Deep-Sequencing of HIV-1

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1 Deep-Sequencing of HIV-1 The quest for true variants Alexander Thielen, Martin Däumer

2 Limitations of drug resistance testing by standard-sequencing Blood plasma RNA extraction RNA Reverse Transcription/ Polymerase Chain Reaction cdna M Sample 2. Nested PCR PCR products (optional) Purification (optional) Sequencing Sensitivity Reaction for minor variants: >20% Purification Sequencer

3 Ultra-deep-sequencing Standard Sanger-sequencing...PQIYMDDHTRE... Ultra-deep-sequencing...PQIYMDDHTRE......PQIYMDDHTRE......PQIYVDDHTRE......PQIYMDDHTRE......PQIYMDDHTRE......PQIYMDDHTRE...

4 454 Sequencing / Roche GS Junior System GS FLX+ System Illumina HiSeq Systems Genome analyzer IIx MiSeq, NextSeq Life Technologies SOLiD 5500 System SOLiD 5500xl System Ion Torrent PGM Proton Next Generation Sequencing, Amplified Single Molecule Sequencing Helicos Helicos Genetic Analysis System Pacific Biosciences PacBio RS Oxford Nanopore Technologies GridION System MinION Third Generation Sequencing, Single Molecule Sequencing

5 The players

6 Most important... iphone docking station

7 Specifications

8 MinION Nanopore technology

9 MinION Nanopore technology

10 MinION Nanopore technology HIV-1 pol amplicon, 1.35kb

11 outlook

12 Illumina systems

13 MiSeq sequencing instrument Illumina s benchtop sequencer easy sample preparation no homopolymer problems current specification: ~ up to 15 Gb output (10 x FLX+, >100 x 454 GS Junior) 2x20mio reads of up to 2x300 nts length 37h run time (2x250 nts)

14 MiSeq Personal Sequencing System

15 Library preparation using Nextera TM XT tagmentation Easy library preparation Fast - less than 20 minutes hands-on time Only 1ng DNA per sample needed Indices for up to 96 samples Normalization step included

16 Drug resistance testing using Illumina s MiSeq

17 Experimental setup HIV genome PRRT IN ENV whole genome

18 Fragmentation

19 putting things together mapping PRRT IN ENV Reference sequences

20 Coverage PR/RT ENV ~7500 full V3 loops

21 Reproducibility

22 PR/RT # resistance mutations found

23 PR/RT resistance interpretation ATV/r limited susceptibility Intermediate Resistance % 2% 5% 10% 15% 20% SANGER interpretation according to

24 Number of sequencing errors, substitutions, deletions, and insertions Archer J et al., 2012, PLoS ONE 7: e doi: /journal.pone

25 Workflow NGS Library preparation: fragmentation & indexing RNA/DNA Total NA extraction rt-pcr/ nested pcr Sanger Sequencing reaction Taq-cycle reaction, sequencing analysis analysis

26 Workflow NGS RNA/DNA Total NA extraction Sanger Library preparation: fragmentation & indexing Sequencing reaction analysis PCR errors Sequencing errors rt-pcr/ nested pcr RNA vs. DNA: viable vs non-viable PCR errors, recombination Potential error sources editing Taq-cycle reaction, sequencing analysis

27 PCR errors in clones 7,00% 6,00% 5,00% 4,00% 3,00% 2,00% 1,00% 0,00% I47V I50V N83D I84V D30N G73D L76V G48V K20M M46L I54M T74P V82L L89V K20V G73T V82C M230V D67G K219E K219R K70E K101E V118I V179I V189I H221Y K238T T215S T215N A B C D E1 E2 V179D K219N A98G Y115F K101Q F227C K101H P225H T215F K103S T69D T215D V179T Y181I A

28 PCR errors in clones

29 Effect of high fideltity enzymes 3,50% 3,00% 2,50% 2,00% 1,50% 1,00% 0,50% 0,00% G48V N83D I50V F53L I54V G73D L10I K20M M46L I50L K43T I54L L10V G48A G73T V82M D67G M230V E138G P225H Y188H V75A V106A V90I G190E E138K D67N V179E Qiagen One step RT-PCR kit / HotStarTaq Invitrogen One step RT-PCR Superscript III / HiFi Platinum Taq M41L L74V L74I Y115F T215N K101Q V179I K101P T215F G190Q V75M T215Y Q151M K103S V179F Y181I

30 Ultra-deep sequencing of proviral DNA

31 C.,J. *1980 1st line ART: TDF/FTC/EFV Resistance test from proviral DNA Viruslast Kop/ml CD4 + /µl

32 Proviral DNA

33 Plasmavirus RNA

34 C.,J. *1980 Proviral DNA Standard Sanger

35 ... but things may turn out diffenrently...

36 Resistence testing from proviral DNA and low-abundance variants D

37 Deep-Sequencing of HIV-1 The quest for true proviral variants Alexander Thielen

38 Resistance testing from proviral DNA viral archive interesting what about defective viruses? => not preferred but sometimes required

39 Resistance testing from proviral DNA what means defective? stop codons => definitively hypermutated => probably other suggestions? do we see these viruses in our data? do we have a big problem?

40 Stop codons in the routine samples: RNA : 528 DNA: 169 samples with stop codons: cutoff DNA RNA 1% % % 2% % % 5% % % 10% % % 15% % % 20% % % 30% % %

41 Where are the stop codons are coming from? PR: W42* RT: W88*, W153*, W229*, W266*, W212*, W71*, W212*, W252*, W153*, W239*, W24* TAG HXB2 reference codon: TGG TGA stop codons mostly: TAG TAA Apobec? Apobec3F: GA AA Apobec3G GG AG, further preference for TGG, TGGG motifs!

42 Are there further motifs found in DNA? other reading frames, e.g. ATGG ATAG (M I) odds ratio DNA / RNA > cutoff, N at least 5: PR: 42*, 90M, 73S,... RT: 135V, 184V, 88*, 51R, 41L, 153*, 36A*, 212*, 230I, 70R, 196R, 35M, 266*, 16I, 210W, 41I, 45E,... can these be explained by Apobec? not all, e.g. M184V: ATG GTG

43 Covariation analysis Are there further motifs found in DNA?

44 Covariation analysis Are there further motifs found in DNA? M184V, 41L, 70R, 210W, 181C, 215Y, 65R, 190A, PR-90M, 219Q, 215F,... no G A! Wx*, 51R, 230I, 196R, 16I, 41I, 45E, 184I, 190R, 276I, 186N,... G A!

45 Covariation within patient sample D 190R 3.6% 230I 3.6% 184I 3.5% 252* 3.35% 153* 4.35% 230I w/o 252* 1.23% 184I w/o 153* 1.38% 190R w/o 153* 1.62%

46 Covariation within patient sample D 190R 8.95% 230I 9.35% 184I 0.17%? 252* 0.52% 153* 0.52% 230I w/o 252* 9.22% also no correlations with other stop-codons but high correlation between 190R and 230I

47 Covariation within patient sample R 0.37% 230I 4.31% 184I 0.24% no correlations with stop-cluster mutations

48 Acknowledgments Kirsten Becker Nina Engel Anna Memmer Benjamin Racké Bettina Spielberger Steffi Wenzel Bernhard Thiele

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