Web Apollo Workshop University of Exeter

An Introduction to Web Apollo
Manual Annotation Workshop at University of Exeter
Monica Munoz-Torres, PhD | @monimunozto
Berkeley Bioinformatics Open-Source Projects (BBOP)
Genomics Division, Lawrence Berkeley National Laboratory
At University of Exeter. October 8, 2014

TEACHING MATERIALS FOR TODAY
Demo
instance
1:
h/p://genomes.missouri.edu:8080/Amel_4.5_demo_1/selectTrack.jsp
Demo
instance
2:
h/p://genomes.missouri.edu:8080/Amel_4.5_demo_2/selectTrack.jsp
Recommended
Browser:
Chrome

OUTLINE
• MANUAL
ANNOTATION
working
concept
• COMMUNITY
BASED
CURATION
in
our
experience
• APOLLO
empowering
collaboraRve
curaRon
• APOLLO
on
THE
WEB
becoming
acquainted
• PRACTICE
demonstraRon
and
exercises
Web
Apollo
CollaboraRve
CuraRon
and
InteracRve
Analysis
of
Genomes
3

DURING THIS WORKSHOP
you will
v Learn
to
idenRfy
homologs
of
known
genes
of
interest
in
a
newly
sequenced
genome
of
interest.
v Become
familiar
with
the
environment
and
funcRonality
of
the
Web
Apollo
genome
annotaRon
ediRng
tool.
v Learn
how
to
corroborate
and
/
or
modify
automaRcally
annotated
gene
models
using
available
biological
evidence
in
Web
Apollo.
v Understand
the
process
of
curaRon
in
the
context
of
genome
annotaRon:
from
the
assembled
genome
to
manual
curaRon
via
automated
annotaRon.
4

I INVITE YOU TO:
v Observe
the
figures
v Listen
to
the
explanaRons
v Interrupt
me
at
any
Rme
to
ask
quesRons
v Use
Twi/er
&
share
your
thoughts:
I
am
@monimunozto
Some
tags
&
users:
#WebApollo
#AnnotaRon
#CuraRon
#GMOD
#genome
@JBrowseGossip
v Take
brakes:
LBL’s
ergo
safety
team
suggests
I
should
not
work
at
the
computer
for
>45
minutes
without
a
break;
neither
should
you!
We
will
be
here
for
2.5
hours:
please
get
up
and
stretch
your
neck,
arms,
and
legs
as
oeen
as
you
need.
5

I kindly ask that you refrain from:
v Reading
all
that
text
I
wrote!
Think
of
the
text
on
these
slides
as
your
“class
notes”.
You
will
use
them
during
exercises.
v Checking
email.
I’d
like
to
kindly
ask
for
your
undivided
a/enRon.

MANUAL ANNOTATION
working concept
v Automated
genome
analyses
remain
an
imperfect
art
that
cannot
yet
resolve
all
elements
of
the
genome.
v Precise
elucidaRon
of
biological
features
encoded
in
the
genome
requires
careful
examinaRon
and
review.
Schiex
et
al.
Nucleic
Acids
2003
(31)
13:
3738-‐3741
Automated Predictions
Experimental Evidence
cDNAs,
HMM
domain
searches,
RNAseq,
genes
from
other
species.
Manual Curation 8

9
Nucleic Acids 2003 vol. 31 no. 13 3738-3741
Manual Curation
GENE PREDICTION
v IdenRficaRon
of
protein-‐coding
genes,
tRNAs,
rRNAs,
regulatory
moRfs,
repeRRve
elements
(masked),
etc.
• Ab
ini-o
(DNA
composiRon):
Augustus,
GENSCAN,
geneid,
fgenesh
• Homology-‐based:
e.g:
SGP2,
fgenesh++

