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&

25. Solutions for assembly

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

30. Shameless self-promotion
flxlexblog.wordpress.com

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…

40. The approach
Error-correction

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