1. Phusion2 and The Genome Assembly of Tasmanian Devil
Zemin Ning
The Wellcome Trust Sanger Institute 1

2. Outline of the Talk:
Challenges in genome assemblies from pure Illumina reads
The Phusion2 pipeline
The Tasmanian devil genome project
The Devil genome assembly
Other assemblies: human cancer, zebrafish, rice, etc

3. Challenges in Whole Genome Assembly using Pure Illumina Reads
Large genome and huge datasets
For human: 100Gb at 30x
Repetitive/Duplication structures, Alus, LINES, SVAs
30-40% such as human, mouse; 50-60% such as rice and other plant genomes.
Tandem repeats: how many copies they have?
TATATATATATATATATATATATATATA
GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCG
GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG
AGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGT

4. De Bruijn vs Read overlap Missing from de Bruijn contigs
Missing sequences
Missing from de Bruijn contigs

5. Phusion2 Assembly Pipeline
Solexa
Reads
Assembly
Reads Group
Data Process
Long Insert Reads
Supercontig
Contigs
PRono
Fuzzypath
Velvet
Phrap
2x75 or 2x100
Base
Correction

6. Grouped Reads by Phusion
Repetitive Contig and Read Pairs
Depth
Grouped Reads by Phusion

7. Kmer Word Hashing Contiguous Base Hash K = 12 Gap-Hash 4x3
ATGGCGTGCAGT
TGGCGTGCAGTC
GGCGTGCAGTCC
GCGTGCAGTCCA
CGTGCAGTCCAT
ATGGCGTGCAGTCCATGTTCGGATCA
Contiguous
Base Hash
K = 12
ATGGGCAGATGT
TGGCCAGTTGTT
GGCGAGTCGTTC
GCGTGTCCTTCG
ATGGCGTGCAGTCCATGTTCGGATCA
Gap-Hash
4x3

8. Word use distribution for the mouse sequence data at ~7.5 fold
Useful Region
Poisson Curve
Real Data Curve

9. High bits
Low bits
Sorted List of Each k-Mer and Its Read Indices
ACAGAAAAGC
10h06.p1c
12a04.q1c
13d01.p1c
16d01.p1c
26g04.p1c
33h02.q1c
37g12.p1c
40d06.p1c
ACAGAAAAGG
16a02.p1c
20a10.p1c
22a03.p1c
26e12.q1c
30e12.q1c
47a01.p1c
64 -2k 2k

10. Relation Matrix: R(i,j) – number of kmer words shared between read i and read j1 2 3 4
Group 2: (4,6) 5 6 i
R(i,j)
Group 1: (1,2,3,5) N

11. Relation Matrix: R(i,j) – Implementation1 2 3 4
Number of shared kmer words (< 63) 5 .
Read index
R(i,j) N

12. Break contigs without read pair coverage

13. Tasmanian devil
Tasmanian devil
Opossum
Wallaby

14. Tasmanian devil facial tumour disease (DFTD)
Transmissible cancer characterised by the growth of large tumours on the face, neck and mouth of Tasmanian devils
Transmitted by biting
Commonly metastasises
First observed in 1996
Primarily affects adults >1yr
Death in 4 – 6 months

15. DFTD samples Area still DFTD free DFTD originated here c.1996
Narawntapu
Mt William (2)
Upper Natone
2006
Wisedale (?)
Frankford
Railton
2007
West Pencil Pine (3)
St Mary’s (2)
Reedy Marsh
Trowunna (2)
2008
Bronte Park
Coles Bay
Tarraleah
Kempton (2)
Mangalore
Fentonbury (no host)
Nugent (2) 4 14
Forestier (33) 13

16. DFTD samples for sequencing
Area still DFTD free
DFTD originated here c.1996
Narawntapu 2007
Mt William 2007 or 2008
Upper Natone 2007
Strain 1, tetraploid
Strain 2
Reedy Marsh 2007
Strain 3
“Evolved”
Unknown strain
Coles Bay
Mangalore 2007
Forestier 2007

17. Sequencing T. Devil on Illumina: Strategy
Tumour or normal genomic DNA
Fragments of defined size
0.5, 5, 7 kb
Sequencing
100 bp reads short insert
75 bp reads long insert
Sequencing performed at Illumina
Alignment using bwa, ssaha2
De novo Assembly
Somatic mutations
Germline variants

18. Paired Reads Separated by “NN”

19. Error Bases Correction

20. Genome Assembly – T. Devil
Solexa reads:
Number of read pairs: Million; Finished genome size: GB;
Read length: 2x100bp;
Estimated read coverage: ~30X;
Insert size: / bp;
Number of reads clustered: 458 Million
Assembly features: - contig stats
Phusion2 ABySS Total number of contigs: ,420,262 7,796,722
Total bases of contigs: Gb 2.28 Gb
N50 contig size: ,618 2,013
Largest contig: 76,
Averaged contig size: ,
Contig coverage on genome: ~94 % 65%
Mis-assembly errors: ? ?

22. Dog
Brown Bear
Macropus eugenii
(Wallaby)
Monodelphis domestica
( Opossum )
Sminthopsis macroura
(Dunnart)

23. Tasmanian devil
Tasmanian devil
Opossum
Wallaby

24. Melanoma cell line COLO-829
Paul Edwards, Departments of Pathology and Oncology, University of Cambridge

25. Human Cancer Genome Assembly – Normal Cell
Solexa reads:
Number of read pairs: Million; Finished genome size: GB;
Read length: 2x75bp;
Estimated read coverage: ~25X;
Insert size: / bp;
Number of reads clustered: 458 Million
Assembly features: - contig stats Total number of contigs: ,020,346;
Total bases of contigs: Gb
N50 contig size: ,344;
Largest contig: ,613
Averaged contig size: ,659;
Contig coverage over the genome: ~90 %;
Mis-assembly errors: ?

26. Genome Assembly – Tumour Cell
Solexa reads:
Number of read pairs: Million; Finished genome size: GB;
Read length: 2x75bp;
Estimated read coverage: ~25X;
Insert size: / bp;
Number of reads clustered: 449 Million
Assembly features: - contig stats Total number of contigs: ,249,719;
Total bases of contigs: Gb
N50 contig size: ,073;
Largest contig: 72,123
Averaged contig size: ,152;
Contig coverage over the genome: ~90 %;
Mis-assembly errors: ?

27. One Of the most difficult Genomes on earth?
Rice Genome Assembly
One Of the most difficult Genomes on earth?
Solexa reads:
Number of read pairs: Million; Finished genome size: MB;
Read length: 2x76bp;
Estimated read coverage: ~33X;
Insert size: / bp;
Number of reads clustered: Million
Assembly features: - contig stats Total number of contigs: ,713;
Total bases of contigs: Mb
N50 contig size: ,639;
Largest contig: 72,321
Averaged contig size: ;
Contig coverage over the genome: ~83 %;
Mis-assembly errors: ?

28. Acknowledgements: Elizabeth Murchuson Erin Preasance Mike Stratton
Dirk Evers
Ole Schulz-Trieglaff
Qi Feng
Bin Han