GENE ANNOTATION
IntegraRon
of
data
from
predicRon
tools
to
generate
a
consensus
set
of
predicRons
or
gene
models.
v Models
may
be
organized
using:
v automaRc
integraRon
of
predicted
sets;
e.g:
GLEAN
v packaged
tools
from
pipeline;
e.g:
MAKER
v All
available
biological
evidence
(e.g.
transcriptomes)
further
informs
the
annotaRon
process.
In
some
cases
algorithms
and
metrics
used
to
generate
consensus
sets
may
actually
reduce
the
accuracy
of
the
gene’s
representaRon;
in
such
cases
it
is
usually
be/er
to
use
an
10
ab
ini-o
model
to
create
a
new
annotaRon.
Manual Curation

MANUAL ANNOTATION
is necessary
v Evaluate
all
available
evidence
and
corroborate
or
modify
genome
element
predicRons.
v Determine
funcRonal
roles
through
comparaRve
analysis
using
literature,
databases,
and
experience*.
v Resolve
discrepancies
and
validate
automated
gene
model
hypotheses.
v Desktop
version
of
Apollo
was
designed
to
fit
the
manual
annotaRon
needs
of
genome
projects
such
as
fruit
fly,
mouse,
zebrafish,
human,
etc.
Manual Curation 11
Automated Predictions
Curated Gene Models
Official Gene Set
“Incorrect
and
incomplete
genome
annota-ons
will
poison
every
experiment
that
uses
them”.
-‐
M.
Yandell

BUT, MANUAL CURATION
did not always scale well
Too
many
sequences
and
not
enough
hands
to
approach
curaRon.
A
small
group
of
highly
trained
experts;
e.g.
GO
1
Museum
2
Jamboree
A
few
very
good
biologists
and
a
few
very
good
bioinformaRcians
camp
together,
during
intense
but
short
periods
of
Rme.
3
Co8age
Researchers
work
by
themselves,
then
may
or
may
not
publicize
results;
…
may
be
a
dead-‐end
with
very
few
people
ever
aware
of
these
results.
Elsik
et
al.
2006.
Genome
Res.
16(11):1329-‐33.
Manual Curation 12

POWER TO THE CURATORS
augment existing tools
Give
more
people
the
power
to
curate!
Fill
in
the
gap
for
all
the
things
that
won’t
be
easy
to
cover
with
these
approaches;
this
will
allow
researchers
to
be/er
contribute
their
efforts.
Big
data
are
not
a
subsRtute
for,
but
a
supplement
to
tradiRonal
data
collecRon
and
analysis.
The
Parable
of
Google
Flu.
Lazer
et
al.
2014.
Science
343
(6176):
1203-‐1205.
v Enable
more
curators
to
work
v Enable
be/er
scienRfic
publishing
v Credit
curators
for
their
work
Manual Curation 13

IMPROVING TOOLS FOR MANUAL ANNOTATION
our plan
“More
and
more
sequences”:
more
genomes,
within
populaRons
and
across
species,
are
now
being
sequenced.
This
begs
the
need
for
a
universally
accessible
genome
curaRon
tool:
To
produce
accurate
sets
of
genomic
features.
Manual Curation 14
To
address
the
need
to
correct
for
more
frequent
assembly
and
automated
predicRon
errors
due
to
new
sequencing
technologies.

GENOME ANNOTATION
an inherently collaborative task
Researchers
oeen
turn
to
colleagues
for
second
opinions
and
insight
from
those
with
experRse
in
parRcular
areas
(e.g.,
domains,
families).
To
facilitate
and
encourage
this,
we
conRnue
to
improve
Apollo.
New
Javascript-‐based
Apollo
:
h/p://GenomeArchitect.org
v Web
based
for
easy
access.
v Concurrent
access
supports
real
Rme
collaboraRon.
v Built-‐in
support
for
standards
(transparently
compliant).
v AutomaRc
generaRon
of
ready-‐made
computable
data.
v Client-‐side
applicaRon
relieves
server
bo/leneck
and
supports
privacy.
v Supports
annotaRon
of
genes,
pseudogenes,
tRNAs,
snRNAs,
snoRNAs,
ncRNAs,
miRNAs,
TEs,
and
repeats.
APOLLO 15

