Whole metashotgun sequencing analysis Workflow

Quality Control

(FastQC, Trimmomatic, TagCleaner)

Remove host contamination

(bowtie2, samtools, bedtools)

Taxonomic Classification

(kraken, kaiju, mOTU, BLAST)

Genome Assembly

(MEGAHIT, metaSPAdes, MetaVelvet)

Contig binning

(CONCOCT, MetaBAT2, MaxBin2)

16S Sequencing Analysis

Quality control (QC)

reads > 20,000(분변검체), 5,000(그 외 검체)

ampliicon size(bp) > 1,200(Long read), 400(Short read)

16S amplicon sequencing analysis Workflow : QIIME2를 사용하여 FASTQ file을 분석

Demultiplex samples

Sequence Quality check with FastQC

Decontaminate and Trim sequences and cut adapters, primers, etc

ASV Assignment with DADA2

Filter and Trim

Merge Forward + Reverse Reads

Create Sequence Table and Remove Chimeras

Remove Chimeras

Assign Taxonomy to ASVs (Database: NCBI, SILVA, Greengenes를 권장)

Save DADA2 Output for future analysis

Statistical Analysis using R programs, etc.

시퀀싱 플랫폼

Illumina Miseq/Novaseq

(Short read)

Pacbio Sequel2

(Long read)

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