視頻推薦
請先在B站觀看視頻，畢竟看視頻比看書學(xué)習(xí)快多了
1.B站檢索（第一天：illumina、PacBio、Nanopore測序原理）
2.B站檢索（Nanopore平臺宏基因組測序的優(yōu)點）
標(biāo)準(zhǔn)流程
本流程復(fù)現(xiàn)自Nature communication的文章“Short- and long-read metagenomics expand individualized structural variations in gut microbiomes”
數(shù)據(jù)來源 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA820119
腳本來源 https://github.com/chen318liang/Gut-Metagenome-Pipeline-Based-on-Nanopore-Sequencing
數(shù)據(jù)下載
按圖示下載數(shù)據(jù)
也可采用sra-toolkit批量下載方法，具體見文
http://m.itdecent.cn/p/6f8f9a6d1a56
SRA數(shù)據(jù)轉(zhuǎn)fastq
使用fasterq-dump轉(zhuǎn)換數(shù)據(jù)
fasterq-dump SRR5318040 -t . -e 24;
###-e 24 是24線程
腳本安裝
本分析涉及到的分析軟件很多，不同的軟件安裝方法差異很大，以下是安裝所需軟件的下載源或安裝源。
提前熟悉linux的基本命令，awk, sed，grep等等
git 安裝
OPERA-MS v0.9.0 mumandco mcaller guppy Prokka CD-hit iqtree2
conda 安裝
IGV seqkit dRep gtdbtk samtools MAFFT CAT blast Nanopolish Canu Flye minimap2 MetaWRAP prokka salmon
pip install drep clustalw Emboss ViennaRNA
網(wǎng)頁安裝
rnammer
https://services.healthtech.dtu.dk/software.php
網(wǎng)頁或UI軟件可視化
ont-tombo
nanodisco
crsprdetect
prophagehunter
itol
單腳本
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
測序質(zhì)量評估
注意設(shè)置好對應(yīng)變量，注意提前測試所需軟件及腳本是否可用
#!/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
基因組組裝
使用的5種策略,Canu, Flye, OPERA-MS, Spades, MetaSPAdes.
使用fastANI計算ANI時需要用到參考基因組，請?zhí)崆跋螺d
https://zenodo.org/record/3935737#.Y1rh37ZBxD8
突變分析
這一步?jīng)]啥特別的，注意提前測試軟件和腳本就行。觀察腳本，提前將文件置于對應(yīng)位置的話能省不少事情。
minimap2 samtools make_IGV_snapshots.py emapper.py prokka
預(yù)測原噬菌體、去除冗余、基因注釋和構(gòu)建進(jìn)化樹
這步用到了CD-hit需要編譯安裝，而iqtree2可在github下載，用于化系統(tǒng)發(fā)育樹。
#####################
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
從所有樣品的contigs預(yù)測CRISPR區(qū)
CRISPRDetect.pl安裝比較繁瑣，現(xiàn)在github下載安裝，之后要安裝依賴環(huán)境，根據(jù)對應(yīng)報錯提示安裝就行，我花了不少時間。crisprdetectparser.py直接在github下載就行。
#!/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
這一步要用到原始fast5格式，暫時不用做。
文獻(xiàn)擴展
1.Complete, closed bacterial genomes from microbiomes using nanopore sequencing.
DOI: 10.1038/s41587-020-0422-6
- Integrating metagenomic binning with flux balance analysis to unravel syntrophies in anaerobic CO2 methanation.
DOI: 10.1186/s40168-022-01311-1
- Long-Read-Resolved, Ecosystem-Wide Exploration of Nucleotide and Structural Microdiversity of Lake Bacterioplankton Genomes.
DOI: 10.1128/msystems.00433-22
簡書優(yōu)秀文章推薦
1.Nanopore測序筆記
http://m.itdecent.cn/p/5d011720bc10
2.原核生物基因組三代數(shù)據(jù)（pacbio/nanopore）組裝
http://m.itdecent.cn/p/2e9d9feed61c
3.centos7安裝perl-XML-Simple（可能用到）
http://m.itdecent.cn/p/ec7d8f35b095
4.fasterq-dump 人多力量大（SRA數(shù)據(jù)轉(zhuǎn)換教程）
http://m.itdecent.cn/p/e9f6e16e2c8a
5.使用libmamba庫來加速canda環(huán)境的解決（加速conda軟件安裝）
http://m.itdecent.cn/p/904d5ed1d841
6.CD-hit安裝及使用
http://m.itdecent.cn/p/4e217eba4e96
7.perl 模塊安裝與使用（中間有需要裝模塊的步驟）