WEB APOLLO
v Integrated
with
JBrowse.
v Two
new
tracks:
“AnnotaRons”
and
“DNA
Sequence”
v IntuiRve
annotaRon,
gestures
and
pull-‐down
menus
to
create
and
edit
transcripts
and
exons
structures,
insert
comments
(CV,
freeform
text),
etc.
v Customizable
look,
feel
&
funcRonality.
v Edits
in
one
client
are
instantly
pushed
to
all
other
clients:
CollaboraRve!
16
APOLLO

WEB APOLLO
v Provides
dynamic
access
to
genomic
analysis
results
from
UCSC
and
Chado
databases,
as
well
as
database
storage
of
user-‐created
annotaRons.
v All
user-‐created
sequence
annotaRons
are
automaRcally
uploaded
to
a
server,
ensuring
reliability.
17
Chado
UCSC
(MySQL)
Ensembl
(DAS)
BAM
BED
BigWig
GFF3
MAKER
output
APOLLO

WEB APOLLO
architecture
1
APOLLO 18
2
3

DISPERSED COMMUNITIES
collaborative manual annotation efforts
We
conRnuously
train
and
support
hundreds
of
geographically
dispersed
scienRsts
from
many
research
communiRes
to
conduct
manual
annotaRons,
recovering
coding
sequences
in
agreement
with
all
available
biological
evidence
using
Web
Apollo.
v Gate
keeping
and
monitoring.
v Tutorials,
training
workshops,
and
geneborees.
v Personalized
user
support.
19
APOLLO

CURATION
in this context
20
1
IdenRfies
elements
that
best
represent
the
underlying
biology
(including
missing
genes)
and
eliminates
elements
that
reflect
systemic
errors
of
automated
analyses.
2
Assigns
funcRon
through
comparaRve
analysis
of
similar
genome
elements
from
closely
related
species
using
literature,
databases,
and
researchers’
lab
data.
Examples
Comparing
7
ant
genomes
contributed
to
be/er
understanding
evoluRon
and
organizaRon
of
insect
socieRes
at
the
molecular
level;
e.g.
division
of
labor,
mutualism,
chemical
communicaRon,
etc.
Libbrecht
et
al.
2012.
Genome
Biology
2013,
14:212
Queen
Bee
Insect
Methylome
Worker
Bee
Castes
Larva
Dnmt
Royal
jelly
RNAi
Kucharski
et
al.
2008.
Science
(319)
5871:
1827-‐1830
Anchoring
molecular
markers
to
reference
genome
pointed
to
chromosomal
rearrangements
&
detecRng
signals
of
adapRve
radiaRon
in
Heliconius
bu/erflies.
APOLLO Joron
et
al.
2011.
Nature,
477:203-‐206

WORKING TOGETHER
we have obtained better results
ScienRfic
community
efforts
bring
together
domain-‐specific
and
natural
history
experRse
that
would
otherwise
remain
disconnected.
Breaking
down
large
amounts
of
data
into
manageable
porRons
and
mobilizing
groups
of
researchers
to
extract
the
most
accurate
representaRon
of
the
biology
from
all
available
data
disRlls
invaluable
knowledge
from
genome
analysis.
21
APOLLO

CURRENT COLLABORATIONS
training and contributions
Partnerships
UNIVERSITY
of MISSOURI
Phlebotomus
papatasi
Wasmania
auropunctata
WEB APOLLO 22
National
Agricultural
Library
Nature
Reviews
Gene-cs
2009
(10),
346-‐347
Norwegian
Spruce
h/p://congenie.org/
Tallapoosa
darter
hGp://dendrome.ucdavis.edu/treegenes/browsers/
h/p://darter2.westga.edu/
Pinus
taeda
Homo
sapiens
hg19

