{"id":1436,"date":"2023-02-13T17:35:47","date_gmt":"2023-02-13T08:35:47","guid":{"rendered":"https:\/\/bdsl.jbnu.ac.kr\/blog\/?p=1436"},"modified":"2023-02-13T17:35:48","modified_gmt":"2023-02-13T08:35:48","slug":"taxonomy-annotation-for-amplicon-sequencing","status":"publish","type":"post","link":"https:\/\/bdsl.jbnu.ac.kr\/blog\/taxonomy-annotation-for-amplicon-sequencing\/","title":{"rendered":"Taxonomy annotation for amplicon sequencing"},"content":{"rendered":"\n<h2 class=\"wp-block-heading\">Taxonomy annotation<\/h2>\n\n\n\n<h4 class=\"wp-block-heading\">\uc18c\uac1c<\/h4>\n\n\n\n<p class=\"wp-block-paragraph\">\ubbf8\uc0dd\ubb3c\uc758 \uc720\uc804\uc790\ub294 \uc9c4\ud654 \uacfc\uc815\uc744 \ud1b5\ud574 \ub04a\uc784 \uc5c6\uc774 \ubcc0\ud654\ud55c\ub2e4. \ub3d9\uc2dc\uc5d0, \uc120\uc870\uc758 \uc720\uc804 \uc815\ubcf4 \ud639\uc740 \uc11c\uc5f4\uc740 \ud6c4\ub300\ub85c \uc774\uc5b4\uc9c4\ub2e4. \uc9c4\ud654\uc801\uc73c\ub85c \uadfc\uc5f0 \uad00\uacc4\uc5d0 \uc788\ub294 \ubbf8\uc0dd\ubb3c\uc740 \ube44\uc2b7\ud55c \uc720\uc804\uc790 \uc11c\uc5f4\uc744 \uac00\uc9c0\uace0 \uc788\uc73c\uba70, \uc5ed\uc73c\ub85c \uc720\uc804\uc790 \uc11c\uc5f4\uc744 \uc774\uc6a9\ud558\uc5ec \ubbf8\uc0dd\ubb3c\uc758 \uacc4\ud1b5\uc744 \uc608\uce21\ud560 \uc218 \uc788\ub2e4. rRNA\ub294 \ubaa8\ub4e0 \ubbf8\uc0dd\ubb3c\uc774 \uac00\uc9c4 \uc720\uc804\uc790\ub85c, \uc774 \uc720\uc804\uc790\uc758 \uc11c\uc5f4\uc744 \uc774\uc6a9\ud574 \ubbf8\uc0dd\ubb3c\uc758 taxon(\ubd84\ub958\uad70)\uc744 \uc720\ucd94\ud560 \uc218 \uc788\ub2e4.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\uc774\ub7ec\ud55c \uc608\uce21\uc744 \uc704\ud574\uc11c\ub294 \ubbf8\uc0dd\ubb3c\uc758 (1) rRNA \uc11c\uc5f4\uacfc (2) taxon \uc5d0 \ub300\ud55c \ub370\uc774\ud130\uac00 \ud544\uc694\ud558\ub2e4. \uc774 \ub370\uc774\ud130\ub97c \uc774\uc6a9\ud558\uc5ec rRNA \uc11c\uc5f4\ub85c \ubd80\ud130 taxon\uc744 \uc608\uce21\ud558\ub294 \ubaa8\ub378\uc744 \uad6c\uc131\ud560 \uc218 \uc788\uace0, \uadf8 \ubaa8\ub378\uc744 \uc774\uc6a9\ud558\uc5ec \uc11c\uc5f4 \uc815\ubcf4\ub85c \ubd80\ud130 taxon \uc815\ubcf4\ub97c \uc720\ucd94\ud560 \uc218 \uc788\ub2e4. \uc5ec\uae30\uc11c\ub294 QIIME2 package\ub97c \uc774\uc6a9\ud55c taxon \uc608\uce21 \ubc29\ubc95\uc744 \uc124\uba85\ud55c\ub2e4. \uadf8\ub9ac\uace0, \uc774\uc5b4\uc11c \uc11c\uc5f4\ub85c \ubd80\ud130 taxon\uc744 \uc608\uce21\ud558\ub294 Naive Bayesian \ubaa8\ub378\uc744 \ud559\uc2b5\ud558\ub294 \ubc29\ubc95\uc744 \uc124\uba85\ud55c\ub2e4.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Annotate Taxonomy<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">Taxonomy\ub97c \uc608\uce21\ud560 \uc218 \uc788\ub294 classification model\uc744 \uc774\uc6a9\ud558\uc5ec taxonomy\ub97c \uc608\uce21\ud560 \uc218 \uc788\ub2e4. Classification model\uc740 rRNA\uc758 \uc11c\uc5f4 &#8211; taxonomy \uc30d \uc815\ubcf4\ub97c \uc774\uc6a9\ud558\uc5ec \uad6c\uc131\ud560 \uc218 \uc788\uc73c\uba70, Naive Bayesian model\uc774 \uc88b\uc740 \uc608\uce21 \uc131\ub2a5\uc744 \ubcf4\uc5ec \ub110\ub9ac \ud65c\uc6a9\ub41c\ub2e4. rRNA-taxonomy database\ub85c\ub294 SILVA\ub098 greengene \uac00 \ub110\ub9ac \uc774\uc6a9\ub41c\ub2e4. Classification model\uc774 \ud655\ubcf4\ub418\uba74 \uc544\ub798 Qiime2 \uba85\ub839\uc5b4\ub97c \uc774\uc6a9\ud558\uc5ec taxonomy\ub97c \uc608\uce21\ud55c\ub2e4.