An introduction to Phage Genome Annotation (Viruses of Microbes 2018)

From Sequence to Knowledge
Assembly, Annotation, and Analysis
of Phage genomes from Isolated
Phages and Metagenomic Data Sets
A helping hand through
The Annotation Bottleneck
Ramy K. Aziz
Professor & Chair, Microbiology and Immunology, Faculty of
Pharmacy, Cairo University
(Twitter @azizrk)

PRELUDE
9 July 2018 Phage Genomics - VoM 2018

A bit of history…
• Since 2009, the Genomics Workshop has
become an essential part of the Evergreen
phage meeting
• The challenge always is: how to meet
needs/expectations that are so many and
so diverse, in ~4 hours
• The answer is:
…….

A bit of history…

Past workshops

MOTIVATION

“The analysis bottleneck”
• Observation:
– We generate more data than we can analyze.
– We generate sequence data faster
than we can analyze them.
• Opinion:
– Bottlenecks are not
created equal!
– It is important to define the question(s)
before working on the answer(s)!

9 July 2018 Phage Genomics - VoM 2018 Roux et al. Submitted

• The Lavigne paradox (2013)

• The Lavigne paradox (2015)

EXPECTATIONS
Audience

Attendees’ expectations
• Who (how many) among you have:
– annotated at least a phage genome?
– worked on a viral metagenome?
– used the command line (Unix, Linux, Mac
Terminal) for sequence analysis?
• To optimize the content, let’s
take this survey on SOCRATIVE
(http://socrative.com)
– Enter ROOM: AZIZ15

Activity: think, pair, share!
Defining the question(s):
• Introduce yourself, your institution, and your
favorite phage/virus
• Do you have a genome sequenced? Planning to?
– Why have you sequenced your phage genome?
– Why you want to sequence your phage genome?
• What is the single most pressing question you
want to have answered from genome analysis?
• What’s your top wish(es) for analysis tools that are
not in the current programs?

Input from group activity

DEFINING THE QUESTION(S)
“Begin with the end in mind”

What you want …... is
from genome from metagenome
Incomplete
frameshift
- complete
- accurate
Credit: Andrew Kropinski Credit: Bas Dutilh
faulty assembly

What you want …... is
from genome from metagenome
9 July 2018
Incomplete faulty assembly
frameshift
- complete
- accurate
Phage Genomics - VoM 2018
Credit: Andrew Kropinski Credit: Bas Dutilh

A process of reconstruction

• Experimentally
DNA
TGATTGTGTGTTTGCGCAATGCG
ATGTGTATATATAGTGAGCTTGCCC
GTCTCTCTNNNTCTCTTG
TGATTGGTCTNNNTCTCTTGCGCAATGCG

• Experimentally
• Computationally
GTCTCTCTNNNTCTCTTG
DNA
GTCTCTCTNNNTCTCTTG

• Experimentally
• Computationally
GTCTCTCTNNNTCTCTTG
“Any phage
one can get!”
“eDNA”
GTCTCTCTNNNTCTCTTG

What will be covered?
1. Annotation overview
2. Using the RAST family for genome annotation:
– Optimizing RAST for phages
– Command line/ Batch options
3. Introducing PATRIC and resources in
development
– Therapeutic phage database
– Assembly
– Variation analysis
– Metagenome binning

Assembly
Gene finding/
ORF calling
tRNA calling
Annotation
(Assigning
functions)
orienting
Validation
Fixing frameshifts
Introns and Inteins Subsystem
assignment
Refinement/
Secondary
annotation
loop
Special purpose:
toxins, morons, integrases,
lifestyle prediction
Regulatory elements
(promoters, terminators)
Output: files and graphics
From raw sequence data to
genome submission/ publication

Countless tools

Authority figures
Andrew Kropinski Rob Lavigne
Rob Edwards

Authority figures
Bonnie Hurwitz
Rodney Brister

The Kropinski wisdom

The Kropinski wisdom
1. Always use more than one tool.
2. Never blindly trust any automated (or manual)
process.
3. Use your eyes and hands: visual inspection/
manual proofreading, re-annotation
– Every apparently complicated file is still editable on
your favorite text editor (e.g., NotePad).
4. If you don’t know a gene’s function (if you
can’t convince your grandma), better keep it
unnamed than contribute to error propagation.

Material/Resources

Material/Resources
• Data & links:
– http://egybio.net/tutorial
• Slides
– http://bit.ly/annotation2018, http://bit.ly/phageRAST2018
– http://bit.ly/phagePATRIC
– Old tutorials (more detailed, but missing latest ):
• Evergreen 2011: http://slidesha.re/phantome1
• http://slidesha.re/phiRAST1 (by Karin Holmfeldt)
• Evergreen 2013: http://bit.ly/phantome2
• Evergreen 2015: http://bit.ly/phantome3
• VoM 2016: http://bit.ly/annotation2016, http://bit.ly/phantome4
• Evergreen 2017:
http://bit.ly/phigenomics2017 , http://bit.ly/phantome2017

