache:kraken_hits_reads
    --verbose
```

```{seealso}
[Bracken](https://ccb.jhu.edu/software/bracken/) is a related tool that additionally estimates relative abundances of species or genera to adjust for
the genome size the organisms from which each read originated. In order to use this tool we need the Bracken database that was fetched in the first step.
```

```{code-cell}
mosh annotate estimate-bracken \
    --i-kraken2-reports ./cache:kraken_reports_reads \
    --i-db ./cache:bracken_db \
    --p-threshold 5 \
    --p-read-len 150 \
    --o-taxonomy ./cache:bracken_taxonomy \
    --o-table ./cache:bracken_ft \
    --o-reports ./cache:bracken_reports
```

To remove the unclassified read fraction we can use the `filter-table` action from the `q2-taxa` QIIME 2 plugin:
```{code-cell}
mosh taxa filter-table \
    --i-table ./cache:bracken_ft \
    --i-taxonomy ./cache:bracken_taxonomy \
    --p-exclude Unclassified \
    --o-filtered-table ./cache:bracken_ft_filtered
```

## Approach 2: Kaiju
Similarly to Kraken 2, Kaiju requires a reference database to perform taxonomic classification. We will use the `fetch-kaiju-db` 
action to download the [nr_euk](https://bioinformatics-centre.github.io/kaiju/downloads.html) database that includes both 
prokaryotes and eukaryotes (more info on the taxa [here](https://github.com/bioinformatics-centre/kaiju/blob/master/util/kaiju-taxonlistEuk.tsv)).
```{code-cell}
mosh annotate fetch-kaiju-db \
    --p-database-type nr_euk \
    --o-db ./cache:kaiju_nr_euk
```

We run Kaiju with the confidence of 0.1 using the paired-end reads as a query and the database artifact that was generated in the previous step:
```{code-cell}
mosh annotate classify-kaiju \
    --i-seqs ./cache:reads_paired \
    --i-db ./cache:kaiju_nr_euk \
    --p-z 16 \
    --p-c 0.1 \
    --o-taxonomy ./cache:kaiju_taxonomy \
    --o-abundances ./cache:kaiju_ft
```

Finally, we filter the table to remove the unclassified reads:
```{code-cell}
mosh taxa filter-table \
    --i-table ./cache:kaiju_ft \
    --i-taxonomy ./cache:kaiju_taxonomy \
    --p-exclude unclassified,belong,cannot \
    --o-filtered-table ./cache:kaiju_ft_filtered
```

## Visualization
You can try to generate a taxa bar plot with either of these results now! We will continue with the Kaiju results - to
generate a taxa bar plot, you can run:
```{code-cell}
mosh taxa barplot \
    --i-table ./cache:kaiju_ft_filtered \
    --i-taxonomy ./cache:kaiju_taxonomy \
    --m-metadata-file ./metadata.tsv \
    --o-visualization ./results/kaiju_barplot.qzv
```
Your visualization should look similar to [this one](https://view.qiime2.org/visualization/?src=https://raw.githubusercontent.com/bokulich-lab/moshpit-docs/main/moshpit_docs/data/kaiju-filtered.qzv).