TRAINING CURATORS
a little training goes a long way!
Provided
with
the
right
tools,
wet
lab
scienRsts
make
excepRonal
curators
who
can
easily
learn
to
maximize
the
generaRon
of
accurate,
biologically
supported
gene
models.
23
APOLLO

WEB APOLLO
the sequence selection window
Sort
Becoming Acquainted with Web Apollo. 25
25

WEB APOLLO
graphical user interface (GUI) for editing annotations
NavigaRon
tools:
pan
and
zoom
Grey
bar
of
coordinates
indicates
locaRon.
You
can
also
select
here
in
order
to
zoom
to
a
sub-‐region.
Search
box:
go
to
a
scaffold
or
a
gene
model.
‘View’:
change
color
by
CDS,
toggle
strands,
set
highlight.
‘File’:
Upload
your
own
evidence:
GFF3,
BAM,
BigWig,
VCF*.
Add
combinaRon
and
sequence
search
tracks.
‘Tools’:
Use
BLAT
to
query
the
genome
with
a
protein
or
DNA
sequence.
Available Tracks
‘User-‐created
AnnotaRons’
Track
Evidence
Tracks
Area
Login
26
Becoming Acquainted with Web Apollo.

WEB APOLLO
additional functionality
In
addiRon
to
protein-‐coding
gene
annotaRon
that
you
know
and
love.
• Non-‐coding
genes:
ncRNAs,
miRNAs,
repeat
regions,
and
TEs
• Sequence
alteraRons
(less
coverage
=
more
fragmentaRon)
• VisualizaRon
of
stage
and
cell-‐type
specific
transcripRon
data
as
coverage
plots,
heat
maps,
and
alignments
27
27

GENERAL PROCESS OF CURATION
steps to remember
1. Select
a
chromosomal
region
of
interest,
e.g.
scaffold.
2. Select
appropriate
evidence
tracks.
3. Determine
whether
a
feature
in
an
exisRng
evidence
track
will
provide
a
reasonable
gene
model
to
start
working.
-‐ If
yes:
select
and
drag
the
feature
to
the
‘User-‐created
AnnotaRons’
area,
creaJng
an
iniJal
gene
model.
If
necessary
use
ediRng
funcRons
to
adjust
the
gene
model.
-‐ If
not:
let’s
talk.
4. Check
your
edited
gene
model
for
integrity
and
accuracy
by
comparing
it
with
available
homologs.
Always
remember:
Becoming Acquainted 28 | with Web Apollo
when
annotaRng
gene
models
using
Web
Apollo,
you
are
looking
at
a
‘frozen’
version
of
the
genome
assembly
and
you
will
not
be
able
to
modify
the
assembly
itself.
28

Choose
(click
or
drag)
appropriate
evidence
tracks
from
the
list
on
the
lee.
Click
on
an
exon
to
select
it.
Double
click
on
an
exon
or
single
click
on
an
intron
to
select
the
enRre
gene.
Select
&
drag
any
elements
from
an
evidence
track
into
the
curaRon
area:
these
are
editable
and
considered
the
curated
version
of
the
gene.
Other
opRons
for
elements
in
evidence
tracks
available
from
right-‐click
menu.
If
you
select
an
exon
or
a
gene,
then
every
track
is
automaRcally
searched
for
exons
with
exactly
the
same
co-‐ordinates
as
what
you
selected.
Matching
edges
are
highlighted
red.
Hovering
over
an
annotaRon
in
progress
brings
up
an
informaRon
pop-‐up.
29 | Becoming Acquainted with Web Apollo. 2
9
USER NAVIGATION

USER NAVIGATION
Right-‐click
menu:
• With
the
excepRon
of
deleRng
a
model,
all
edits
can
be
reversed
with
‘Undo’
opRon.
‘Redo’
also
available.
All
changes
are
immediately
saved
and
available
to
all
users
in
real
Rme.
• ‘Get
sequence’
retrieves
pepRde,
cDNA,
CDS,
and
genomic
sequences.
• You
can
select
an
exon
and
select
‘Delete’.
You
can
create
an
intron,
flip
the
direcRon,
change
the
start
or
split
the
gene.
30 | Becoming Acquainted with Web Apollo.
30

