新冠病毒数据分析

anneng

https://www.frontiersin.org/articles/10.3389/fmicb.2021.665041/full
A Novel SARS-CoV-2 Viral Sequence Bioinformatic Pipeline Has Found Genetic Evidence That the Viral 3′ Untranslated Region (UTR) Is Evolving and Generating Increased Viral Diversity

这个文章对新冠的UTR部分的突变做了分析

这个文章的附件有一张表里面有SRR号序列数很多对我们来说需要支持这种批量下载数据的情况

anneng

https://www.cdc.gov/amd/pdf/slidesets/toolkitmodule_3.5-508c.pdf
新冠病毒的仓库 gisaid 和ncbi

anneng

https://www.mdpi.com/2075-1729/12/1/69/htm
Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical Material from Swabs
直接用Nanopore RNA测序来检测新冠

basecalling
Nanopore Guppy base caller (v3.4.4) tool
“flow cell = FLO-MIN106” and “kit = SQK-RNA002”.
质控
PycoQC (v2.5.0.21) software
过滤
NanoFilt (v2.7.0)
minimum read length ≥500 nt and read quality ≥8.
去宿主和其他微生物(去污染)
去除人GRCh38 (hg38) fungal and bacterial genome
minimap2 (v2.17–r941)
提取新冠病毒
samtools (v1.7) view unmapped reads and reads with mapping quality lower than 10
对齐 call 突变
minimap2
BCFtools mpileup\call
使用Integrative Genomic Viewer (IGV) (v2.8.2) 查看突变

anneng

新冠的命名
https://covariants.org/
https://cov-lineages.org/ Pango的官网

anneng

https://www.nature.com/articles/s41467-020-20075-6

anneng

https://terra.bio/workflow-updates-to-the-covid-19-workspace-better-viral-assembly-and-phylogenetics-with-nextstrain/
terra 对sars 流程的更新说明
公开流程仓库
https://app.terra.bio/#workspaces/pathogen-genomic-surveillance/COVID-19

https://support.terra.bio/hc/en-us/articles/360041068771

2020.08.23.20178236v1.full.sars.kraken2.terra.pdf

这个文章里面提到使用kraken2检测其他病毒主要用于排除新冠和其他病毒的交叉感染

We used Kraken2 (46) to identify other viral taxa present in NP swab samples from COVID
positive patients, excluding those removed by filters i and ii described above. To do so, we ran
the classify_single workflow on all reads from all samples (with
kraken2_db_tgz=”gs://pathogen-public-dbs/v1/kraken2-broad-20200505.tar.zst”,
krona_taxonomy_db_kraken2_tgz=”gs://pathogen-public-dbs/v1/krona.taxonomy-20200505.tab.
zst”, ncbi_taxdump_tgz=”gs://pathogen-public-dbs/v1/taxdump-20200505.tar.gz”,
trim_clip_db=”gs://pathogen-public-dbs/v0/contaminants.clip_db.fasta”,
spikein_db=”gs://pathogen-public-dbs/v0/ERCC_96_nopolyA.fasta”). Our kraken2 database was

Terra的这个流程是针对illumina二代的情况
https://app.terra.bio/#workspaces/pathogen-genomic-surveillance/COVID-19_Broad_Viral_NGS

anneng

https://dockstore.org/organizations/BroadInstitute/collections/pgs

anneng

https://artic.readthedocs.io/en/latest/primer-schemes/
artic的引物设计

该图来自该nature的文献对多重PCR做了详细说明
https://www.nature.com/articles/nprot.2017.066
Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples
文档中提到了这个方法主要是用于在临床中宏基因测序的病毒载量很低
genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads.
这个文章还提到了一个在线引物设计工具（引物设计是实验的一个关键环节这类工具我们可以做到系统里面甚至做成一个app）

anneng

https://bugseq.com/demo/metagenomic

anneng

https://training.galaxyproject.org/training-material/topics/variant-analysis/tutorials/sars-cov-2-variant-discovery/tutorial.html