A hierarchical approach to building contig scaffolds

1
A hierarchical approach to building contig
scaffolds

• Mihai Pop
• Dan Kosack
• Steven L. Salzberg
• Genome Research 14(1), pp. 149-159, 2004.

2
Sequencing pipeline

• Random sequencing
• un-related reads 500-700 base-pairs
• Assembly
• un-related contigs 5,000-10,000 base-pairs
• Scaffolding
• un-related scaffolds 30,000-50,000 base-pairs
• Finishing/gap closure
• completed genomes millions-billions of
  base-pairs

3
Scaffolding

• Given a set of non-overlapping contigsorder and
  orient them along a chromosome

4
Clone-mates
Insert
R
F
Clone
5
Scaffolder output
Physical gaps


Sequencing gaps

6
Problems with the data

• Incorrect sizing of inserts
• cut from gel sizing is subjective
• error increases with size
• Chimeras (ends belong to different inserts)
• biological reasons (esp. for large sized inserts)
• sample tracking (human error)
• Software must handle a certain error rate.

7
Theoretical abstraction

• Given a set of entities (reads/contigs) and
  constraints between them (overlaps/mate pairs)
  provide a linear/circular embedding that
  preserves most constraints.

8
Graph representation

• Nodes contigs
• Directed edges constraints on relative
  placement of contigs relative order and
  relative orientation
• Embedding order (coordinate along chromosome)
  and orientation (strand sampled)

9
Challenges

• Orientation node coloring problem
  (forward/reverse)
• feasibility no cycles with odd number of
  reversal edges (blue edges)
• optimality remove minimum number of edges such
  that a solution exists (NP-hard)

10
Challenges

• Ordering generate a linear embedding
• feasibility lengths of parallel DAG paths are
  consistent
• optimality remove minimum number of edges such
  that DAG is feasible (NP-hard)

11
The real world

• Use of scaffolds
• Analysis longest unambiguous sub-graphs
• Finishing present all reliable relationships
  between contigs
• Sources of error
• mis-assemblies
• sizing errors (increases with library size)
• chimeras

12
Ambiguous scaffold
13
Hierarchical scaffolding

• For each contig pair, consolidate all linking
  data into a single relationship 2 correct
  links required

14
Hierarchical scaffolding

• Use most reliable links to build scaffolds
• Repeatedly build super-scaffolds based on less
  reliable linking data

15
Rationale
Problem complexity
problem size (nodes)
error rate
Hierarchical step
16
Linking information

• Overlaps
• Mate-pair links
• Similarity links
• Physical markers
• Gene synteny

reference genome
physical map
17
BAMBOO (BAMBUS)
Best effort Attempt Multiple Branches
allowed Order, Orient
18
Inputs

• Set of contigs names and lengths
• Groups of contig links
• groups correspond to quality of links
• link relative distance between contig origins
  relative orientation of contigs
• Priorities for each group specify order in
  which links are considered

19
Outputs

• XML representation of layout
• contig orientations
• contig position (x-coordinate of contig origin)
• links used to construct layout
• Graphical display of the layout
• uses GraphViz package from ATT

20
1.0 release

• XML input not yet supported
• All scaffold placed in the same output file
• Only Linux executable released
• Hacker friendly

21
Current release 2.33

• XML input and more general output module
• Collection of input modules from common assembly
  formats
• Better handling of priority data
• Repeat masking features
• More platforms supported and source code released
  as open source
• http//amos.sourceforge.net

22
Future enhancements

• Option to generate un-ambiguous (non-branching)
  scaffolds
• Better layout algorithms
• Specialized drawing tools
• Interactive browser
• Represent/handle multiple haplotypes

23
Acknowledgements

• Dan Kosack
• Steven Salzberg
• Martin Shumway
• Hean Koo
• Luke Tallon
• Jessica Vamathevan