<\/p>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">qiime feature-classifier classify-sklearn \\\n        --i-classifier silva-138.1-ssu-nr99-v3v4-classifier.qza \\\n        --i-reads sequences.qza \\\n        --o-classification taxonomy.qza\n<\/code><\/pre>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Construct taxonomy classifier<\/h2>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">QIIME2 package\uc640 RESCRIPt plugin\uc744 \uc774\uc6a9\ud558\uc5ec Naive Bayesian classification model\ub97c \uad6c\uc131\ud558\ub294 \ubc29\ubc95\uc744 \uc124\uba85\ud558\uba70, \ud2b9\ud788, 16S rRNA V3-V4 region\uc744 \ub300\uc0c1\uc73c\ub85c \ud558\ub294 classifier \uad6c\ucd95\ud558\ub294 \ubc29\ubc95\uc744 \uc124\uba85\ud55c\ub2e4. <\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Taxonomy prediction\uc744 \uc704\ud574\uc11c SILVA database\ub97c \uc774\uc6a9\ud55c\ub2e4. SILVA\ub294 \ubbf8\uc0dd\ubb3c\uc758 rRNA \uc11c\uc5f4\uacfc taxon \uc815\ubcf4\ub97c \ud3ec\ud568\ud558\ub294 \ub370\uc774\ud130\ubca0\uc774\uc2a4\uc774\ub2e4 (<a href=\"https:\/\/www.arb-silva.de\/\">SILVA<\/a>). \uc624\ub798\ub3d9\uc548 greengene database\uac00 \ub110\ub9ac \uc774\uc6a9\ub418\uc5c8\uc73c\ub098, \ucd5c\uadfc \uc790\ub8cc\ub97c \ubc18\uc601\ud558\uc9c0 \ubabb\ud558\ub294 \ub2e8\uc810\uc774 \uc788\uc5b4 SILVA database\uac00 \ub110\ub9ac \ud65c\uc6a9\ub41c\ub2e4.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\uc5ec\uae30\uc11c \uc124\uba85\ud558\ub294 \ub0b4\uc6a9\uc740 \uc544\ub798\uc758 \ub9c1\ud06c\uc758 \ub0b4\uc6a9\uc5d0 \ubc14\ud0d5 \ud558\uc5ec \uc791\uc131\ud558\uc600\ub2e4. \uc774 \ub0b4\uc6a9\uc740 Conda\ub97c \ud1b5\ud574 QIIME2 package\ub97c \uc124\uce58\ud558\uc600\uc74c\uc744 \uc804\uc81c\ub85c \ud55c\ub2e4. <\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Based on: <a href=\"https:\/\/forum.qiime2.org\/t\/processing-filtering-and-evaluating-the-silva-database-and-other-reference-sequence-data-with-rescript\/15494\">https:\/\/forum.qiime2.org\/t\/processing-filtering-and-evaluating-the-silva-database-and-other-reference-sequence-data-with-rescript\/15494<\/a><\/p>\n\n\n\n<h4 class=\"wp-block-heading\">Step 1. Install RESCRIPt within QIIME2 environment<\/h4>\n\n\n\n<p class=\"wp-block-paragraph\">SILVA database\ub85c \ubd80\ud130 \ub370\uc774\ud130\ub97c \ubc1b\uace0, \ub370\uc774\ud130\ub97c \uc815\ub9ac\ud558\uace0 \uc704\ud574\uc11c RESCRIPt\ub97c \uc774\uc6a9\ud55c\ub2e4. RESCRIPt\ub294 SILVA\uc640 \uac19\uc740 sequence database\ub97c \uad00\ub9ac\ud558\uae30 \uc704\ud55c Python package\uc774\ub2e4 (<a href=\"https:\/\/github.com\/bokulich-lab\/RESCRIPt\/\">RESCRIPt<\/a>). \uc5ec\uae30\uc11c\ub294 QIIME2 package\ub0b4\uc5d0 RESCRIPt plugin\uc744 \uc124\uce58\ud558\ub294 \uacfc\uc815\uc744 \uc124\uba85\ud55c\ub2e4. <\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\uc544\ub798 \uba85\ub839\uc5b4 \uc2e4\ud589\uc744 \ud1b5\ud574 QIIME2 \ud658\uacbd\uc744 \ud65c\uc131\ud654 \uc2dc\ud0a4\uace0, \ud544\uc694\ud55c library\ub97c \uc124\uce58\ud558\uace0, RESCRIPt package\ub97c \uc124\uce58\ud55c\ub2e4. <\/p>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">conda activate qiime2-2022.8\n\n# Installation of libraries required for RESCRIPt\nconda install -c conda-forge -c bioconda -c qiime2 -c defaults xmltodict\n\n# Installation of RESCRIPt\npip install git+https:\/\/github.com\/bokulich-lab\/RESCRIPt.git<\/code><\/pre>\n\n\n\n<h4 class=\"wp-block-heading\">Step 2. Download SILVA database<\/h4>\n\n\n\n<p class=\"wp-block-paragraph\">SILVA database\ub85c \ubd80\ud130 \ub370\uc774\ud130\ub97c \ubc1b\uae30 \uc704\ud574 \uc544\ub798 \uba85\ub839\uc5b4\ub97c \uc218\ud589\ud55c\ub2e4. Version 138.1\uc744 \ubc1b\uc73c\uba70 rRNA small subunit (SSU)\uc5d0 \ub300\ud55c \uc11c\uc5f4\uc744 \ubc1b\ub294 \uba85\ub839\uc5b4\uc774\uba70, 99% sequence identify\ub85c \uc11c\uc5f4\uc744 clustering\ud55c \uacb0\uacfc\uc774\ub2e4. <\/p>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">qiime rescript get-silva-data \\\n    --p-version '138.1' \\\n    --p-target 'SSURef_NR99' \\\n    --p-include-species-labels \\\n    --o-silva-sequences silva-138.1-ssu-nr99-rna-seqs.qza \\\n    --o-silva-taxonomy silva-138.1-ssu-nr99-tax.qza<\/code><\/pre>\n\n\n\n<h4 class=\"wp-block-heading\">Step 3. Preprocess SILVA database<\/h4>\n\n\n\n<p class=\"wp-block-paragraph\">\ub2e4\uc6b4 \ubc1b\uc740 database\uc758 \ub370\uc774\ud130\uc758 \uc9c8\uc744 \uc810\uac80\ud558\uace0, \ub0ae\uc740 \ud488\uc9c8\uc758 \ub370\uc774\ud130\ub97c \uc0ad\uc81c\ud55c\ub2e4. <\/p>\n\n\n\n<ol class=\"wp-block-list\">\n<li>Convert RNA sequences into DNA sequences<\/li>\n\n\n\n<li>Screen out low-quality sequences<\/li>\n\n\n\n<li>Filter out short sequences<\/li>\n\n\n\n<li>Remove redundant sequence-taxonomy pairs <\/li>\n<\/ol>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\"># 1. Convert RNA sequence into DNA sequence\nqiime rescript reverse-transcribe \\\n    --i-rna-sequences silva-138.1-ssu-nr99-rna-seqs.qza \\\n    --o-dna-sequences silva-138.1-ssu-nr99-seqs.qza\n\n# 2. Culling low-quality sequences\nqiime rescript cull-seqs \\\n    --i-sequences silva-138.1-ssu-nr99-seqs.qza \\\n    --o-clean-sequences silva-138.1-ssu-nr99-seqs-cleaned.qza \\\n    --p-n-jobs 20\n\n# 3. Filter sequence by minimum length\nqiime rescript filter-seqs-length-by-taxon \\\n    --i-sequences silva-138.1-ssu-nr99-seqs-cleaned.qza \\\n    --i-taxonomy silva-138.1-ssu-nr99-tax.qza \\\n    --p-labels Archaea Bacteria Eukaryota \\\n    --p-min-lens 900 1200 1400 \\\n    --o-filtered-seqs silva-138.1-ssu-nr99-seqs-filt.qza \\\n    --o-discarded-seqs silva-138.1-ssu-nr99-seqs-discard.qza\n\n# 4. Remove redundant sequence\nqiime rescript dereplicate \\\n    --i-sequences silva-138.1-ssu-nr99-seqs-filt.qza  \\\n    --i-taxa