研究方法：ZOTU 与 OTU 和 ASV 的比较

特性	OTU（97% 聚类）	ZOTU（UNOISE3）	ASV（DADA2/Deblur）
定义方法	相似性聚类（阈值依赖）	100% 相似性，基于去噪	精确去噪，单碱基分辨率
分辨率	较低，可能合并相似物种	高，可区分单碱基差异	高，可区分单碱基差异
嵌合体处理	可能保留部分嵌合体	自动检测并移除嵌合体	自动检测并移除嵌合体
跨项目可比性	受阈值和工具影响	高，基于精确序列定义	高，基于精确序列定义
计算需求	较低	中等	较高
常用工具	VSEARCH、USEARCH、Mothur	UNOISE3（USEARCH）	DADA2、Deblur、QIIME 2
去噪方法	不去噪（基于聚类）	去噪（规则和经验方法）	去噪（统计模型）

Operational Taxonomic Units:OTUs

Amplicon Sequence Variants:ASVs, also known as Exact Sequence Variants (ESVs)

zero-radius OTUs:ZOTUs

bioinformatics pipeline

Li Z, Zhao W, Jiang Y, et al. New insights into biologic interpretation of bioinformatic pipelines for fish eDNA metabarcoding: A case study in Pearl River estuary[J]. Journal of Environmental Management, 2024, 368: 122136.

Hakimzadeh A, Abdala Asbun A, Albanese D, et al. A pile of pipelines: An overview of the bioinformatics software for metabarcoding data analyses[J]. Molecular Ecology Resources, 2024, 24(5): e13847.

ASV

MiFish pipeline

https://mitofish.aori.u-tokyo.ac.jp/mifish/help/

https://scikit.bio

conda install -c conda-forge scikit-bio

dada2_custom_fungal

https://github.com/thierroll/dada2_custom_fungal

nemabiome(dada2_ITS)

https://www.nemabiome.ca

ITS：https://www.nemabiome.ca/sequencing

16S rDNA V3-V4 amplicon sequencing analysis using dada2, phyloseq, LEfSe, picrust2 and other tools.16S rDNA V3-V4 amplicon sequencing analysis using dada2, phyloseq, LEfSe, picrust2 and other tools.

https://github.com/ycl6/16S-rDNA-V3-V4/

PICRUSt2

(Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) is a software for predicting functional abundances based only on marker gene sequences.

https://benjjneb.github.io/dada2/training.html

IDTAXA is part of the DECIPHER package

https://github.com/billzt/MiFish/tree/main

Build docker images

cd Docker/
docker build -t edna ./

Build database

16s rRNA demo data

These fastq files were generated by 2x250 Illumina Miseq amplicon sequencing of the V4 region of the 16S rRNA gene from gut samples collected longitudinally from a mouse post-weaning.

Download link:https://mothur.s3.us-east-2.amazonaws.com/wiki/miseqsopdata.zip