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Accurate Assembly of Maize BACs Patrick S. Schnable Srinivas Aluru Iowa State University.

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Presentation on theme: "Accurate Assembly of Maize BACs Patrick S. Schnable Srinivas Aluru Iowa State University."— Presentation transcript:

1 Accurate Assembly of Maize BACs Patrick S. Schnable Srinivas Aluru Iowa State University

2 Motivation Maize genome is more complex than previously sequenced genomes – Many high-copy, long, highly conserved repeats – Genome contains many NIPs (Nearly Identical Paralogs, low-copy genes that are expressed and >98% identical; Emrich et al., 2007) (= CNPs and CNV) Hence, assembling this genome presents new challenges Are existing assembly programs up to the task?

3 Evidence of Assembly Errors Wash U noticed examples of collapse of repeats ISU identified examples of NIP collapse

4 AC AT GC B73 Mo17 SNP: single nucleotide polymorphism between alleles of a single gene Paramorphism (PM): a single nucleotide substitution between paralogs Nearly Identical Paralogs (NIPs): paralogous sequences with >99% identity Terms

5 Paramorphisms Provide Evidence of NIPs

6 Frequency of NIPs Conservatively ~1% of maize genes have NIPs (Emrich et al., 2007) Inspection of assembled BACs reveals NIP clusters But in addition also detect examples of NIP collapse CNPs/CNV associated with adaptive evolution in humans (Perry et al., Nat. Genetics, 2007)

7 BAC Assembly, Example 1 MAGI3.1 ID: MAGI_18749 (Emrich et al., 2007) BAC ID: CH C17 Paramorphic Sites: C/T (1,175), C/T (1,293), C/T (1,359) CH C17: gi| |gb|AC (152,054 bp) GenBank 56,57255, bp

8 BAC Assembly Example 1 - Site #1 BAC ID: CH C17 GI: GB: AC ,054 bp MAGI_18749 Paramorphic Site #1: C/T (1,175) 2 C vs 2 T Consensus Base Paramorphic Site #1 2/7 assembled BACs known to contain NIPs exhibit evidence of NIP collapse (conservative)

9 Traditional Assembly Sequence alignments between reads are identified Construct contigs – Start at a good alignment – Extend ends of contig one sequence at a time Clone pair information is used to scaffold contigs after contig construction.

10 Our Approach Integrate clone pair data into contig assembly process Model sequence alignments & clone pairs as a graph. First, construct an alignment graph Sequence reads are nodes A black edge is drawn between a pair of nodes if there is a valid sequence alignment

11 Clone Pair Informed Assembly Second, introduce two addl types of edges into the graph Clone pair edges (red) Path edges (green) A path edge exists between two nodes if: they are close together in the graph AND their clone pairs are also close together Identifies assembly-relevant sequence alignments

12 Repeat Example

13 Our Approach Series of graph transformations to ensure black edges (sequence alignments) represent correct genomic overlaps, and resolve entries into and exits out of repeats. – Use clone pairs to validate alignments in repeat regions if the corresponding mate pairs are anchored to unique regions and exhibit alignment. – Use paramorphisms to break spurious alignments due to NIPs. – Use clone pairs to match entries into and exits out of repeats. – Use clone pairs and validated alignments to guide contigs. – Use graph min-cuts to find correct assignment of reads to the complementary strands. – Use graph reductions and visualization for further analysis.

14 Example: Use Paramorphisms to Break Spurious Alignments GTCT A CAG GTCT C CAG GTCT A CAG GTCT C CAG

15 Three Random Stage 3 BACs Shotgun sequences extracted from Genbank and trimmed NameReadsPost TrimCorrupt Quality Info 273D N H

16 273D22 Annotate paths via walking through the graph. Make use of three levels of pointers: – Black edges: show what steps are available – Green edges: indicate the best path – Red edges: indicate our final destination

17 273D22: Incorrect Contiging Contig 0 Contig 1 Contig 1 is a small contig in the finished BAC that contains sequences that should be attached to the end of Contig 0.

18 273D22: Missing Scaffold

19 306N19: Mis-assembly Contig 3 Contig 5 Contig 0 Contig 4 Contig 3

20 306N19: Complex Repeat

21 D396H10: Missed Scaffolding Contig 6 Contig 8 Contig 5

22 D396H10: Missed Scaffolding Contig 7 Contig 2 Contig 3

23 Identifying Assembly Errors ???

24 273D22: Weak Link not Corroborated by Clone Pairs Contig 3

25 Conclusions & Future Directions Discovered misassembled regions in all three randomly chosen BACs – Conclusions supported by multiple lines evidence (clone pair + overlap) – Mis-assemblies (e.g., repeat-induced knots; collapsed repeats & NIPs) and missed scaffolding Benefits of our approach – Can provide better assemblies Can navigate through repeats Can correctly assemble NIPs – With development could output contigs and perform scaffolding in one step – Could provide refined finishing advice – Could include a community-accessible visualization of assembled BAC contigs and supporting data (confidence levels) Longer term – Our assembly approach could be applied to whole genome assembly of maize and other complex genomes – Could incorporate paired next generation sequencing data (e.g. 454, Solexa, Solid) Needed research – Random collection of finished BACs (truth) – Develop algorithms for navigating paths through the graph – Accurately construct final contigs that contain multiple copies of repeats – Create BAC re-assembly pipeline (inform finishing efforts in future sequencing projects) – Scale approach to whole genome level

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