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    • A
      anneng 最后由 anneng 编辑

      https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC7564258/
      9e3c2efc-187d-41c6-80e7-0894c75be13a-image.png
      分型的方法：
      Sanger sequencing：
      Sanger sequencing is often restricted to analyzing the S gene and is inaccurate for detecting minority genetic variants
      reverse hybridization：
      reverse hybridization detects only known mutations

      这个文档提到的分型方法
      The analyzed data was deposited in the NCBI SRA database under the bioproject number PRJNA659786 and the accession numbers SRR12535947, SRR12535946, SRR12535938, SRR12535937 and SRR12535936. The read quality was accessed using FastQC and reads of <150 nt were excluded using Trimmomaticv0.36 [22]; the latter program was also used to filter the reads (Illuminaclip, Headcrop = 14, Leading = 3, Trailing = 3, Slidingwindow = 4:30, Minlen = 18).

      The high-quality reads (all samples with minimum Q-Score = 30) were mapped, independently, over all genomes of all eight HBV genotypes obtained from HBVdb [21] using Bowtie2 v2.3.2. [23] with stringent parameters (–very-sensitive, –no-discordant –N 0 –L 4, –gbar 10). The aligned read counting was performed using Bed tools multiBamCov v2.25.0 [24] (default) and was loaded into an in-house Python script for mean coverage plot and genotype determination; moreover, genomic coverage throughout the A, D, and G genomes was compared using Circos plot [25].

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      • A
        anneng 最后由 编辑

        https://www.news-medical.net/health/Hepatitis-B-Structure-Capsid-Flexibility-and-Function.aspx
        表达的抗原
        2e17c577-2f17-4507-8619-e3cdd7f547af-image.png

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        • A
          anneng 最后由 anneng 编辑

          https://www.ncbi.nlm.nih.gov/nuccore/AB981583.1
          参考基因组

          https://hbvdb.lyon.inserm.fr/HBVdb/
          HBV数据库 可以找到各种分型好的序列
          https://academic.oup.com/nar/article/41/D1/D566/1051781
          HBVdb: a knowledge database for Hepatitis B Virus

          dfbfa074-495d-4f7e-87c7-411930e98eba-image.png

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          • A
            anneng 最后由 编辑

            https://pubmed.ncbi.nlm.nih.gov/25034481/
            Genotypes and genetic variability of hepatitis B virus
            Based on an intergroup divergence of greater than 7.5%
            across the complete genome, HBV has been classified phylogenetically into 9 genotypes, A–I, with a putative 10th genotype ‘J’, isolated from a single individual.
            基因组之间差异7.5%作为分类的边界值

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            • A
              anneng 最后由 anneng 编辑

              https://github.com/DrTrevorBell/CCT

              http://hbv-glue.cvr.gla.ac.uk/#/home
              GLUE
              http://hbv-glue.cvr.gla.ac.uk/#/aboutGlueProject

              https://github.com/LosicLab/ViralMine
              A pipeline tool that mines unmapped RNA/DNA reads for viral sequences and predicts viral genotype (built for HBV)

              https://www.ncbi.nlm.nih.gov/projects/genotyping/formpagex.cgi
              b2c14f82-f146-4762-bd98-df3bb39e5f20-image.png
              https://pubmed.ncbi.nlm.nih.gov/15215470/

              https://github.com/tacatanach/Hepatitis_B_Virus_alignment
              NCBI的分型曲线解释：
              1.矩形是按照窗口作为宽度 每个窗口内步进的各个矩形堆叠展示
              2.曲线Y是score值 X是每个窗口的起始点坐标
              这些数据可以下载下来 用excel处理 如下：
              bb80e687-e881-4e69-a854-64158d0c093a-image.png

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              • A
                anneng 最后由 anneng 编辑

                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
                临床样本的突变分析
                immune escape,
                antiviral resistance
                hepatocellular carcinoma (HCC)

                该文章提到了一些突变的临床意义
                https://www.sciencedirect.com/science/article/abs/pii/S001650850901556X

