1. Early changes of hepatitis B virus quasispecies during lamivudine treatment and the correlation with antiviral efficacy. J Hepatol. 2009 May;50(5):895-905. 20130701 StatGenet Journal Yosuke Fujii

2. もう 7 月 !?!? Now July!?!?

4. Evaluation of genomic diversity and complexity Advice on calculation from base frequency data A viral quasispecies is a group of viruses related by a similar mutation or mutations, competing within a highly mutagenic environment.

5. Hepatitis B virus (HBV) 3.2kb, double strand, circular DNA virus. HBV is the second most cause of hepatocellular carcinoma (HCC). HCC is the 3 rd cause of cancer death in Japan. A nucleoside analog (lamivudine) therapy is available but we often encounter drug-resistance.

6. n=25 n=14 n=11 Experimental design and result Baseline (0 week) 4 weeksLamivudine (anti-HBV drug) DNA cloning Responder Low HBV-DNA Low AST, ALT Non-responder High HBV-DNA High AST, ALT ? What dynamic changes could occur during anti-viral therapy? ? DNA cloning

7. n=25 n=14 n=11 Experimental design and result Baseline (0 week) 4 weeksLamivudine (anti-HBV drug) DNA cloning Responder Low HBV-DNA Low AST, ALT Non-responder Low HBV-DNA High AST, ALT ? What dynamic changes could occur during anti-viral therapy? ?

8. n=14 n=11 Experimental design and result 4 weeks Responder Low HBV-DNA Low AST, ALT Non-responder Low HBV-DNA High AST, ALT → Responders were low complexity and diversity. Non-responders were high. ↓ Responders were monoclonal. Non-responders were polyclonal.

9. Evaluation of quasispecies heterogeneity complexitydiversity Shannon entropy (Sn)Genetic distance (d) Synonymous substitution/site (dS or Ks) Non-Synonymous substitution/site (dN or Ka)

10. 4/8 2/8 1/8 Evaluation of quasispecies heterogeneity complexity Shannon entropy (Sn) Frequency of clones in viral population Total # of clones p <- c(1,2,4)/8 - sum(log(p)*p)/log(8) [1] 0.4583333 0 ~ monoclonal 1~ polyclonal

11. Evaluation of quasispecies heterogeneity diversity Genetic distance (d) Synonymous substitution/site (dS or Ks) Non-Synonymous substitution/site (dN or Ka) Hamming distance AATGCT ACTGTT Dist 2 Levenshtein distance AATG-CT ACTGGTT Transversion 1 Transition 1 Insertion 1 (any cost can be set) Dist 3

12. Evaluation of quasispecies heterogeneity diversity Genetic distance (d) Synonymous substitution/site (dS or Ks) Non-Synonymous substitution/site (dN or Ka) CGA CAA CCA CTA GGA TGA CGG CGC CGT Arg(R) Synonymous mutation Possible dS sites CGA 1/30/33/3 Methods Miyata & Yasunaga Nei & Gojobori Li Maximum Likelihood AGA

13. Evaluation of quasispecies heterogeneity diversity Genetic distance (d) Synonymous substitution/site (dS or Ks) Non-Synonymous substitution/site (dN or Ka) CGA CAA CCA CTA GGA TGA CGG CGC CGT Arg(R) Methods Miyata & Yasunaga Nei & Gojobori Li Maximum Likelihood All are non-synonymous. 1/3 is synonymous. All are synonymous. # of transition at # of transversion at CGA AGA Synonymous mutation

14. Evaluation of quasispecies heterogeneity diversity Genetic distance (d) Synonymous substitution/site (dS or Ks) Non-Synonymous substitution/site (dN or Ka) CGA CAA CCA CTA GGA TGA CGG CGC CGT Arg(R) Synonymous mutation Methods Miyata & Yasunaga Nei & Gojobori Li Maximum Likelihood more than one mutation synonymous transversion synonymous transition nonsynonymous transversion nonsynonymous transition Transition rate Transversion rate Substitution frequency from codon i to j. Equilibrium frequency of codon j. distribution (df=1) AGA

15. Clustering Sorry!!!

17. n=14 n=11 Experimental design and result 4 weeks Responder Low HBV-DNA Low AST, ALT Non-responder Low HBV-DNA High AST, ALT → Responders were low complexity and diversity. Non-responders were high. ↓ Responders were monoclonal. Non-responders were polyclonal.

18. Genotype estimation from frequency? CloningNGS 4/82/81/8 ATTGCGATGCACTGCT ATTCGGATGCGCTGCT ATTGCGAAGCGCTGCT ?? ATTGCGATGCACTGCT ATT??GATGC?CTGCT ATTGCGA?GC?CTGCT A G C T 0.95 0.05 0.98 0.02 0.90 0.10

19. Monte Carlo simulation? Random sampling Generate pseudosequence Rare mutation ?? Linkage ?? NGS ?? ATTGCGATGCACTGCT ATT??GATGC?CTGCT ATTGCGA?GC?CTGCT A G C T 0.95 0.05 0.98 0.02 0.90 0.10