Taxonomy classification is a crucial component of metagenome analysis. Since next-generation sequencing has been the majority method to generate those data, accurately identifying these sequences has become fundamental to research in a microbial community study. 16S amplicon analysis is one of the standard processes to identify metagenome taxonomy. However, only a few databases are available for 16S amplicon analysis, and most popular and extensive databases, such as Silva, have their limitations. To improve classification efficiency, we use the computational method to integrate those popular databases and establish a meta-metagenome database called MetaSquare.

1. Download Metasquare_Kraken2_database from https://hub.docker.com/r/lsbnb/metasquare_db
2. Transfer Metasquare_Kraken2_database to the output_dir
3. Run Porechop to clean up the sequences before classification

$docker run --rm -v $(pwd):/data --name=porechop -it robegan21/porechop
$cd data
$porechop -i input_reads.fastq.gz -o output_reads_1.fastq.gz
$porechop -i output_reads_1.fastq.gz -b output_dir/seq.fastq

4. Install Kraken2
5. Run Kraken2 to Classification at output_dir

kraken2 -db Metasquare_Kraken2_database --threads N --report seq.fasta.report seq.fastq

6. The .report file could be vizualized and analyzed through online-tool Pavian

1. Download combine6DB_V3V4_classifier.qza to target folder from https://hub.docker.com/r/lsbnb/metasquare_db
2. Initialize QIIME2 2019.10 version

P.S. Because our Classifier is trained with QIIME2 2019.10 version, it is necessary to use QIIME2 2019.10 version for the following classification.

$docker run -t -i -v $(pwd):/data quay.io/qiime2/core:2019.10 qiime
$cd data

3. Processed data through QIIME2 pipline
4. Run QIIME2 clasaification

qiime feature-classifier classify-sklearn \
--i-classifier Metasquare_QIIME2_V3V4_classifier.qza \
--i-reads rep-seqs.qza \
--p-n-jobs N \  
--o-classification taxonomy.qza

5. Export QIIME2 visualized file

qiime taxa barplot \
--i-table table.qza \
--i-taxonomy taxonomy.qza \
--m-metadata-file sample-metadata.tsv \
--o-visualization taxa-bar-plots.qzv

6. QZV file could be analysis on website QIIME 2 View

Version history:

First Version: v1.0, 2, June, 2021

Reference:

1.https://github.com/rrwick/Porechop

2.Wood, D.E., Lu, J. & Langmead, B. Improved metagenomic analysis with Kraken 2. Genome Biol 20, 257 (2019). https://doi.org/10.1186/s13059-019-1891-0

3.Bolyen E, et. al 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature Biotechnology 37: 852–857. https://doi.org/10.1038/s41587-019-0209-9

Citation:

1. Chun-Chieh Liao, Po-Ying Fu, Chih-Wei Huang, Chia-Hsien Chuang, Yun Yen, Chung-Yen Lin, Shu-Hwa Chen, MetaSquare: an integrated metadatabase of 16S rRNA gene amplicon for microbiome taxonomic classification, Bioinformatics, Volume 38, Issue 10, May 2022, Pages 2930–2931, https://doi.org/10.1093/bioinformatics/btac184