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                • A
                  anneng 最后由 编辑

                  分型方法研究：
                  https://www.sciencedirect.com/science/article/pii/S1201971210024239

                  https://onlinelibrary.wiley.com/doi/abs/10.1002/rmv.400
                  Hepatitis B virus genotypes: comparison of genotyping methods
                  35b0b9d6-f965-41ee-8204-e75835e9c3e9-image.png

                  https://journals.asm.org/doi/pdf/10.1128/cmr.00009-07
                  Molecular Testing in the Diagnosis and Management of
                  Chronic Hepatitis B

                  https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-020-05269-z
                  Distribution of hepatitis B virus genotypes in the general population of Myanmar via nationwide study

                  https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC4431363/
                  Sequence-based genotyping of hepatitis B virus in general population

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                  • A
                    anneng 最后由 编辑

                    Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)
                    https://www.nature.com/articles/s41598-019-43524-9#Sec11

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                    • A
                      anneng 最后由 编辑

                      https://bmcinfectdis.biomedcentral.com/track/pdf/10.1186/s12879-017-2697-x.pdf
                      Molecular characterization of hepatitis B
                      virus in Vietnam
                      The Illumina fastq sequence files were assembled
                      using Genious 8.0.5, software package (Biomatters
                      Ltd, AK, New Zeland) utilizing a reference-based
                      mapping tool after primer sequence clipping (i.e. the
                      consensus sequence was obtained by mapping individual reads of each sample to a reference sequence).
                      Finally, screening of minor (sub-consensus) variants
                      was performed using the SNP detection tool available
                      in Geneious. A minimum variant frequency of 5% and
                      500-fold coverage were chosen as cut-off values.

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                      • A
                        anneng 最后由 编辑

                        https://sci-hub.st/10.1007/978-1-4939-6700-1_17
                        Deep Sequencing of the Hepatitis B Virus Genome: Analysis
                        of Multiple Samples by Implementation of the Illumina
                        Platform
                        A BLASTN comparison analysis is performed between clean
                        reads and the HBV genomic reference sequence, to eliminate
                        non-HBV sequences (anything with similarity<70%). After
                        this filtering step, the average coverage of HBV sequence site
                        should be above 2600× (Fig. 1).
                        6. Shan entropy and Relative entropy calculations are carried out.

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                        • A
                          anneng 最后由 编辑

                          Identification and Comparative Analysis of Hepatitis B Virus Genotype D/E Recombinants in Africa
                          https://stacks.cdc.gov/view/cdc/50437

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                          • A
                            anneng 最后由 编辑

                            https://sci-hub.st/10.1016/j.jviromet.2013.06.015
                            08373011-71d4-4b31-9a33-846b89b5eebb-image.png

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                            • A
                              anneng 最后由 编辑

                              https://github.com/hr283

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                              • A
                                anneng 最后由 编辑

                                The Study of Hepatitis B Virus Using Bioinformatics
                                https://www.intechopen.com/chapters/50624

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                                • A
                                  anneng 最后由 anneng 编辑

                                  https://hbv.geno2pheno.org/
                                  http://www.hiv-grade.de/hbv_grade/deployed/grade

                                  分型工具和耐药分型

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                                  • A
                                    anneng 最后由 编辑

                                    https://www.niid.go.jp/niid/images/JJID/58/244.pdf
                                    Analysis of genomic-length HBV sequences to determine genotype and subgenotype reference sequences

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                                    • A
                                      anneng 最后由 anneng 编辑

                                      https://www.sciencedirect.com/science/article/pii/S1567134821004846#bb0040
                                      Analysis of entire hepatitis B virus genomes reveals reversion of mutations to wild type in natural infection, a 15 year follow-up study