USER NAVIGATION
Right-‐click
menu:
• If
you
select
two
gene
models,
you
can
join
them
using
‘Merge’,
and
you
may
also
‘Split’
a
model.
• You
can
select
‘Duplicate’,
for
example
to
annotate
isoforms.
• Set
translaRon
start,
annotate
selenocysteine-‐containing
proteins,
match
edges
of
annotaRon
to
those
of
evidence
tracks.
31

32
AnnotaRons,
annotaRon
edits,
and
History:
stored
in
a
centralized
database.
32
USER NAVIGATION

The
AnnotaRon
InformaRon
Editor
DBXRefs
are
database
crossreferences:
if
you
have
reason
to
believe
that
this
gene
is
linked
to
a
gene
in
a
public
database
(including
your
own),
then
add
it
here.
33
33
USER NAVIGATION

The
AnnotaRon
InformaRon
Editor
34
• Add
PubMed
IDs
• Include
GO
terms
as
appropriate
from
any
of
the
three
ontologies
• Write
comments
staRng
how
you
have
validated
each
model.
34
USER NAVIGATION

• ‘Zoom
35 |
to
base
level’
opRon
reveals
the
DNA
Track.
• Change
color
of
exons
by
CDS
from
the
‘View’
menu.
• The
reference
DNA
sequence
is
visible
in
both
direcRons
as
are
the
protein
translaRons
in
all
six
frames.
You
can
toggle
either
direcRon
to
display
only
3
frames.
Zoom
in/out
with
keyboard:
shie
+
arrow
keys
up/down
35
USER NAVIGATION

Web Apollo User Guide
(Fragment)
http://genomearchitect.org/web_apollo_user_guide

ANNOTATING SIMPLE CASES
In
a
“simple
case”
the
predicted
gene
model
is
correct
or
nearly
correct,
and
this
model
is
supported
by
evidence
that
completely
or
mostly
agrees
with
the
predicRon.
Evidence
that
extends
beyond
the
predicted
model
is
assumed
to
be
non-‐coding
sequence.
The
following
secRons
describe
simple
modificaRons.
37

ADDING EXONS
Select
and
drag
the
putaRve
new
exon
from
a
track,
and
add
it
directly
to
an
annotated
transcript
in
the
‘User-‐created
AnnotaRons’
area.
• Click
the
exon,
hold
your
finger
on
the
mouse
bu/on,
and
drag
the
cursor
unRl
it
touches
the
receiving
transcript.
A
dark
green
highlight
indicates
it
is
okay
to
release
the
mouse
bu/on.
• When
released,
the
addiRonal
exon
becomes
a/ached
to
the
receiving
transcript.
• A
38 |
confirmaRon
box
will
warn
you
if
the
receiving
transcript
is
not
on
the
same
strand
as
the
feature
where
the
new
exon
originated.
38

ADDING EXONS
Each
Rme
you
add
an
exon
region,
whether
by
extension
or
adding
an
exon,
Web
Apollo
recalculates
the
longest
ORF,
idenRfying
‘Start’
and
‘Stop’
signals
and
allowing
you
to
determine
whether
a
‘Stop’
codon
has
been
incorporated
aeer
each
ediRng
step.
39 |
Web
Apollo
demands
that
an
exon
already
exists
as
an
evidence
in
one
of
the
tracks.
You
could
provide
a
text
file
in
GFF
format
and
select
File
à
Open.
GFF
is
a
simple
text
file
delimited
by
TABs,
one
line
for
each
genomic
‘feature’:
column
1
is
the
name
of
the
scaffold;
then
some
text
(irrelevant),
then
‘exon’,
then
start,
stop,
strand
as
+
or
-‐,
a
dot,
another
dot,
and
Name=some
name
Example:
scaffold_88
Qratore
exon
21
2111
+
.
.
Name=bob
scaffold_88
Qratore
exon
2201
5111
+
.
.
Name=rad
39

