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Combining PacBio with short read technology for improved de novo genome assembly
- 1. The best of both worlds Combining PacBio with short read technology for improved de novo genome assembly Lex Nederbragt, NSC and CEES lex.nederbragt@bio.uio.no
- 2. This talk
- 3. Why does everybody want longer reads? … for genome assemblies
- 4. What is a genome assembly Hierarchical structure reads contigs scaffolds
- 5. Sequence data Reads reads contigs scaffolds original DNA fragments original DNA fragments Sequenced ends http://www.cbcb.umd.edu/research/assembly_primer.shtml
- 6. Contigs Building contigs reads contigs scaffolds ACGCGATTCAGGTTACCACG GCGATTCAGGTTACCACGCG GATTCAGGTTACCACGCGTA TTCAGGTTACCACGCGTAGC CAGGTTACCACGCGTAGCGC Aligned reads GGTTACCACGCGTAGCGCAT TTACCACGCGTAGCGCATTA ACCACGCGTAGCGCATTACA CACGCGTAGCGCATTACACA CGCGTAGCGCATTACACAGA CGTAGCGCATTACACAGATT TAGCGCATTACACAGATTAG Consensus contig ACGCGATTCAGGTTACCACGCGTAGCGCATTACACAGATTAG
- 7. Contigs Building contigs reads contigs scaffolds Repeat copy 1 Repeat copy 2 Contig orientation? Contig order? Collapsed repeat consensus http://www.cbcb.umd.edu/research/assembly_primer.shtml
- 8. Mate pairs Other read type reads contigs scaffolds Repeat copy 1 Repeat copy 2 (much) longer fragments mate pair reads
- 9. Scaffolds Ordered, oriented contigs reads contigs scaffolds mate pairs contigs gap size estimate
- 10. What is a genome assembly Hierarchical structure reads ACGCGATTCAGGTTACCACG GCGATTCAGGTTACCACGCG GATTCAGGTTACCACGCGTA TTCAGGTTACCACGCGTAGC CAGGTTACCACGCGTAGCGC Aligned reads GGTTACCACGCGTAGCGCAT TTACCACGCGTAGCGCATTA ACCACGCGTAGCGCATTACA CACGCGTAGCGCATTACACA CGCGTAGCGCATTACACAGA contigs CGTAGCGCATTACACAGATT TAGCGCATTACACAGATTAG Consensus contig ACGCGATTCAGGTTACCACGCGTAGCGCATTACACAGATTAG scaffolds
- 11. Genome assembly So, what’s so hard about it?
- 12. 1) Repeats reads contigs scaffolds Repeat copy 1 Repeat copy 2 Repeats break up contigs Collapsed repeat consensus http://www.cbcb.umd.edu/research/assembly_primer.shtml
- 13. 2) Heterozygosity Differences between sister * chromosomes * * http://commons.wikimedia.org/wiki/File:Chromosome_1.svg
- 14. 2) Heterozygosity Polymorphic contig 2 Contig 1 Contig 4 Polymorphic contig 3
- 15. 2) Heterozygosity http://www.astraean.com/borderwars/wp-content/uploads/2012/04/heterozygoats.jpg and many other sites
- 16. 3) Many programs to choose from Zhang et al. (2011) doi:10.1371/journal.pone.0017915.g001
- 17. Assembly: challenges Repeat copy 1 Repeat copy 2 Knowing how to use the programs Heterozygosity Polymorphic contig 2 Contig 1 Contig 4 Polymorphic contig 3
- 18. So, why does everybody want longer reads? http://www.autobizz.com.my/forum/forum/General-Chat/944-The-worlds-longest-car.html
- 19. Longer reads? Repeat copy 1 Repeat copy 2 Long reads can span repeats and heterozygous regions Polymorphic contig 2 Contig 1 Contig 4 Polymorphic contig 3
- 20. PacBio to the rescue?
