1. 1 DNA Sequencing Achim Tresch UoC / MPIPZ Cologne treschgroup.de/OmicsModule1415.html tresch@mpipz.mpg.de

2. - DNA sequencing in the last century - Current technologies (Illumina, Ion Torrent) - New developments (PacBio, Nanopore) Topics

3. T Sanger sequencing - Random incorporation of blocked nucleotides  at any position, reaction stops in a small fraction of the reads TTGCACTTGAGTCGT AACGTGAACTCAGCATAGGCTCAGATAGAT A-Reaction: add dATP (elongation) and ddATP (block) Analogous: C-, G-, T-Reaction ddATP - Developed by Fred Sanger in the 70ies (1918-2013, 2*Nobel laureate: 1958 – protein structure of insulin, 1980 – sequencing of nucleic acids) - Sequencing by synthesis: DNA polymerase is synthesizing a complementray strand by adding single nucleotides TTGCACTGAGTCG AACGTGACTCAGCATAGGCTCAGATAGAT

4. TTGCACTTGAGTCG AACGTGAACTCAGCATAGGCTCAGATAGAT A-Reaction: TTGCA TTGCACTTGA C-Reaction: TTGC TTGCAC TTGCACTTGAGTC G-Reaction: TTG TTGCACTTG TTGCACTTGAG TTGCACTTGAGTCG T-Reaction: TT TTGCACT TTGCACTT TTGCACTTGAGT ddNTP Sanger sequencing ladder of DNA fragments  electrophoresis  sequence T G C A

5. GATTGATAGTTGC CTAACTATCAACGTATAGGCTCAGATAGAT G GA GAT GATT GATTG GATTGA GATTGAT GATTGATA GATTGATAG GATTGATAGT GATTGATAGTT GATTGATAGTTG GATTGATAGTTGC - labeled ddNTPS, capillary sequencing A Sanger sequencing

6. Pyrosequencing - immobilize DNA on beads, pyrosequencing in microreactors dTTP TTGCACTGAGTCGT AACGTGACTCAGCATAGGCTCAGATAGAT PPi ATP Oxyluciferin + light 454 technology

7. DNA-loaded beads + primer + polymerase + sulfurylase + luciferase flowgram TTGCACTGAGTCGT AACGTGACTCAGCAAGTCTATTCACCCAC... 454 technology Problem: homopolymers difficult to detect

8. increase throughput: - DNA gel electrophoresis, single genes in few days - capillary electrophoresis, 96 capillaries per machine, human genome in a few years - sequencing on microbeads: 454 technology Parallelisation & Miniaturisation

9. Illumina sequencing: - sequencing by synthesis - massive parallelisation and miniaturisation by self-organising DNA microarrays on a glass surface - several hundred Gb, >10 9 reads per run Illumina technology

10. - generate libraries - grow clusters on a flowcell - sequence by addition and imaging of blocked & fluorescence-labeled nucleotides Illumina technology

11. library preparation: DNA fragments Blunting by Fill-in and exonuclease Phosphorylation Addition of A-overhang Ligation to adapters Illumina technology

12. cluster generation: 1. flowcell Illumina technology

13. cluster generation: 1. flowcell 2. hybridize template Illumina technology

14. cluster generation: 1. flowcell 2. hybridize template 3. immobilize template Illumina technology

15. cluster generation: 1. flowcell 2. hybridize template 3. immobilize template 4. bridge amplification Illumina technology

16. cluster generation: 1. flowcell 2. hybridize template 3. immobilize template 4. bridge amplification Illumina technology

17. cluster generation: 1. flowcell 2. hybridize template 3. immobilize template 4. bridge amplification Illumina technology

18. cluster generation: 1. flowcell 2. hybridize template 3. immobilize template 4. bridge amplification Illumina technology

19. cluster generation: 1. flowcell 2. hybridize template 3. immobilize template 4. bridge amplification Illumina technology

20. cluster generation: 1. flowcell 2. hybridize template 3. immobilize template 4. bridge amplification 5. linearisation Illumina technology

21. cluster generation: 1. flowcell 2. hybridize template 3. immobilize template 4. bridge amplification 5. linearisation 6. cleave reverse strand Illumina technology

22. cluster generation: 1. flowcell 2. hybridize template 3. immobilize template 4. bridge amplification 5. linearisation 6. cleave reverse strand 7. block 3‘-ends Illumina technology

23. cluster generation: 1. flowcell 2. hybridize template 3. immobilize template 4. bridge amplification 5. linearisation 6. cleave reverse strand 7. block 3‘-ends 8. hybridize primer Illumina technology

24. Imaging & Sequencing: Illumina technology Nucleotide + fluorescent dye + terminator

25. reversible terminators: Illumina technology

26. fluorescently labelled clusters: Illumina technology

27. data output: Hiseq: - ca. 250 Mio reads * 8 lanes - 2*100 bp paired end-> 400 Gb / 8 days Hiseq rapid run: - ca. 200 Mio reads * 2 lanes - 2*150 bp paired end-> 120 Gb / 40 hours - (2*250 bp paired end)-> 200 Gb / 60 hours) Miseq: - ca. 25 Mio reads * 1 lane - 2*300 bp paired end-> 15 Gb / 65 hours Illumina technology

28. Fastq quality scores good quality quality drops towards the end 0.1 % error 1 % error Data quality of short reads

29. Amplification Artifacts Duplicate reads

30. Ion torrent: semiconductor sequencing - detect H+ release upon nucleotide incorporation by DNA polymerase Ion torrent

31. work flow: Ion torrent

32. data output: Ion Proton: - up to 80 mio reads - up to 10 Gb (200 base read length) - 4 hours runtime Ion Torrent PGM: - up to 5 mio reads - up to 2 Gb (400 base read length) - 8 hours runtime Ion torrent

33. homopolymer problem? Ion torrent - nonlinear increase of signal

34. what can we do with short reads? RNA-seq, identify transcripts, count reads per transcript  assessment of differential expression problem: reads are too short to establish connectivity of all exons, difficult/impossible to quantify multiple isoforms of a gene Sequencing Applications

35. Stefan Krebs, 30.09.2013 Single end: ambiguous mapping Paired end sequencing: read fragment from both ends -> resolve ambiguities Improvements: Paired end Reads

36. further improvements long jumping mate-pair libraries: circularize large fragment and reads junctions (2-10 kb) resolve large repeats in genome assembly Improvements: Circularization

37. Third generation Sequencing

38. - single molecule detection -several kilobases read length -moderate output (150.000 wells) -expensive instrument and high cost per base Pacific Biosciences

41. Read length distribution

42. Pacific Biosciences Read quality

43. Pacific Biosciences

44. - DNA polymerase coupled to pore releases tags when incorpotating labeled nucleotides -tags passing through nanopore change ion current - read length = length of DNA fragment Oxford Nanopore

45. everything that can be converted to a DNA strand can be sequenced - even long-term data storage by encoding in synthetic DNA is possible BIOLOGICAL APPLICATIONS: sequencing of genomes, transcriptomes, population diversity, composition of microbial communities, ChIPseq, methyl-Seq, translating RNA from ribosomes,... MEDICAL APPLICATIONS: whole genome sequencing, exome sequencing, tumor diagnostics, sequencing of T-cell receptor diversity, identification of pathogens,... FORENSICS, FOOD SAFETY, ARCHEOLOGY, … Applications

46. Other Approaches

47. Summary third generation Sequencing

48. Acknowledgements Stefan Krebs Gene Center LMU Munich