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    • A
      anneng 最后由 编辑

      https://www.nature.com/articles/srep28274
      A fast and low-cost genotyping method for hepatitis B virus based on pattern recognition in point-of-care settings

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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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