                                      2.6. NGS data preprocessing and sample genotyping
                                      Quality control and preprocessing of each sample's raw NGS short reads was performed by fastp v0.20.1 (Chen et al., 2018). The adapter sequences were removed and 15 bases of each read were trimmed from the 5’end. Any reads with an average quality score lower than 30, or length shorter than 50 nt, were filtered further. The remaining high quality reads from each sample were then mapped to a common reference sequence (accession no. X02763) using bowtie2 v2.3.4.1 (Langmead and Salzberg, 2012). After sorting and removing the duplications using Samtools v1.7 (Li et al., 2009), the consensus sequence was generated using CliqueSNV v1.5.3 (Knyazev et al., 2021). Consensus sequences of all samples were then multi-aligned with the reference sequences from HBVdb (Hayer et al., 2013) and three additional sequences, including FJ023664 of genotype I, AB486012 of genotype J, and AM117397 from Africa chimpanzees as the outgroup. A maximum-likelihood tree was then constructed using MEGA 7 (Kumar et al., 2016) and each sample was genotyped accordingly.

                                      这个文章里面做consensus序列的时候 用的是 cliqueSNV 该软件和samtools consensus 的对比需要研究下：
                                      48ce066a-c93b-45f1-8f55-f569eb742f4d-image.png

                                      2.7. Haplotype construction and diversity analysis
                                      分型完毕后 每个样本对应的参考序列就可以更具体 然后进行突变分析
                                      bowtie2's very-sensitive-local 比对
                                      Sambamba 去重
                                      CliqueSNV haplotype reconstruction
                                      haplotypes with a minimum abundance of 1%

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                                      • A
                                        anneng 最后由 anneng 编辑

                                        二代数据分析过程
                                        对比分型
                                        使用比对 直接将reads比对到参考基因组上 然后进行统计

                                        blast分型
                                        使用blast直接和hdvdb进行对比查询（ncbi自己的分型工具对查询序列进行了分段 类似kmer 我们一般拿到的reads双端文件 感觉和这种分段很类似 可以直接查询 也不用组装了）ncbi宣称好处是可以找出“重组”过的病毒类型 即一个病毒由多个病毒拼接而成 这种情况很难和混合样本（一个样本有多个分型的感染）区分开
                                        e76e0106-c88e-4c78-80c6-d707708cc59b-image.png
                                        直接使用reads 来分型 可以参考这个文章
                                        https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC4382110/
                                        进化树方式进行分型：
                                        1.fastp ：质控和预处理
                                        2.去接头（可选）
                                        3.过滤：碱基质量
                                        4.比对：bowtie2、bwa-mem 和通用株X02763
                                        5.排序、去重：Samtools
                                        6.生成一致性序列：CliqueSNV
                                        7.多序列比对：和hdvdb数据库进行多序列比对（blast方法不需要这个步骤 由于病毒的变化很多 很可能在多序列比对时无法有效对齐）
                                        8.构建进化树：MEGA 7 maximum-likelihood方法 该步骤就可以得到每个样本的分型

                                        构建单倍型 Haplotype construction
                                        1.比对：和该样本的对应分型参考序列进行比对
                                        bowtie2's very-sensitive-local
                                        2.去重：Sambamba
                                        3.构建单倍型：CliqueSNV

                                        diversity analysis
                                        香农商Shannon entropy (Sn)：
                                        S = −pilnpi, where pi is the frequency of each haplotype in the viral quasispecies population
                                        genetic diversity (D) ：MEGA X v10.1.8 genetic distance of the haplotypes
                                        病毒进化率 viral evolutionary rates：MEGA 7 HKY substitution model 、BEAST v2.6.3、Tracer

                                        统计分析
                                        SPSS

                                        突变分析
                                        Samtools mpileup：

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                                        • A
                                          anneng 最后由 编辑

                                          https://www.rivm.nl/mpf/typingtool/hev/introduction
                                          一个在线的hev的分型工具 有参考报告

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                                          • A
                                            anneng 最后由 编辑

                                            https://livedataoxford.shinyapps.io/1510659619-3Xkoe2NKkKJ7Drg/
                                            一个shinyapps 展示了非洲的HBV耐药性
                                            https://github.com/martinjhnhadley/hbv-alignment-viz

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