Genome Annotation

Masking:

• Done to facilitate conventional gene annotation efforts.
• Helps avoid false SNP calls and mapping ambiguities.
• Hard Masking: replacing repeats with Ns {ACGTNNNNNNNNNATGG}
• Soft Masking: replacing repeats with lowercase {ACGTtagtagtagATGG}

Repeat Annotation:

• Different types of repeats can be studied along with their levels of activity (evolutionary analyses)

Types of Repeats:

• low-complexity sequence: microsatellites, homopolymers, etc.
• Transposable Elements:
• * class 1: retrotransposon; “copy & paste”; LTR, LINES, SINES
• * class 2: DNA transposons; “cut & paste”; subclass 1 and subclass 2

Repeat Content

• Does not necessarily correlate with genome size
• some correlation within the same group

Tools

• Homology: RepeatMasker
• denovo: RepeatModeler, WindowMasker, RepeatScout, Piler
• denovo from reads: REPdenovo, TEDNA
• NOTE: denovo tools run risk of false positives from highly conserved protein-coding genes.

Evidence-driven Annotation

• protein information, EST, RNA-Seq

Ab initio Gene Prediction

• doesn't require evidence data
• requires training for organism of interest
• most find single most likely CDS
• do not report UTR's (incomplete gene model)
• does not accommodate spliceoforms
• requires high-quality assembly (scaffold N50 ≈ avg gene size)

Combined Approach

• challenge of collating different models and sources of evidence.

Annotation Metrics

• Sensitivity, specificity, accuracy, AED
• AED = 1 - ACC = 1 - .5(Sensitivity+specificity)
• AED useful for identifying low quality inconsistent annotations (can be manually curated later)

Tools

• Pipelines: Maker2, Pasa, Ensembl, NCBI
• Evidence Mapping: BLAST/BLAT, Exonerate (computationally expensive)
• ab initio gene predictors: Augustus, SNAP, GeneMark
• Choosers and Combiners: JigSaw, Glean
• Visualization & Curation: Artemis, Apollo, JBROWSE, IGV