- 21. High-throughput sequencing Library preparation SMRTBell'template' SMRTBell'template' Standard'Sequencing' Standard'Sequencing' Generates& pass& each& one& on& molecule& Insert& Large& Sizes& Generates& pass& each& one& on& molecule& Large Insert& Sizes Large& Sizes& Insert sequenced& Single pass sequenced& Circular'Consensus'Sequencing' Circular'Consensus'Sequencing' Continued generations of reads Small Insert Sizes& Small& Small& Insert& Sizes Insert& Sizes& Multiple mul8ple& passes passes& each& Generates& on& molecule& Generates& mul8ple& sequenced& passes& each& on& molecule& sequenced&
- 22. High-throughput sequencing Raw read length
- 23. High-throughput sequencing SMRTBell'template' Raw reads and subreads Standard'Sequencing' Generates& pass& each& one& on& molecule& Large Insert& Sizes Large& Sizes& Insert Single pass sequenced& ‘Subreads’ Circular'Consensus'Sequencing' Small Insert Sizes& Small&Insert& Sizes Multiple mul8ple& passes passes& each& Generates& on& molecule& sequenced&
- 24. PacBio: uses SMRTBell'template' Long reads low quality Standard'Sequencing' Generates& pass& each& one& on& molecule& Large Insert& Sizes Large& Sizes& Insert Single pass sequenced& 85-87% accuracy Circular'Consensus'Sequencing' Useful for assembly? Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& sequenced&
- 26. Solutions for assembly (1) Designed by Pacific Biosciences http://www.clker.com/clipart-4245.html
- 27. Solutions for assembly (2) Broad Institute Need a special recipe for sequencing
- 28. Solutions for assembly (3) PacBioToCA Error correct with short reads Celera assembler http://schatzlab.cshl.edu/presentations/2012-01-17.PAG.SMRTassembly.pdf
- 29. PacBioToCA Koren et al, 2012
- 31. Shameless self-promotion @lexnederbragt
- 32. The Atlantic cod genome project
- 33. First draft Fragmented assembly - short contigs - many gap bases http://en.wikipedia.org
- 34. First draft 6467 scaffolds 35% gap bases
- 35. The causes Short Tandem Repeats (>20% of gaps)
- 36. The causes Heterozygosity? Polymorphic contig 2 Contig 1 Contig 4 Polymorphic contig 3
- 37. The goal 23 pseudochromosomes Longer contigs Below 5% gap bases PacBio to the rescue?
- 38. The approach SMRTBell'template' Libraries Standard'Sequencing' Generates& pass& ea one& on& Large Insert& Sizes Large& Sizes& Insert sequenced& Aim for looooong insert sizes Circular'Consensus'Sequencing' Small& Insert& Sizes& Generates& mul8ple& passes sequenced&
- 39. SMRTBell'template' The approach Sequencing Standard'Sequencing' Generates& pass& each& one& on& molecule& Large Insert& Sizes Large& Sizes& Insert Single pass sequenced& Sequence with 90 minute movies Circular'Consensus'Sequencing' Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& 10 x coverage in reads of at least 3000 bp sequenced& No, we don’t throw this away…
- 41. PacBio results 100 Relative throughput at different minimum length cutoffs 10kb lib 2 Fraction of bases at minimum 10kb lib 1 length 4kb lib 80 Percentage of total sequence 60 40 20 0 0kbp 3kbp 5kbp 10kbp 15kbp Length cutoff longest subread Large library insert size important!
- 42. chnology PacBio results SMRTBell'template' 64 SMRT Cells 3.2 Gigabytes in raw reads at least 3kb 3.8 x coverage 3 Standard'Sequencing' Generates& pass& each& one& on& molecule& Large Insert& Sizes Large& Sizes& Insert sequenced& 2.2 Gigabytes in longest subreads reads Circular'Consensus'Sequencing' Largest 15 kbp Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule&
- 43. PacBio results Mapping to the cod genome 11.4 kbp subread 10.6 kbp subread 10.9 kbp subread
- 44. Example 1 ACACAC repeat 232 bp Gap TGTGTG repeat
- 45. Example 1
- 46. Example 1
- 47. Example 1 Scaffold ...ACACAC TGTGTG... PacBio reads Unplaced contig
- 48. Example 2 TGTGTG repeat 344 bp Gap
- 49. Example 2
- 50. Example 2 Scaffold ...TGTGTG PacBio reads Heterozygosity?
- 51. Example 3 Scaffold PacBio reads 300 bp misassembly?
- 52. Error-correction Work In Progress http://openclipart.org/
- 53. Outlook Will PacBio solve our problems?
- 54. Outlook Or
- 55. Outlook Polymorphic contig 2 Contig 1 Contig 4 Polymorphic contig 3 Will we find the heterozygous regions?
- 56. Outlook http://www.pasteur.fr/recherche/unites/Bbi/ en.wikipedia.org and Martin Malmstrøm