advanced practical course in genome...

Practical course in genome bioinformatics

Day 6 – lectureManual annotation (Web Apollo & automatic tools)

24 Feb 2017http://ekhidna.biocenter.helsinki.fi/downloads/teaching/spring2017

26.2.2016 [email protected] http://padlet.com/juhana_kammonen/bioinfo

http://padlet.com/juhana_kammonen/bioinfo

Genome project “roadmap”

• After experimental design and preparations a genome project can be roughly split into the following steps:

1. Sequencing2. (de novo) assembly, scaffolding3. RNA-sequencing and mapping4. Gene prediction5. Manual & functional annotation6. Submission and publication of the genome in a biodatabase7. Further downstream analysis

26.2.2016 [email protected]

You are here !



Manual annotation - outline

• Repetitive elements prediction (ab initio) finds and masks repeats in the genome

• Automated gene prediction reveals potential gene content from the genome• A challenging task especially in eukaryotic genomes

• After gene prediction the genome must be manually curated• Apply a set of trained human eyes to evaluate different tracks

of evidence and find potential errors in gene predictions



The ”rocky path” from de novoassembly to manual annotation



Evidence tracks not covered well by gene prediction

• RNA-sequencing coverage• Relatively weak correlation with

actual gene location but admittedly is an indication of expression level

• Should be included as an evidence track in Web Apollo

• Splicing in eukaryotes• Exons may be incorrectly linked /

separated in the gene models



Gene prediction and splicing


Cantarel BL, Korf I, Robb SMC, Parra G, Ross E, Moore B, Holt C, Sanchez Alvarado A, Yandell M (2008). MAKER: An easy-to-use annotation pipeline designed for emerging model organism genomes. Genome Research, 18(1), 188–196.


Gene prediction accuracy revisited



Web Apollo manual annotation tool


Lee E, Helt GA, Reese JT, Munoz-Torres MC, Childers CP, Buels RM, Stein L, Holmes IH, Elsik CG, Lewis SE (2013). Web Apollo: a web-based genomic annotation editing platform. Genome Biology, 14(8), R93.


Web Apollo annotation view



Basic recipe for manually annotating a single gene• Use as many relevant tracks of evidence as possible to

verify a predicted gene

• Align the predicted sequence against various databases

• BLAST

• Possible databases of related species

• Add comments on the annotation of your findings

• If the predicted annotation was modified, specify the reason

• Web Apollo allows the annotations to be marked e.g. as ”needs revision”



Today’s features

• Web Apollo – collaborative online manual annotation tool• Lee E, Helt GA, Reese JT, Munoz-Torres MC, Childers CP, Buels RM, Stein L,

Holmes IH, Elsik CG, Lewis SE (2013). Web Apollo: a web-based genomic annotation editing platform. Genome Biology, 14(8), R93.

• BLAST – Basic Local Alignment Search Tool• ”Swiss army knife” of a bioinformatician

• Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3):403-10

• MAFFT – Multiple Alignment Using Fast Fourier Transform• Katoh K, Misawa K, Kuma K, & Miyata T (2002). MAFFT: a novel method

for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research, 30(14), 3059–3066.



Manual annotation efforts at Viikki campus• Betula pendula (silver birch) genome annotation in

spring of 2014• 1000 genes curated and annotated during 3 weeks

• Taphrina betulina genome annotation in 2015• 800 genes curated and annotated during 2 weeks

• P. Hispida saimensis genome annotation coming up later this year



Next: Computer exercises

• Getting familiar with Web Apollo http://apollo.berkeleybop.org

• Example annotation of a gene in Apis mellifera (honeybee) genome with Web Apollo

• Confirmation of proper annotation using BLAST and MAFFT

• Download exercise sheet from: http://ekhidna.biocenter.helsinki.fi/downloads/teaching/spring2017/Exercises_day6.pdf


http://apollo.berkeleybop.org/

http://ekhidna.biocenter.helsinki.fi/downloads/teaching/spring2016/Exercises_day6.pdf


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