ADDING UTRs
Gene
predicRons
may
or
may
not
include
UTRs.
If
transcript
alignment
data
are
available
and
extend
beyond
your
original
annotaRon,
you
may
extend
or
add
UTRs.
1. PosiRon
the
cursor
at
the
beginning
of
the
exon
that
needs
to
be
extended
and
‘Zoom
to
base
level’.
2. Place
the
cursor
over
the
edge
of
the
exon
unRl
it
becomes
a
black
arrow
then
click
and
drag
the
edge
of
the
exon
to
the
new
coordinate
posiRon
that
includes
the
UTR.
View
zoomed
to
base
level.
The
DNA
track
and
annotaRon
track
are
visible.
The
DNA
track
includes
the
sense
strand
(top)
and
anR-‐sense
strand
(bo/om).
The
six
reading
frames
flank
the
DNA
track,
with
the
three
forward
frames
above
and
the
three
reverse
frames
below.
The
User-‐
created
AnnotaRon
track
shows
the
terminal
end
of
an
annotaRon.
The
green
rectangle
highlights
the
locaRon
of
the
nucleoRde
residues
in
the
‘Stop’
signal.
40 |
To
add
a
new
spliced
UTR
to
an
exisRng
annotaRon
follow
the
procedure
for
adding
an
exon.
40

EXON STRUCTURE INTEGRITY
1. Zoom
in
sufficiently
to
clearly
resolve
each
exon
as
a
disRnct
rectangle.
2. Two
exons
from
different
tracks
sharing
the
same
start
and/or
end
coordinates
will
display
a
red
bar
to
indicate
the
matching
edges.
3. SelecRng
the
whole
annotaRon
or
one
exon
at
a
Rme,
use
this
‘edge-‐
matching’
funcRon
and
scroll
along
the
length
of
the
annotaRon,
verifying
exon
boundaries
against
available
data.
Use
square
[
]
brackets
to
scroll
from
exon
to
exon.
4. Note
if
there
are
cDNA
/
RNAseq
reads
that
lack
one
or
more
of
the
annotated
exons
or
include
addiRonal
exons.
41

EXON STRUCTURE INTEGRITY
To
modify
an
exon
boundary
and
match
data
in
the
evidence
tracks:
select
both
the
offending
exon
and
the
feature
with
the
expected
boundary,
then
right
click
on
the
annotaRon
to
select
‘Set
3’
end’
or
‘Set
5’
end’
as
appropriate.
42 |
In
some
cases
all
the
data
may
disagree
with
the
annotaRon,
in
other
cases
some
data
support
the
annotaRon
and
some
of
the
data
support
one
or
more
alternaRve
transcripts.
Try
to
annotate
as
many
alternaRve
transcripts
as
are
well
supported
by
the
data.
42

EDITING LOGIC
Flags
non-‐canonical
splice
sites.
SelecRon
of
features
and
sub-‐
features
Edge-‐matching
‘User-‐created
AnnotaRons’
Track
Evidence
Tracks
Area
The
ediRng
logic
in
the
server:
§ selects
longest
ORF
as
CDS
§ flags
non-‐canonical
splice
sites
43

SPLICE SITES
Zoom
to
base
level
to
review
non-‐
canonical
splice
site
warnings.
These
do
not
necessarily
need
to
be
corrected,
but
should
be
flagged
with
the
appropriate
comment.
Curated
model
Exon/intron
juncRon
possible
error
44 |
Original
model
Non-‐canonical
splices
are
indicated
by
an
orange
circle
with
a
white
exclamaRon
point
inside,
placed
over
the
edge
of
the
offending
exon.
Most
insects,
have
a
valid
non-‐canonical
site
GC-‐AG.
Other
non-‐canonical
splice
sites
are
unverified.
Web
Apollo
flags
GC
splice
donors
as
non-‐canonical.
Canonical
splice
sites:
forward
strand
5’-‐…exon]GT
/
AG[exon…-‐3’
reverse
strand,
not
reverse-‐complemented:
3’-‐…exon]GA
/
TG[exon…-‐5’
44

