HBV分析
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https://jbiomedsci.biomedcentral.com/articles/10.1186/s12929-018-0442-4
Applications of next-generation sequencing analysis for the detection of hepatocellular carcinoma-associated hepatitis B virus mutations
这篇文章分析了HBV突变和肝癌的相关性
1.对于组装 文章提到有参组装更好 相对de novo 组装而言 毕竟illumina的reads比较短2.文章里面提到了一个sample-specific 参考序列、同基因型的参考序列、其他不兼容参考序列对假阳性的影响。
这个文章很水 没有提到生信是怎么做的 -
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四医大HBV分析记录
1.使用bbmerge合并R1 R2 下面脚本的意思是使用find找到样本名称 然后使用这个样本名称传递给parallel并发处理find ../all_data/*_L001_R1_001.fastq.gz | sed 's/_L001_R1_001.fastq.gz$//' | parallel 'bbmerge.sh in1=../all_data/{}_L001_R1_001.fastq.gz in2=../all_data/{}_L001_R2_001.fastq.gz out={}.fastq outu1={}.R1.umerged outu2={}.R2.unmerged'发现327个样本中 有几个样本 R1 和 R2 的数量不一致 针对这些样本 使用spades进行组装 取最长的序列进行第二步
因为涉及到组装 无法进行混合样品的分析 把这些样本当作单样本处理
将所有的fastq转成fasta(blast只识别fasta)parallel 'seqtk seq -a {}> {.}.fasta' ::: *.fastq2.使用blast 对样本中的序列进行分型 得到每个样本中各种分型的序列数量
构建blast数据库
从hbvdb下载的参考序列 有一个类别是RF 例如 https://www.ncbi.nlm.nih.gov/nucleotide/EU871985.1?report=genbank&log$=nuclalign&blast_rank=1&RID=Z8DW1MY8016 这个序列 NCBI没有标识类型 hbvdb将其注释为了BC重组型 我们当前先把这种RF的去掉makeblastdb -in all_hbvdb_Genomes.fas -dbtype nuclblastn -task blastn -max_target_seqs 1 -query ../0-merging-pe/100_S42.fasta -db ../hbvdb/all_hbvdb_Genomes.fas -num_threads 10 -out 100_S42.m8 -outfmt 6nohup bash -c "find ../0-merging-pe/*.fasta | sed 's/.fasta$//' | parallel --joblog ./logs -j40 blastn -task blastn -max_target_seqs 1 -query ../0-merging-pe/{}.fasta -db ../hbvdb/A-H/HBV_A_H.fas -out {/}.m8 -outfmt 6 " &3.比对
nohup bash -c "find ../all_data/*_L001_R1_001.fastq.gz | sed 's/_L001_R1_001.fastq.gz$//' | parallel 'bwa mem -M AB033556_hbc_type_C.fasta {}_L001_R1_001.fastq.gz {}_L001_R2_001.fastq.gz > {/}.sam' " &nohup parallel "samtools view -bF 4 {} > {/.}.bam" ::: ./sam/*.sam & parallel samtools sort {} -o {.}.sorted.bam ::: *.bam4.call
nohup parallel "lofreq indelqual {} --dindel -f ../3-mapping/AB033556_hbc_type_C.fasta -o {/.}.sorted.dindel.bam " ::: ../3-mapping/bam/*.sorted.bam & nohup parallel "lofreq call {} --call-indels -f ../3-mapping/AB033556_hbc_type_C.fasta -o {/.}.vcf " ::: *.bam &5.分析单倍型
find /ceph_disk2/siyida_327_sample/3-mapping/sam/ -name "*.sam" -exec basename \{} .sam \; | sed 's/.sam$//' |parallel 'java -jar clique-snv.jar -m snv-illumina -in /ceph_disk2/siyida_327_sample/3-mapping/sam/{}.sam' -
spades的组装
/home/bioinfo/miniconda2/envs/assembly/bin/spades.py -1 /ceph_disk3/hbv/HBV_illumina/106/106_S46_L001_R1_001.fastq -2 /ceph_disk3/hbv/HBV_illumina/106/106_S46_L001_R2_001.fastq -o /ceph_disk3/hbv/HBV_illumina/106/spades -
https://www.sciencedirect.com/science/article/pii/S1386653218300970
Frequency of hepatitis B surface antigen variants (HBsAg) in hepatitis B virus genotype B and C infected East- and Southeast Asian patients: Detection by the Elecsys
HBsAg II assay
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https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172101
Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population
https://github.com/spabinger/HBV_data_publication_2016_07
an MHR variant was defined as a nucleotide sequence change in the S gene region (encoding amino acids 99 to 170) with an allele frequency >5% (in both sequencing directions) and at least 3 variant reads present on the forward as well as on the reverse strand.
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https://sci-hub.st/10.1159/000361076
Hepatitis B Virus Drug Resistance Tools:
One Sequence, Two Predictions
www.genafor.org/services.phpHIV-GRADE HBV
文章提到了一些工具 用于分型、耐药、免疫逃逸的分析
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Genetic Diversity of Hepatitis B Virus
Strains Derived Worldwide: Genotypes,
Subgenotypes, and HBsAg Subtypeshttps://sci-hub.st/10.1159/000080872
对HBV进行进化树分析 里面也提到血清型和基因型之间的复杂的对应关系。
涉及的软件：
DNADIST and NEIGHBOR from the Phylip program package version 3.53PUZZLE
Bootstrap on 1,000 replicas was performed with SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE from the Phylip package.
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https://www.aimspress.com/article/doi/10.3934/microbiol.2020024?viewType=HTML
突变可能造成的影响 这个论文做了一个总结
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https://www.nature.com/articles/s41598-019-43524-9
Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)
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https://www.frontiersin.org/articles/10.3389/fmicb.2020.616023/full
Comprehensive Analysis of Clinically Significant Hepatitis B Virus Mutations in Relation to Genotype, Subgenotype and Geographic Region
使用公开数据分析HBV的突变
Table_1_Comprehensive Analysis of Clinically Significant Hepatitis B Virus Mutations in Relation to Genotype, Subgenotype and Geographic Region.XLSX这个表格的格式可以作为分析的模板
行是样本 列是突变的位置或者重要图标的代号 -
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229894/
四医大肖老师提供的一个文章 这个使用clone测序方法对HBV的全长进行了测序
组装：Contig-Express 和Codon Code Aligner
序列对齐：MEGAX Clustal X -
Inference with viral quasispecies diversity indices: Clonal and
NGS approaches对突变频率 香农熵做了详细分析
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https://elifesciences.org/articles/61803
The haplotypes for each sample were reconstructed for each gene segment using a previously published pipeline (Cacciabue et al., 2020). In brief, FastQC (Andrews, 2010) was used for quality assurance of the NGS paired-end raw reads followed by BBtools (Bushnell, 2014), for removing and filtering adapters and low-quality reads. Bowtie2 (Langmead and Salzberg, 2012), an aligner tool to align the trimmed reads to the selected reference of the influenza strain (i.e. the inoculum), was then used. Samtools suite (Li et al., 2009) was used to sort, index, and generate depth and coverage statistics for read alignment files. Next, CliqueSNV (Knyazev, 2020) was used to infer the haplotypes and frequencies for all eight gene segments for each sample.
