This document summarizes work using PacBio long reads to improve the Atlantic cod genome assembly. Error-corrected and raw PacBio reads were used with different assembly programs. Both helped increase contig and scaffold lengths over the previous assembly, with raw reads performing best. Bridgemapper validation found misassemblies corrected by PacBio. The improved assembly met goals of <5% gaps and scaffold N50 over 1 Mbp. Lessons included developing programs to handle cod's heterozygosity and structural variation better. The new assembly version aims to have 23 pseudochromosomes and improved annotation.

1. Improving and validating the Atlantic Cod
genome assembly using error-corrected
as well as raw PacBio reads
Lex Nederbragt, NSC and CEES
lex.nederbragt@ibv.uio.no
@lexnederbragt
OK

2. Acknowledgements
University of Oslo
Sequencing team NSC
Ole Kristian Tøressen
Kjetill Jakobsen
Sissel Jentoft
Cod genome group
Jason Miller, JCVI
Pacific Biosciences

3. The Atlantic cod genome project

4. Cod: the genome
850 million bases (Mbp )
‘Wild-caught’
Heterozygote
*
*
*

5. Cod: phase 1
454 sequencing
(Sanger sequencing)

6. N50
50% of the genome is in contigs as large as the N50 value
1000 bp genome
Sum
400
445
490
520
N50
Courtesy of Michael Schatz, CSHL

7. Cod: phase 1
(Sanger sequencing)
454 sequencing
Phase 1 assembly
157 887 sequences
753 Mbp of 830 Mbp
N50 460 kbp
Scaffold
N50 2.8 kbp
contig
gap

8. Cod: phase 1
6467 scaffolds
35% gap bases

9. The causes
Short Tandem Repeats (>20% of gaps)

10. The causes
Heterozygosity?
Polymorphic contig 2
Contig 1
Contig 4
Polymorphic contig 3

11. Cod: phase 2
New data
Illumina sequencing
Paired end
>200x
Mate Pair 5kb >100x
Improved/new software

12. Cod: phase 2 goal
23 pseudochromosomes
Longer contigs
Below 5% gap bases
Phase 2 goal
Scaffold N50 1 Mbp
Contig N50 15 kbp

13. Cod: phase 2 programs
Zhang et al. PLoSOne 2011

14. Cod phase 2: status
Contig
N50
gaps
scaffold
N50
15 kbp
<5%
1.5 Mbp
Celera, 454 + Ilmn
9 kbp
5%
too short
Newbler, 454
6 kbp
24%
OK
Goal

15. Enter PacBio

16. SMRTBell'template'
Sequencing
Standard'Sequencing'
Large&
Sizes&
Large Insert& Sizes
Insert
Aim for looooong insert sizes
Circular'Consensus'Sequencing'
SMRT® Technology
Small&
Insert&
Sizes&
Chemistry Coverage
Av. Raw
length
C2
Photo: Tore Oldeide Elgvin
3.2x
4.6 kb
XL-XL
3.5x
5.1 kb
TOTAL
3
3.0 kb
C2-XL
147 SMRT Cells
9.2x
15.9x

17. Error-correction
Celera Assembler merTrim
PacBioToCa (Koren et al)
13.7x
27x
+
+
234x
27x
 9x (67%) recovered

18. Using PacBio reads

19. PacBio reads for cod
Error-corrected
reads
Assembly improvement
Celera
Assembly validation
blasr
De novo assembly
Celera
Raw
reads
PBJelly
blasr
bridgemapper

20. PacBio reads for cod
Error-corrected
reads
Assembly improvement
PBJelly
Assembly validation
blasr
De novo assembly
Celera
Raw
reads
PBJelly
blasr
bridgemapper

21. PacBio reads for cod
Error-corrected
reads
Assembly improvement
PBJelly
Assembly validation
blasr
De novo assembly
Celera
Raw
reads
PBJelly
blasr
bridgemapper

22. PacBio reads for cod
Error-corrected
reads
Assembly improvement
PBJelly
Assembly validation
blasr
De novo assembly
Celera
Raw
reads
PBJelly
blasr
bridgemapper

23. Assembly improvement: corrected reads
N50
Goal
Celera, 454 reads
+ corrected PacBio + PBJelly
gaps
15 kbp
<5%
9 kbp
5%
11 kbp
1.5%

24. PacBio reads for cod
Error-corrected
reads
Assembly improvement
PBJelly
Assembly validation
blasr
De novo assembly
Celera
Raw
reads
PBJelly
blasr
bridgemapper

