Additional Information:

I am planning to conduct the downstream analysis based on the methods described in the following paper: DOI: 10.1186/s42523-023-00229-9.

https://github.com/smdabdoub/kraken-biom

follow these steps:

1. Install the necessary tools: Ensure that you have the required dependencies installed, including Kraken2, Krona, and biom-format. You can refer to the respective documentation for installation instructions.

2. Generate Krona report: Run the following command to generate a Krona report from your Kraken2 output:

```

ktImportTaxonomy -q 1 kraken-output01.txt kraken-output02.txt ... kraken-output12.txt -o krona_report.html

```

This command will generate a Krona HTML report (`krona_report.html`) representing the taxonomic composition of your samples.

3. Convert Krona report to biom format: Next, use the following command to convert the Krona report to the biom format:

```

ktImportText -n Krona_Report krona_report.html -o biom_file.biom

```

This command will generate the biom file (`biom_file.biom`) in the desired format.

4. Import biom file into QIIME2: You can then import the biom file into QIIME2 for downstream analysis. Use the appropriate QIIME2 command to import the biom file based on your analysis requirements.

It's important to note that the biom format is commonly used in QIIME1, but QIIME2 generally uses the newer QZA or QZV formats. If you'reIf you're using QIIME2 for downstream analysis, it is recommended to convert the Kraken2 output into the QIIME2-compatible format (QZA or QZV) instead of the biom format. You can do this using the QIIME2 command-line interface or Python API. Here's a general outline of the steps:

1. Install QIIME2: Follow the installation instructions provided by QIIME2 (https://docs.qiime2.org/2021.8/install/) to set up QIIME2 on your system.

2. Convert Kraken2 output to QIIME2 format: Use the `qiime tools import` command to convert the Kraken2 output to QIIME2-compatible format. Here's an example command:

```

qiime tools import \

--input-path kraken-output01.txt \

--output-path kraken-output01.qza \

--type 'FeatureData[Taxonomy]'

```

Repeat this step for each Kraken2 output file to convert them all to the QIIME2 format.

3. Perform diversity analysis: Once you have the QZA files, you can perform diversity analysis using various QIIME2 plugins. For example, you can use the `qiime diversity` plugin to calculate alpha and beta diversity metrics. Refer to the QIIME2 documentation and tutorials for more information on available plugins and analysis options.

Regarding the identification of variable species, Kraken2 provides taxonomic assignments for each sequence read. You can analyze the Kraken2 output directly to identify the taxonomic groups that show differential abundance between your diseased cohort and control group. This can be done using statistical analysis tools such as DESeq2, edgeR, or LEfSe. These tools can help you identify taxonomic groups that are significantly differentially abundant between the two groups.

Alternatively, if you decide to convert your data to OTU or ASV format for downstream analysis, you can use tools like QIIME2, mothur, or DADA2 to perform OTU or ASV clustering. These tools provide options for denoising, quality filtering, chimera removal, and clustering to generate OTU or ASV tables that can be further analyzed for diversity analysis, differential abundance testing, and other downstream analyses.

Hope it helps