视频推荐

请先在B站观看视频，毕竟看视频比看书学习快多了

1.B站检索（第一天：illumina、PacBio、Nanopore测序原理）

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2.B站检索（Nanopore平台宏基因组测序的优点）

视频推荐2.png

标准流程

本流程复现自Nature communication的文章“Short- and long-read metagenomics expand individualized structural variations in gut microbiomes”

数据来源 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA820119
脚本来源 https://github.com/chen318liang/Gut-Metagenome-Pipeline-Based-on-Nanopore-Sequencing

数据下载

按图示下载数据

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也可采用sra-toolkit批量下载方法，具体见文
https://www.jianshu.com/p/6f8f9a6d1a56

SRA数据转fastq

使用fasterq-dump转换数据

fasterq-dump  SRR5318040 -t . -e 24;
###-e 24 是24线程

脚本安装

本分析涉及到的分析软件很多，不同的软件安装方法差异很大，以下是安装所需软件的下载源或安装源。
提前熟悉linux的基本命令，awk, sed，grep等等

git 安装

OPERA-MS v0.9.0 mumandco mcaller guppy Prokka CD-hit iqtree2

conda 安装

IGV seqkit dRep gtdbtk samtools MAFFT CAT blast Nanopolish Canu Flye minimap2 MetaWRAP prokka salmon

pip install drep clustalw Emboss ViennaRNA

网页安装

rnammer
https://services.healthtech.dtu.dk/software.php

网页或UI软件可视化

ont-tombo
nanodisco
crsprdetect
prophagehunter
itol

单脚本

CRISPRDetect (https://github.com/davidchyou/CRISPRDetect_2.4)
crisprdetectparser.py (https://github.com/hwalinga/crisprdetect-parser)
~/miniconda3/envs/metawrap/bin/metawrap-scripts/split_salmon_out_into_bins.py

分析流程

测序质量评估

注意设置好对应变量，注意提前测试所需软件及脚本是否可用

#!/bin/bash

threads="20"
assembly="/data/chenliang/Zymo_Community_Standards_data_assembly/spades_hybird_assembly/Zymo-GridION-EVEN_spades_hybird_assembly/scaffolds.fasta"
out="quant_bins"
reads_1="/data/chenliang/MC.Hiseq/MC_1.fastq"
reads_2="/data/chenliang/MC.Hiseq/MC_2.fastq"
tmp=${reads_1##*/}
sample=${tmp%_*}
bin_folder="bin_refinement/metawrap_70_10_bins/"


salmon index -p $threads -t $assembly -i ${out}/assembly_index
salmon quant -i ${out}/assembly_index --libType IU -1 $reads_1 -2 $reads_2 -o ${out}/alignment_files/${sample}.quant --meta -p $threads

home=$(pwd)
cd ${out}/alignment_files/
/software_users/chenliang/miniconda3/envs/metawrap/bin/metawrap-scripts/summarize_salmon_files.py
cd $home
mkdir ${out}/quant_files
#for f in $(ls ${out}/alignment_files/ | grep .quant.counts); do mv ${out}/alignment_files/$f ${out}/quant_files/; done

#n=$(ls ${out}/quant_files/ | grep counts | wc -l)
/software_users/chenliang/miniconda3/envs/metawrap/bin/metawrap-scripts/split_salmon_out_into_bins.py ${out}/quant_files/ $bin_folder $assembly > ${out}/bin_abundance_table.tab

基因组组装

使用的5种策略,Canu, Flye, OPERA-MS, Spades, MetaSPAdes.

使用fastANI计算ANI时需要用到参考基因组，请提前下载
https://zenodo.org/record/3935737#.Y1rh37ZBxD8

详细脚本见
https://github.com/chen318liang/Gut-Metagenome-Pipeline-Based-on-Nanopore-Sequencing/blob/main/scripts/0_Zymo_community_standard_data_assembly.sh

突变分析

这一步没啥特别的，注意提前测试软件和脚本就行。观察脚本，提前将文件置于对应位置的话能省不少事情。
minimap2 samtools make_IGV_snapshots.py emapper.py prokka

详细脚本见
https://github.com/chen318liang/Gut-Metagenome-Pipeline-Based-on-Nanopore-Sequencing/blob/main/scripts/2_Gut_metagenome_SV_analysis.sh

预测原噬菌体、去除冗余、基因注释和构建进化树

这步用到了CD-hit需要编译安装，而iqtree2可在github下载，用于化系统发育树。

#####################
Part III. Prediction of prophage, redundancy removal, gene annotation, and construction of evolutionary trees
###################
# prophage dientification using the machine-learning-based tool ProphageHunter
# Webserver address
https://pro-hunter.genomics.cn/

# Candidate propages clustering by CDhit
CDhit-est -i Candidate_propahge.fasta -c 0.95 -o Candidate_propahge_clusted.fasta

# Viral genomic CDS prediction using multiphate2 (https://github.com/carolzhou/multiPhATE2)
#Set up the configuration file as required and then run cmd as follow
python3 multiPhate.py multiPhate.config


# Create phylogenetic tree using iqtree2 (https://github.com/iqtree/iqtree2)
#Join_MCP_TLS.faa is joined protein sequence with major capsid protein and terminal large subunit 
iqtree2 -s Join_MCP_TLS.faa -m MFP -B 1000 --bnni -T 40

从所有样品的contigs预测CRISPR区

CRISPRDetect.pl安装比较繁琐，现在github下载安装，之后要安装依赖环境，根据对应报错提示安装就行，我花了不少时间。crisprdetectparser.py直接在github下载就行。

#!/usr/bin/bash
## CRISPRDetect (https://github.com/davidchyou/CRISPRDetect_2.4)
## crisprdetectparser.py (https://github.com/hwalinga/crisprdetect-parser)

path="/data/nano_meta_ref/result/spades/hybird_temp/spades"

perl CRISPRDetect.pl -f $path/contigs.fasta -o CRISPRDetect_result \
-check_direction 0 -array_quality_score_cutoff 3 -T 20

python crisprdetectparser.py --spacers-directory spacer_dir --spacers-extension fna CRISPRDetect_result > metadata.tsv

甲基化分析

这一步要用到原始fast5格式，暂时不用做。

文献扩展

1.Complete, closed bacterial genomes from microbiomes using nanopore sequencing.

DOI: 10.1038/s41587-020-0422-6

2. Integrating metagenomic binning with flux balance analysis to unravel syntrophies in anaerobic CO2 methanation.

DOI: 10.1186/s40168-022-01311-1

3. Long-Read-Resolved, Ecosystem-Wide Exploration of Nucleotide and Structural Microdiversity of Lake Bacterioplankton Genomes.

DOI: 10.1128/msystems.00433-22

简书优秀文章推荐

1.Nanopore测序笔记

https://www.jianshu.com/p/5d011720bc10

2.原核生物基因组三代数据（pacbio/nanopore）组装

https://www.jianshu.com/p/2e9d9feed61c

3.centos7安装perl-XML-Simple（可能用到）

https://www.jianshu.com/p/ec7d8f35b095

4.fasterq-dump 人多力量大（SRA数据转换教程）

https://www.jianshu.com/p/e9f6e16e2c8a

5.使用libmamba库来加速canda环境的解决（加速conda软件安装）

https://www.jianshu.com/p/904d5ed1d841

6.CD-hit安装及使用

https://www.jianshu.com/p/4e217eba4e96

7.perl 模块安装与使用（中间有需要装模块的步骤）

https://www.jianshu.com/p/17ed0e5ff031