silva-138.1-ssu-nr99-tax.qza \\\n    --p-mode 'uniq' \\\n    --o-dereplicated-sequences silva-138.1-ssu-nr99-seqs-derep-uniq.qza \\\n    --o-dereplicated-taxa silva-138.1-ssu-nr99-tax-derep-uniq.qza \\\n    --p-threads 20\n\n<\/code><\/pre>\n\n\n\n<h4 class=\"wp-block-heading\">Step 4. Extract V3-V4 region<\/h4>\n\n\n\n<p class=\"wp-block-paragraph\">\uc5ec\uae30\uc11c\ub294 V3-V4 region\uc744 \ub300\uc0c1\uc73c\ub85c amplicon sequencing\ud55c \uacbd\uc6b0\ub97c \uac00\uc815\ud55c\ub2e4. \ub530\ub77c\uc11c classification model\uc744 \uad6c\uc131\ud560 \ub54c\ub3c4 \ud574\ub2f9 \ubd80\uc704\uc758 \uc11c\uc5f4 \ub9cc\uc744 \uc774\uc6a9\ud55c\ub2e4. <\/p>\n\n\n\n<ol class=\"wp-block-list\">\n<li>Extract V3-V4 region<\/li>\n\n\n\n<li>Remove redundant sequence-taxonomy pairs<\/li>\n<\/ol>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\"># 1. Extract V3-V4 region\nqiime feature-classifier extract-reads \\\n    --i-sequences silva-138.1-ssu-nr99-seqs-derep-uniq.qza \\\n    --p-f-primer CCTACGGGNGGCWGCAG \\\n    --p-r-primer GACTACHVGGGTATCTAATCC \\\n    --p-n-jobs 10 \\\n    --p-read-orientation 'forward' \\\n    --o-reads silva-138.1-ssu-nr99-seqs-v3v4.qza\n\nqiime rescript dereplicate \\\n    --i-sequences silva-138.1-ssu-nr99-seqs-v3v4.qza \\\n    --i-taxa silva-138.1-ssu-nr99-tax-derep-uniq.qza \\\n    --p-mode 'uniq' \\\n    --o-dereplicated-sequences silva-138.1-ssu-nr99-seqs-v3v4-uniq.qza \\\n    --o-dereplicated-taxa  silva-138.1-ssu-nr99-tax-v3v4-derep-uniq.qza\n<\/code><\/pre>\n\n\n\n<h4 class=\"wp-block-heading\">Step 5. Build classifier<\/h4>\n\n\n\n<p class=\"wp-block-paragraph\">\ub9c8\uc9c0\ub9c9\uc73c\ub85c classificatio model\uc744 \uad6c\uc131\ud55c\ub2e4. QIIME2 \uc758 Naive Bayesian classifier \ubaa8\ub378\uc744 \uc774\uc6a9\ud558\uba70, SILVE nr99\uc758 V3-V4 \ubd80\uc704 \ub370\uc774\ud130\ub97c \uc774\uc6a9\ud55c\ub2e4.<\/p>\n\n\n\n<pre class=\"wp-block-code\"><code lang=\"bash\" class=\"language-bash\">qiime feature-classifier fit-classifier-naive-bayes \\\n    --i-reference-reads silva-138.1-ssu-nr99-seqs-v3v4-uniq.qza \\\n    --i-reference-taxonomy silva-138.1-ssu-nr99-tax-v3v4-derep-uniq.qza \\\n    --o-classifier silva-138.1-ssu-nr99-v3v4-classifier.qza<\/code><\/pre>\n","protected":false},"excerpt":{"rendered":"<p>Taxonomy annotation \uc18c\uac1c \ubbf8\uc0dd\ubb3c\uc758 \uc720\uc804\uc790\ub294 \uc9c4\ud654 \uacfc\uc815\uc744 \ud1b5\ud574 \ub04a\uc784 \uc5c6\uc774 \ubcc0\ud654\ud55c\ub2e4. \ub3d9\uc2dc\uc5d0, \uc120\uc870\uc758 \uc720\uc804 \uc815\ubcf4 \ud639\uc740 \uc11c\uc5f4\uc740 \ud6c4\ub300\ub85c \uc774\uc5b4\uc9c4\ub2e4. \uc9c4\ud654\uc801\uc73c\ub85c \uadfc\uc5f0 \uad00\uacc4\uc5d0 \uc788\ub294 \ubbf8\uc0dd\ubb3c\uc740 \ube44\uc2b7\ud55c \uc720\uc804\uc790 \uc11c\uc5f4\uc744 \uac00\uc9c0\uace0 \uc788\uc73c\uba70, \uc5ed\uc73c\ub85c \uc720\uc804\uc790 \uc11c\uc5f4\uc744 \uc774\uc6a9\ud558\uc5ec \ubbf8\uc0dd\ubb3c\uc758 \uacc4\ud1b5\uc744 \uc608\uce21\ud560 \uc218 \uc788\ub2e4. rRNA\ub294 \ubaa8\ub4e0 \ubbf8\uc0dd\ubb3c\uc774 \uac00\uc9c4 \uc720\uc804\uc790\ub85c, \uc774 \uc720\uc804\uc790\uc758 \uc11c\uc5f4\uc744 \uc774\uc6a9\ud574 \ubbf8\uc0dd\ubb3c\uc758 taxon(\ubd84\ub958\uad70)\uc744 \uc720\ucd94\ud560 \uc218 \uc788\ub2e4. \uc774\ub7ec\ud55c \uc608\uce21\uc744 \uc704\ud574\uc11c\ub294 [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_uag_custom_page_level_css":"","site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"default","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","ast-disable-related-posts":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""}},"footnotes":"","_members_access_role":[],"_members_access_error":""},"categories":[32],"tags":[],"class_list":["post-1436","post","type-post","status-publish","format-standard","hentry","category-metagenomics"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v28.4 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Taxonomy annotation for amplicon sequencing - Biomedical Data Science Laboratory<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/bdsl.jbnu.ac.kr\/blog\/taxonomy-annotation-for-amplicon-sequencing\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Taxonomy annotation for amplicon sequencing - Biomedical Data Science Laboratory\" \/>\n<meta property=\"og:description\" content=\"Taxonomy annotation \uc18c\uac1c \ubbf8\uc0dd\ubb3c\uc758 \uc720\uc804\uc790\ub294 \uc9c4\ud654 \uacfc\uc815\uc744 \ud1b5\ud574 \ub04a\uc784 \uc5c6\uc774 \ubcc0\ud654\ud55c\ub2e4. \ub3d9\uc2dc\uc5d0, \uc120\uc870\uc758 \uc720\uc804 \uc815\ubcf4 \ud639\uc740 \uc11c\uc5f4\uc740 \ud6c4\ub300\ub85c \uc774\uc5b4\uc9c4\ub2e4. \uc9c4\ud654\uc801\uc73c\ub85c \uadfc\uc5f0 \uad00\uacc4\uc5d0 \uc788\ub294 \ubbf8\uc0dd\ubb3c\uc740 \ube44\uc2b7\ud55c \uc720\uc804\uc790 \uc11c\uc5f4\uc744 \uac00\uc9c0\uace0 \uc788\uc73c\uba70, \uc5ed\uc73c\ub85c \uc720\uc804\uc790 \uc11c\uc5f4\uc744 \uc774\uc6a9\ud558\uc5ec \ubbf8\uc0dd\ubb3c\uc758 \uacc4\ud1b5\uc744 \uc608\uce21\ud560 \uc218 \uc788\ub2e4. rRNA\ub294 \ubaa8\ub4e0 \ubbf8\uc0dd\ubb3c\uc774 \uac00\uc9c4 \uc720\uc804\uc790\ub85c, \uc774 \uc720\uc804\uc790\uc758 \uc11c\uc5f4\uc744 \uc774\uc6a9\ud574 \ubbf8\uc0dd\ubb3c\uc758 taxon(\ubd84\ub958\uad70)\uc744 \uc720\ucd94\ud560 \uc218 \uc788\ub2e4. \uc774\ub7ec\ud55c \uc608\uce21\uc744 \uc704\ud574\uc11c\ub294 [&hellip;]\" \/>\n<meta property=\"og:url\" content=\"https:\/\/bdsl.jbnu.ac.kr\/blog\/taxonomy-annotation-for-amplicon-sequencing\/\" \/>\n<meta property=\"og:site_name\" content=\"Biomedical Data Science Laboratory\" \/>\n<meta property=\"article:published_time\" content=\"2023-02-13T08:35:47+00:00\" \/>\n<meta property=\"article:modified_time\" content=\"2023-02-13T08:35:48+00:00\" \/>\n<meta name=\"author\" content=\"sphong\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:label1\" content=\"Written by\" \/>\n\t<meta name=\"twitter:data1\" content=\"sphong\" \/>\n\t<meta name=\"twitter:label2\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data2\" content=\"3 minutes\" \/>\n<script type=\"application\/ld+json\" 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