A new system for bacterial genome annotation designed for NGS data

BG7
A new system for bacterial genome
annotation designed for NGS data

www.ohnosequences.com www.era7bioinformatics.com

Motivation
Motivation
Features
The need of a system specially designed for NGS data
annotation with a pipeline unbiased by existing annotation systems
How it works? designed for Sanger sequences

The need of a versatile system able to annotate genes even in the
Comparisons
step of preliminary assembly of the genome

Upcoming features Special focus is given to the detection of “unexpected
proteins” without orthologous in close genomes (horizontally
acquired genes, phage genes, plasmid genes…)

A fast, automated and scalable process to face the
challenge of analyzing the huge amount of genomes that are being
sequenced with NGS technologies


Motivation
Features
Features

1. A new approach
How it works?

2. It’s tolerant to NGS errors
Comparisons

3. It’s based on cloud computing
Upcoming features

4. It uses bio4j


Motivation
Features: Approach
Features

How it works?
ORF prediction
Comparisons
is based on
Upcoming features
protein similarity


Motivation
Features: Approach
Features
Use as much information as you can
(not just start/stop signals)
How it works?

TGGATGTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAAAGGCTGA
Comparisons

Upcoming features


A B C D E


Motivation
Features: Approach
Features
Standard BG7
How it works? Sequence Sequence

Comparisons

Protein searching
Upcoming features ORF prediction (Blast)
(Glimmer)

CDS prediction

Function prediction RNA searching
(Blast) (Blast)


Motivation
Features: NGS errors
Features

Issue Technology
How it works?
Genomes in several contigs All
Comparisons Sequencing errors in start/stop codons Illumina substitutions
454 indels
Upcoming features Frameshifts 454 indels
Horizontal gene transfer None

BG7 system is tolerant to all these issues


Motivation
Features: Cloud computing
Features
AWS (Amazon Web Services)
How it works?

Comparisons Completely Scalable On demand

Upcoming features

Fast Cheap

Useful in tracking outbreaks
1 genome in ~2 hours
100 genomes in ~2 hours once you’ve got the reference proteins


Motivation
Features: bio4j
Features
It uses
How it works?

Comparisons

Upcoming features
Much richer
annotations

www.bio4j.com


Motivation
How it works?
Features

How it works?

Comparisons

Upcoming features


• Expert Manual Selection of reference sequences
1

• Protein search
2 • Blast

• CDS definition
• HSPs merge
3 • Extension of the similarity region searching for start/stop signals

• Solving conflicts
• Solving duplicates
4 • Solving overlaps

• RNA search
5 • Blast

• Incorporation of RNA genes
• Definition of RNA genes
6 • Conflicts with protein coding genes previously annotated are solved


Motivation
Step 2: Protein search with tBlastn
Features
A B C

How it works?

Comparisons

Upcoming features

Reference
Proteins (aa)

are searched in
the contigs sequences Input contigs (aa)


Motivation
Step 3: CDS definition
Features Merging HSPs

How it works?

Several HSPs
Comparisons
Input contigs (aa)
Upcoming features

Protein


Motivation
Features Merging HSPs

How it works?

Several HSPs
Comparisons
Input contigs (aa)
Upcoming features

Protein

We merge the HSPs to form a single similarity region


Motivation
Features Search for start/stop signals

How it works?

Comparisons

Upcoming features

We then search for start/stop signals upstream and
downstream the region with high similarity with the protein


Motivation
Features

Although we don’t find an start/stop codon for a given
How it works?
CDS we keep it

Comparisons We just mark it accordingly

Upcoming features


Motivation
Step 4: Solving conflicts
Features Duplicates

How it works?

Comparisons

Upcoming features


Motivation
Step 4: Solving conflicts
Features Overlapping CDS

How it works?

Comparisons

Upcoming features


Motivation
Step 5: RNA search
Features Blastn
Input contigs (nt)
How it works?

Comparisons

Upcoming features

Reference RNAs (nt) are searched in the contigs


Motivation
Step 6: Incorporation of RNA genes
Features Definition of RNA genes
Input contigs (nt)
How it works?

Comparisons

Upcoming features


Motivation
Step 6: Incorporation of RNA genes
Features Conflicts with protein coding genes are solved

How it works?

Comparisons

Upcoming features

If in a particular region we find a protein coding gene and
a RNA gene. RNA gene is selected over the protein coding
one


Motivation
Finally
Features

How it works?

Comparisons

Upcoming features


A B C D E


Motivation
Comparisons
Features
We’ve compared the NCBI annotations for
How it works?
Escherichia coli str. K-12 substr. MG1655
(Refseq ID NC_000913)
Comparisons

Upcoming features
With BG7 annotations


Motivation
Comparisons
Features
The results we got were:
How it works?

Comparisons
Feature NCBI BG7
Upcoming features Protein coding genes 4145 43701
49512
RNA 175 156

1 Selected genes
2 All detected genes: Selected + dismissed


Motivation
Comparisons
Features

How it works?

Comparisons

Upcoming features


Motivation
Comparisons
Features
Conclusions
How it works?

Even in a not advantageous situation
Comparisons (not a NGS project and a very well annotated genome)

Upcoming features We got in one round annotation step

- ~95% of the NCBI protein coding genes
- ~89% of the NCBI RNA genes
- 419 new proteins detected


Motivation
Upcoming features
Features
Improvements now focused on:
How it works?

- Overlapping solving phase
Comparisons

- Detection of very small proteins
Upcoming features

And any new need we find using it


Motivation
Thanks:
Features
Oh no sequences! team
How it works?
Raquel Tobes: Bioinformatician, main advisor
Comparisons
Pablo Pareja: Main developer
Upcoming features
Eduardo Pareja: Scientific advisor

Eduardo Pareja-Tobes: Mathematician, advisor

Carmen Torrecillas: Junior Bioinformatician

Marina Manrique: Bioinformatician


Thanks for your attention!


A new system for bacterial genome annotation designed for NGS data

Recommandé

Recommandé

Contenu connexe

Similaire à A new system for bacterial genome annotation designed for NGS data

Similaire à A new system for bacterial genome annotation designed for NGS data (20)

Dernier

Dernier (20)

A new system for bacterial genome annotation designed for NGS data