ANNOTATION OVERVIEW

Desired outcome
Well characterized genome, in which, ideally we
know:
 the location & function of all the genes
 the location of promoters & terminators
 the correct taxonomy
PstI PstI
20
21
22
23
24
25
26
26A
27 28 29
30
31
32
33
30.0 kb
Viruses; dsDNA viruses, no RNA stage; Caudovirales; Siphoviridae;
T1virus

Desired outcome:
Create GenBank submission
• Complete, accurate description of the
genome and its taxonomy

Desired outcome (2)

Desired outcome (3)

Desired outcome (4)
 Protein products of concern, particularly
for those interested in phage therapy:
 Integrases
 Toxins
PstI PstI
20
21
22
23
24
25
26
26A
27 28 29
30
31
32
33
30.0 kb

Processes and Steps
I. Primary analysis
(QC/ pre-annotation proofreading: e.g., orient with BLASTN)
II. Genome annotation
– Gene finding (ORF calling)
– Automated annotation
– Massaging (edition, functional assignment)
III. Second dimension (regulatory elements)
IV. Comparative genomics
V. Metadata
VI. Visualization

Assembly
Gene finding/
ORF calling
tRNA calling
Annotation
(Assigning
functions)
orienting
Validation (segmenter)
Fixing frameshifts
Introns and Inteins Subsystem
assignment
Refinement/
Secondary
annotation
loop
Special purpose:
toxins, morons, integrases,
lifestyle prediction
Regulatory elements
(promoters, terminators)
Output: files and graphics
From raw sequence data to
genome submission/ publication

Classification
• The phage sequence space (Lima-Mendez et al.)
• The phage proteomic tree (Edwards & Rohwer)
• New: VIP tree http://www.genome.jp/viptree

AUTOMATED ANNOTATION
II. Genome Annotation

RAST (subsystems-based tools)
• Will be the major focus of this short
tutorial…
• The goal is:
1. Quick demo how to use RAST
2. Optimize RAST for phage annotation
3. New RAST implementation in the PATRIC
database
4. PATRIC features and future development

Nomenclature Sins
 hypothetical protein  DNA polymerase with no
or poor quality evidence is far worse than:
 DNA polymerase  hypothetical protein
 Be cautious about using BLASTP hits in naming
gps – is there additional evidence to back the
designation up

 All of these describe homologs of the
product of the coliphage T4 rIIA gene!
rIIA protector from prophage-induced early lysis
protector from prophage-induced early lysis
protector from prophage-induced early lysis rIIA
membrane-associated affects host membrane ATPase
rIIA membrane-associated affects host membrane ATPase
phage rIIA lysis inhibitor
rIIA protector
rIIA
rIIA protein
membrane integrity protector
hypothetical protein
unnamed protein product !!!!!!
protein of unknown function
Consistent Nomenclature

What do I call my gene product
(i.e. protein)?
 “phage hypothetical protein” – redundant
 “gp87” (gp = gene product)  hypothetical protein
 gp200 describes radically different proteins in
Listeria, Enterococcus, Mycobacterium,
Rhodococcus, Sphingomonas, Pseudomonas,
• Bacillus and Synechococcus phage genomes
 Add /note=“similar to gp43 of Escherichia coli
phage T4”

Bottom line:
Manual vs. Automated
• “Turtles know the road better than
rabbits… ” Khalil Gibran
• “… but they may never reach the end!”
• The best approach?
– Human expert-based annotation

IV. COMPARATIVE GENOMICS

Genomic pairwise comparisons
 EMBOSS Stretcher:http://emboss.bioinformatics.nl/cgi-
bin/emboss/stretcher N.B. genomes must be collinear
 BLASTN - NCBI
 ANI (Average Nucleotide Identity):http://enve-
omics.ce.gatech.edu/ani/
 GGDC 2.0 (Genome to Genome Distance Calculator):
http://ggdc.dsmz.de/distcalc2.php
 jSpeciesWS –
ANI:http://jspecies.ribohost.com/jspeciesws/

Proteomic pairwise
comparisons
 CoreGenes
(http://binf.gmu.edu:8080/CoreGenes3.0/)
 tBLASTX
 Remember protein sequence is more conserved
than DNA sequence; probably useful for more
distant relationships

V. METADATA

Standards: coming soon…
Roux, et al.

VI. “POLISH” IT TO PUBLISH IT

Servers & software
 BLAST Ring Image Generator (http://brig.sourceforge.net)
 CGView (http://wishart.biology.ualberta.ca/cgview)
 CGView Comparison Tool:
http://stothard.afns.ualberta.ca/downloads/CCT
 Circos (http://circos.ca)
 DNAPlotter:
(http://www.sanger.ac.uk/science/tools/dnaplotter)
 Easyfig (http://mjsull.github.io/Easyfig/)
 GenomeVx (http://wolfe.ucd.ie/GenomeVx)
 GView Server (https://server.gview.ca)
 progressiveMauve and ACT

EasyFig

CGView Comparison Tool

BLAST Ring Image Generator

An introduction to Phage Genome Annotation (Viruses of Microbes 2018)

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