{"id":1453,"date":"2023-02-14T02:38:26","date_gmt":"2023-02-13T17:38:26","guid":{"rendered":"https:\/\/bdsl.jbnu.ac.kr\/blog\/?p=1453"},"modified":"2023-02-21T17:12:47","modified_gmt":"2023-02-21T08:12:47","slug":"generate-microbial-feature-table","status":"publish","type":"post","link":"https:\/\/bdsl.jbnu.ac.kr\/blog\/generate-microbial-feature-table\/","title":{"rendered":"Generate microbial feature table"},"content":{"rendered":"\n<h2 class=\"wp-block-heading\">Manifest file <\/h2>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">sample-id     forward-absolute-filepath       reverse-absolute-filepath\nsample-1      $PWD\/some\/filepath\/sample0_R1.fastq.gz  $PWD\/some\/filepath\/sample1_R2.fastq.gz\nsample-2      $PWD\/some\/filepath\/sample2_R1.fastq.gz  $PWD\/some\/filepath\/sample2_R2.fastq.gz\n<\/code><\/pre>\n\n\n\n<p class=\"wp-block-paragraph\">based on: https:\/\/docs.qiime2.org\/2022.2\/tutorials\/importing\/<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Collect files into QIIME2 artifact<\/h2>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">conda activate qiime2_2022.2\nqiime tools import \\\n  --type 'SampleData[PairedEndSequencesWithQuality]' \\\n  --input-path manifest.tsv \\\n  --output-path paired-end-demux.qza \\\n  --input-format PairedEndFastqManifestPhred33V2 <\/code><\/pre>\n\n\n\n<h2 class=\"wp-block-heading\">Trim primers <\/h2>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"python\" class=\"language-python\">qiime cutadapt trim-paired \\\n  --i-demultiplexed-sequences paired-end-demux.qza \\\n  --p-cores 20 \\\n  --p-front-f CCTACGGGNGGCWGCAG \\\n  --p-front-r GACTACHVGGGTATCTAATCC \\\n  --p-minimum-length 100 \\\n  --p-discard-untrimmed \\\n  --o-trimmed-sequences paired-end-demux-trimmed.qza\n<\/code><\/pre>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Denoising with DADA2<\/h2>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">conda activate qiime2_2022.2\nqiime dada2 denoise-paired \\\n  --i-demultiplexed-seqs paired-end-demux-trimmed.qza \\\n  --p-trunc-len-f 270 \\\n  --p-trunc-len-r 220 \\\n  --o-representative-sequences representative_sequences.qza \\\n  --p-n-threads 20 \\\n  --o-table table.qza \\\n  --output-dir .\/dada2_output<\/code><\/pre>\n\n\n\n<h2 class=\"wp-block-heading\">Build phylogenetic trees<\/h2>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">conda activate qiime2_2022.2\nqiime alignment mafft   --i-sequences representative_sequences.qz --o-alignment aligned.qza --p-n-threads 20\nqiime alignment mask --i-alignment aligned.qza --o-masked-alignment masked.qza\nqiime phylogeny fasttree --i-alignment masked.qza --o-tree unrooted-tree.qza --p-n-threads 20\nqiime phylogeny midpoint-root --i-tree unrooted-tree.qza --o-rooted-tree rooted-tree.qza\n<\/code><\/pre>\n\n\n\n<h2 class=\"wp-block-heading\">Rarefaction<\/h2>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">conda activate qiime2_2022.2\nqiime feature-table rarefy --i-table table.qza \\\n  --p-sampling-depth 10000 \\\n  --o-rarefied-table feature-table_rarefied.qza \\\n  --output-dir output_rarefaction\n<\/code><\/pre>\n\n\n\n<h2 class=\"wp-block-heading\">Alpha-diversity<\/h2>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">conda activate qiime2_2022.2\nqiime diversity alpha --i-table feature-table_rarefied.qza --p-metric &lt;MEASURE&gt; --o-alpha-diversity &lt;MEASURE&gt;_vector.qza\n<\/code><\/pre>\n\n\n\n<h2 class=\"wp-block-heading\">Beta-diversity<\/h2>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">conda activate qiime2_2022.2\nqiime diversity beta-phylogenetic --i-table feature-table_rarefied.qza --i-phylogeny rooted-tree.qza --p-metric &lt;MEASURE&gt; --o-distance-matrix &lt;MEASURE&gt;_dmat.qza\n<\/code><\/pre>\n\n\n\n<h2 class=\"wp-block-heading\">Principal coordinate analysis<\/h2>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">conda activate qiime2_2022.2\nqiime diversity pcoa --i-distance-matrix &lt;MEASURE&gt;_dmat.qza --o-pcoa &lt;MEASURE&gt;_pcoa_result.qza\n<\/code><\/pre>\n\n\n\n<h2 class=\"wp-block-heading\">Taxonomy annotation<\/h2>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"python\" class=\"language-python\">conda activate qiime2_2022.2\nqiime feature-classifier classify-sklearn --i-classifier &lt;ClassifierModel&gt;.qza --i-reads representative_sequences.qza --o-classification taxonomy.qza --p-n-jobs 20\n<\/code><\/pre>\n\n\n\n<h2 class=\"wp-block-heading\">LEfSe analysis<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">Input: tab-delimited numeric features, class vector, (optionally the subclass and subject vectors)<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li>features: read counts or abundance floating-point values <\/li>\n\n\n\n<li>the first field: name of the feature <\/li>\n\n\n\n<li>Class, subclass and subject vectors have a name (the first field) and a list of non-numerical strings <\/li>\n<\/ul>\n\n\n\n<figure class=\"wp-block-embed is-type-wp-embed is-provider-the-huttenhower-lab wp-block-embed-the-huttenhower-lab\"><div class=\"wp-block-embed__wrapper\">\nhttps:\/\/huttenhower.sph.harvard.edu\/lefse\/\n<\/div><\/figure>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">conda create -n lefse\nconda install -c bioconda lefse\n<\/code><\/pre>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">conda activate lefse\nrun_lefse.py\nplot_res.py hmp_aerobiosis_small.res hmp_aerobiosis_small.png\nplot_cladogram.py hmp_aerobiosis_small.res hmp_aerobiosis_small.cladogram.png --format png\n<\/code><\/pre>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">docker run -it biobakery\/lefse bash<\/code><\/pre>\n","protected":false},"excerpt":{"rendered":"<p>Manifest file based on: https:\/\/docs.qiime2.org\/2022.2\/tutorials\/importing\/ Collect files into QIIME2 artifact Trim primers Denoising with DADA2 Build phylogenetic trees Rarefaction Alpha-diversity Beta-diversity Principal coordinate analysis Taxonomy annotation LEfSe analysis Input: tab-delimited numeric features, class vector, (optionally the subclass and subject 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