AMOS tools for assembly validation

1. AMOS tools for assembly validation • Automatically scan an assembly to locate misassembly signatures for further analysis and correction • Load Assembly Data into Bank • Evaluate Mate Pairs & Libraries • Evaluate Read Alignments • Evaluate Read Breakpoints • Analyze Depth of Coverage • Identify “Surrogates” • Load Misassembly Signatures into Bank AMOS Bank http://amos.sourceforge.net

2. Assembly QC: mate happiness • Evaluate mate “happiness” across assembly • Happy = Correct orientation and distance • Finds regions with multiple: • Compressed Mates (too close together) • Expanded Mates (too far apart) • Invalid same orientation () • Invalid “outie” orientation () • Missing Mates • Linking mates (mate in a different scaffold) • Singleton mates (mate is not in any contig) • Regions with high C/E statistic

3. Mate happiness • Excision:Skip reads between flanking repeats • Truth • Misassembly: Compressed Mates, Missing Mates

4. Mate happiness • Insertion:Additional reads between flanking repeats • Truth • Misassembly: Expanded Mates, Missing Mates

5. A B B A Mate happiness • Rearrangement: Reordering of reads • Truth • Misassembly: Misoriented Mates Note: if A,B too far apart, mates may all be “happy”

6. Compression/Expansion (C/E) Statistic • The presence of individual compressed or expanded mates is rare but expected • Do the inserts spanning a given position differ from the rest of the library? • Flag large differences as potential misassemblies • Even if each individual mate is “happy” • Compute the statistic at all positions • (Local Mean – Global Mean) / Scaling Factor • Introduced by Jim Yorke’s group at UMD

7. Library size variation 0kb 2kb 4kb 6kb 8 inserts: 3kb-6kb Local Mean: 4048 C/E Stat: (4048-4000) = +0.33 (400 / √8) Near 0 indicates overall happiness

8. C/E statistic: Compression 0kb 2kb 4kb 6kb 8 inserts: 3.2 kb-4.8kb Local Mean: 3488 C/E Stat: (3488-4000) = -3.62 (400 / √8) C/E Stat ≤ -3.0 indicates Compression

9. Read Alignment • Multiple reads with same conflicting base are unlikely • 1x QV 30: 1/1000 base calling error • 2x QV 30: 1/1,000,000 base calling error • 3x QV 30: 1/1,000,000,000 base calling error • Correlated SNPs are likely to be assembly errors, usually collapsed repeats • AMOS Tools: analyzeSNPs & clusterSNPs • Locate regions with high rate of correlated SNPs • Parameterized thresholds: • Multiple positions within 100bp sliding window • 2+ conflicting reads • Cumulative QV >= 40 (1/10000 base calling error) AG C AG C AG C AG C AG C AG C C TA C TAC TAC TAC TA

10. Read breakpoints: compression error ribosomal RNA repeats, B. anthracis • QC METHOD: • Align singleton reads to consensus assembly • Find any breakpoints shared by multiple reads mates “chimeric” reads

11. “Uncompress” by creating new repeat copy Reference: B. anthracis Ames ‘ancestor’ strain B. anthracis Ames Porton Down strain Tandem duplication

12. Read Coverage • Find regions of contigs where the depth of coverage is unusually high • AMOS Tool: analyzeReadDepth • 2.5x mean coverage B A R1 + R2 A R1 R2 B

13. Hawkeye: assembly viewer and debugger

14. Launch Pad

15. Histograms & Statistics Insert Size Read Length GC Content Overall Statistics • Bird’s eye view of data and assembly quality

16. Scaffold View • Statistical Plots • Scaffold • Features • Clone inserts • Overview • Control Panel • Details

17. Standard Feature Types [B] Breakpoint Alignment ends at this position [C] Coverage Location of unusual mate coverage (asmQC) [S] SNPs Location of Correlated SNPs [U] Unitig Used to report location of surrogate unitigs in CA assemblies [X] Other All other Features

18. Insert (mate) Happiness Happy • Oriented Correctly && • |Insert Size – Library.mean| <= Happy-Distance * Library.sd Stretched • Oriented Correctly && • Insert Size > Library.mean + Happy-Distance * Library.sd Compressed • Oriented Correctly && • Insert Size < Library.mean - Happy-Distance * Library.sd Misoriented • Same or Outies Linking • Read’s mate is in some other scaffold Singleton • Read’s mate is a singleton Unmated • No mate was provided for read Both mates present Only 1 read present

19. Discrepancy Navigation Contig Quick Select Discrepancy Regular Expression Consensus Search Consensus & Position Scrollable Read Tiling Summary Read Orientation Discrepancy Highlight Contig View: detailed alignment of reads to contigs

20. Zoom Out SNP View SNP Sorted Reads Polymorphism View

21. SNP Barcode SNP Sorted Reads Colored Rectangle indicate the positions and composition of the SNPs

22. Scaffold View Coverage CE Statistic SNP Feature Happy Stretched Compressed Misoriented Linking

23. Collapsed Repeat Read Coverage Spike -5.5 CE Dip Compressed Mates Cluster 68 Correlated SNPs

24. Example 1: Compression in Prevotella intermedia 17assembly, found by the CE statistic • Green inserts are <=2 standard deviations from the mean, and the orange inserts are compressed by > 2 standard deviations. • Vertical yellow line shows the most likely place of a compression misassembly. • Only one insert in this case is compressed by > 3 standard deviations

25. Example 2: Compression in Prevotella intermedia 17assembly, found by the CE statistic

26. Fixing collapsed repeats with AMOS Original Contig Compression Point Before Patch Contig Resolved “Stitched” Contig After

27. Assemblies can be preserved at NCBI’s Assembly Archivehttp://www.ncbi.nlm.nih.gov/Traces/assembly/assmbrowser.cgi