AMOS tools for assembly validation

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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
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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

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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
Mate happiness

• Rearrangement Reordering of reads
• Truth
• Misassembly Misoriented Mates

Note if A,B too far apart, mates may all be
happy
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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 Yorkes group at UMD

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Library size variation
2kb
4kb
6kb
0kb
8 inserts 3kb-6kb Local Mean 4048 C/E Stat
(4048-4000) 0.33 (400 / v8)
Near 0 indicates overall happiness
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C/E statistic Compression
2kb
4kb
6kb
0kb
8 inserts 3.2 kb-4.8kb Local Mean 3488 C/E
Stat (3488-4000) -3.62 (400
/ v8) C/E Stat -3.0 indicates Compression
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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 gt 40 (1/10000 base calling error)

A G C A G C A G C A G C A G C A G C C T A C T A C
T A C T A C T A
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Read breakpoints compression error
ribosomal RNA repeats, B. anthracis

• QC METHOD
• Align singleton reads to consensus assembly
• Find any breakpoints shared by multiple reads

chimeric reads
mates
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Uncompress by creating new repeat copy
Reference B. anthracis Ames ancestor strain
B. anthracis Ames Porton Down strain
Tandem duplication
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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
B
R2
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Hawkeye assembly viewer and debugger
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Launch Pad
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Histograms Statistics
Insert Size
Read Length
GC Content
Overall Statistics

• Birds eye view of data and assembly quality

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Scaffold View

• Statistical Plots
• Scaffold
• Features
• Clone inserts
• Overview
• Control Panel
• Details

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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

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Insert (mate) Happiness

• Happy
• Oriented Correctly
• Insert Size Library.mean lt Happy-Distance
  Library.sd
• Stretched
• Oriented Correctly
• Insert Size gt Library.mean Happy-Distance
  Library.sd
• Compressed
• Oriented Correctly
• Insert Size lt Library.mean - Happy-Distance
  Library.sd
• Misoriented
• Same or Outies
• Linking
• Reads mate is in some other scaffold

Both mates present
Only 1 read present
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Contig View detailed alignment of reads to
contigs
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SNP View
SNP Sorted Reads
Polymorphism View
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SNP Barcode
SNP Sorted Reads
Colored Rectangle indicate the positions and
composition of the SNPs
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Scaffold View
Coverage
CE Statistic
SNP Feature
Happy
Stretched
Compressed
Misoriented
Linking
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Collapsed Repeat
Read Coverage Spike
-5.5 CE Dip
Compressed Mates Cluster
68 Correlated SNPs
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Example 1 Compression in Prevotella intermedia
17assembly, found by the CE statistic

• Green inserts are lt2 standard deviations from
  the mean, and the orange inserts are compressed
  by gt 2 standard deviations.
• Vertical yellow line shows the most likely place
  of a compression misassembly.
• Only one insert in this case is compressed by gt 3
  standard deviations

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Example 2 Compression in Prevotella intermedia
17assembly, found by the CE statistic
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Fixing collapsed repeats with AMOS
Original Contig
Compression Point
Before
Patch Contig
Resolved Stitched Contig
After
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Assemblies can be preserved at NCBIs Assembly
Archivehttp//www.ncbi.nlm.nih.gov/Traces/assembl
y/assmbrowser.cgi