SPLICE SITES
keep this in mind
Some
gene
predicRon
algorithms
do
not
recognize
GC
splice
sites,
thus
the
intron/exon
juncRon
may
be
incorrect.
For
example,
one
such
gene
predicRon
algorithm
may
ignore
a
true
GC
donor
and
select
another
non-‐canonical
splice
site
that
is
less
frequently
observed
in
nature.
Therefore,
if
upon
inspecRon
you
find
a
non-‐
canonical
splice
site
that
is
rarely
observed
in
nature,
you
may
wish
to
search
the
region
for
a
more
frequent
in-‐frame
non-‐canonical
splice
site,
such
as
a
GC
donor.
If
there
is
an
in-‐frame
site
close
that
is
more
likely
to
be
the
correct
splice
donor,
you
may
make
this
adjustment
while
zoomed
at
base
level.
Curated model
Exon/intron junction possible error
Canonical
splice
sites:
5’-‐…exon]GT
/
AG[exon…-‐3’
reverse
strand,
not
reverse-‐complemented:
3’-‐…exon]GA
/
TG[exon…-‐5’
45 |
Original model
Use
RNA-‐Seq
data
to
make
a
decision.
forward
strand
45

‘START’ AND ‘STOP’ SITES
Web
Apollo
calculates
the
longest
possible
open
reading
frame
(ORF)
that
includes
canonical
‘Start’
and
‘Stop’
signals
within
the
predicted
exons.
If
it
appears
to
have
calculated
an
incorrect
‘Start’
signal,
you
may
modify
it
selecRng
an
in-‐frame
‘Start’
codon
further
up
or
downstream,
depending
on
evidence
(protein
database,
addiRonal
evidence
tracks).
An
upstream
‘Start’
codon
may
be
present
outside
the
predicted
gene
model,
within
a
region
supported
by
another
evidence
track.
46

‘START’ AND ‘STOP’ SITES
keep this in mind
Note
that
the
‘Start’
codon
may
also
be
located
in
a
non-‐predicted
exon
further
upstream.
If
you
cannot
idenRfy
that
exon,
add
the
appropriate
note
in
the
transcript’s
‘Comments’
secRon.
In
very
rare
cases,
the
actual
‘Start’
codon
may
be
non-‐canonical
(non-‐ATG).
In
some
cases,
a
‘Stop’
codon
may
not
be
automaRcally
idenRfied.
Check
to
see
if
there
are
data
supporRng
a
3’
extension
of
the
terminal
exon
or
addiRonal
3’
exons
with
valid
splice
sites.
47

Web Apollo Workshop University of Exeter

COMPLEX CASES
merge two gene predictions on the same scaffold
Evidence
may
support
joining
two
or
more
different
gene
models.
Warning:
protein
alignments
may
have
incorrect
splice
sites
and
lack
non-‐conserved
regions!
1. Drag
and
drop
each
gene
model
to
‘User-‐created
AnnotaRons’
area.
Shie
click
to
select
an
intron
from
each
gene
model
and
right
click
to
select
the
‘Merge’
opRon
from
the
menu.
2. Drag
supporRng
evidence
tracks
over
the
candidate
models
to
corroborate
overlap,
or
review
edge
matching
and
coverage
across
models.
3. Check
the
resulRng
translaRon
by
querying
a
protein
database
e.g.
UniProt.
Record
the
IDs
of
both
starRng
gene
models
in
‘DBXref’
and
add
comments
to
record
that
this
annotaRon
is
the
result
of
a
merge.
Red
lines
around
exons:
‘edge-‐matching’
allows
annotators
to
confirm
whether
the
evidence
is
in
agreement
without
examining
each
exon
at
the
base
level.
49

