新冠病毒数据分析
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https://pubmed.ncbi.nlm.nih.gov/31978945/
A Novel Coronavirus from Patients with Pneumonia in China, 2019
这个文章提到了最初武汉的几个样本采用的二代和三代混合测序的方式 -
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929396/
Bioinformatics resources for SARS-CoV-2 discovery and surveillance
几种实验方法
The workflow of different NGS sequencing approaches currently available for virus discovery and genomic surveillance. The library construction scheme employed in (A) metatranscriptomic sequencing, (B) a hybrid capture-based approach based on a metatranscriptomic library, (C) multiplex PCR amplification for NGS platforms and (D) the Oxford Nanopore sequencing platform.
新病毒发现的基本过程和工具
在去宿主步骤 提到了要去掉rRNA 而且也提到病毒载量比较低的情况下 可以不去宿主 -
covid19.sfb.uit.no
新冠数据库 -
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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
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https://www.cdc.gov/amd/pdf/slidesets/toolkitmodule_3.5-508c.pdf
新冠病毒的仓库 gisaid 和ncbi -
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
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新冠的命名
https://covariants.org/
https://cov-lineages.org/ Pango的官网 -
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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-19https://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 wasTerra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 -
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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）
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