25. Assembly improvement: raw reads
N50
Goal
Newbler, 454
+ raw PacBio + PBJelly
gaps
15 kbp
<5%
6 kbp
24%
30 kbp
20%

26. Assembly improvement: raw reads
N50
15 kbp
+ raw PacBio + PBJelly
Too good to be true?
5%
46 kbp
Celera, 454 + Ilmn
<5%
9 kbp
Goal
gaps
1.5%

27. PacBio reads for cod
Error-corrected
reads
Assembly improvement
PBJelly
Assembly validation
blasr
De novo assembly
Celera
Raw
reads
PBJelly
blasr
bridgemapper

28. Assembly validation
Sequence

29. Assembly validation
Sequence
Coverage
Aligned raw Pacbio reads

30. Assembly validation
Sequence
Coverage
Aligned raw Pacbio reads
Aligned corrected Pacbio reads

31. Assembly validation
Newbler scaffold
Corrected
pacbio reads
Raw
pacbio reads
308 bp gap
(TG)n repeat
(TG)n repeat

32. Assembly validation
Newbler scaffold
Raw
pacbio reads
939 bp gap
(AG)n repeat
Heterozygous region

33. Assembly validation
Raw
pacbio reads
Celera scaffold
Misassembly?

34. PacBio reads for cod
Error-corrected
reads
Assembly improvement
PBJelly
Assembly validation
blasr
De novo assembly
Celera
Raw
reads
PBJelly
blasr
bridgemapper

35. Assembly validation: bridgemapper (beta)
Split alignments
structural variation
misassemblies

36. bridgemapper (beta) on E. coli
Positions in the contig color coded
Illumina + velvet

37. bridgemapper (beta) on cod
s05514
2510 bp gap
Point to a 2350 bp scaffold

38. bridgemapper (beta) on cod
2145 bp gap
s08737
Point to a 3 kbp scaffold

39. PacBio reads for cod
Error-corrected
reads
Assembly improvement
PBJelly
Assembly validation
blasr
De novo assembly
Celera
Raw
reads
PBJelly
blasr
bridgemapper

40. Assembly with error-corrected reads
Contig
N50
15 kbp
CA + corrected PacBio
+ 454 mates
1.4 times genome size
 underassembled
5%
too short
8 kbp
Celera Assembly
<5%
9 kbp
Goal
gaps
2%
very short
scaffolds

41. The improved Atlantic cod genome:
status
http://en.wikipedia.org

42. Newbler plus Celera
Celera: Long contigs, short scaffolds
Scaffold
gap
contig
Slide courtesy of Ole Kristian Tøressen

43. Newbler plus Celera
Celera: Long contigs, short scaffolds
Scaffold
gap
contig
Newbler: Short contigs, long scaffolds
Scaffold
contig
gap
Slide courtesy of Ole Kristian Tøressen

44. Newbler plus Celera
Celera: Long contigs, short scaffolds
Scaffold
gap
contig
Newbler: Short contigs, long scaffolds
Scaffold
contig
gap
Combined: Long contigs, long scaffolds
gap
Scaffold
contig
Slide courtesy of Ole Kristian Tøressen

45. Adding PacBio
Closed gap
Reduced gap
Using PBJelly
PacBio reads
Contig
Scaffold
Slide courtesy of Ole Kristian Tøressen

46. Polishing the assembly
454 and Illumina reads
Contig
Scaffold
Contig N50: 30 - 40 kbp
Scaffold N50: 1 - 1.5 Mbp
Slide courtesy of Ole Kristian Tøressen

47. Imageby Mathieu Thouvenin http://www.flickr.com/photos/mathoov/4681491052/

48. PacBio reads for cod
Error-corrected
reads
Assembly improvement
PBJelly
Assembly validation
blasr
De novo assembly
Celera
Raw
reads
PBJelly
blasr
bridgemapper

49. PacBio reads for cod
Error-corrected
reads
Assembly improvement
PBJelly
Assembly validation
blasr
De novo assembly
Celera
Raw
reads
PBJelly
blasr
bridgemapper
Celera

50. Assembly
Contig
N50
15 kbp
CA + corrected PacBio
+ 454 mates
CA + raw PacBio reads + 454 mates
1.6 times genome size
 underassembled
<5%
8 kbp
Goal
gaps
2%
very short
38 kbp
<1%
very short
scaffolds

51. Lessons learned from PacBio reads

52. Cod genome
Heterozygous:
Large polymorphism
(100’s of bases)
Homozygous
Homozygous
Heterozygous:
Large indel
(100’s of bases)
Homozygous

53. Atlantic cod version 2
23 pseudochromosomes
Longer contigs
Below 5% gap bases
New annotation

54. From observation to insight
We need better programs
Mathias Bigge, Ricordisamoa, others (wikimedia commons)