COMPLEX CASES
split a gene prediction
One
or
more
splits
may
be
recommended
when
different
segments
of
the
predicted
protein
align
to
two
or
more
different
families
of
protein
homologs,
and
the
predicted
protein
does
not
align
to
any
known
protein
over
its
enRre
length.
Transcript
data
may
support
a
split
(if
so,
verify
that
it
is
not
a
case
of
alternaRve
transcripts).
50

frameshifts, single-base errors, and selenocysteines
DNA
Track
COMPLEX CASES
‘User-‐created
AnnotaJons’
Track
51

COMPLEX CASES
frameshifts, single-base errors, and selenocysteines
1. Web
Apollo
allows
annotators
to
make
single
base
modificaRons
or
frameshies
that
are
reflected
in
the
sequence
and
structure
of
any
transcripts
overlapping
the
modificaRon.
Note
that
these
manipulaRons
do
NOT
change
the
underlying
genomic
sequence.
2. If
you
determine
that
you
need
to
make
one
of
these
changes,
zoom
in
to
the
nucleoRde
level
and
right
click
over
a
single
nucleoRde
on
the
genomic
sequence
to
access
a
menu
that
provides
opRons
for
creaRng
inserRons,
deleRons
or
subsRtuRons.
3. The
‘Create
Genomic
InserRon’
feature
will
require
you
to
enter
the
necessary
string
of
nucleoRde
residues
that
will
be
inserted
to
the
right
of
the
cursor’s
current
locaRon.
The
‘Create
Genomic
DeleRon’
opRon
will
require
you
to
enter
the
length
of
the
deleRon,
starRng
with
the
nucleoRde
where
the
cursor
is
posiRoned.
The
‘Create
Genomic
SubsRtuRon’
feature
asks
for
the
string
of
nucleoRde
residues
that
will
replace
the
ones
on
the
DNA
track.
4. Once
you
have
entered
the
modificaRons,
Web
Apollo
will
recalculate
the
corrected
transcript
and
protein
sequences,
which
will
appear
when
you
use
the
right-‐click
menu
‘Get
Sequence’
opRon.
Since
the
underlying
genomic
sequence
is
reflected
in
all
annotaRons
that
include
the
modified
region
you
should
alert
the
curators
of
your
organisms
database
using
the
‘Comments’
secRon
to
report
the
CDS
edits.
5. In
special
cases
such
as
selenocysteine
containing
proteins
(read-‐throughs),
right-‐click
over
the
offending/premature
‘Stop’
signal
and
choose
the
‘Set
readthrough
stop
codon’
opRon
from
the
menu.
52

COMPLETING THE ANNOTATION
Follow
our
checklist
unRl
you
are
happy
with
the
annotaRon!
Then:
– Comment
to
validate
your
annotaRon,
even
if
you
made
no
changes
to
an
exisRng
model.
Your
comments
mean
you
looked
at
the
curated
model
and
are
happy
with
it;
think
of
it
as
a
vote
of
confidence.
– Or
add
a
comment
to
inform
the
community
of
unresolved
issues
you
think
this
model
may
have.
Always
Remember:
53 | 53
Web
Apollo
curaRon
is
a
community
effort
so
please
use
comments
to
communicate
the
reasons
for
your
annotaRon
(your
comments
will
be
visible
to
everyone).

CHECK LIST
for accuracy and integrity
1. Can
you
add
UTRs
(e.g.:
via
RNA-‐Seq)?
2. Check
exon
structures
3. Check
splice
sites:
most
splice
sites
display
these
residues
…]5’-‐GT/AG-‐3’[…
4. Check
‘Start’
and
‘Stop’
sites
5. Check
the
predicted
protein
product(s)
– Align
it
against
relevant
genes/gene
family.
– blastp
against
NCBI’s
RefSeq
or
nr
6. If
the
protein
product
sRll
does
not
look
correct
then
check:
– Are
there
gaps
in
the
genome?
– Merge
of
2
gene
predicRons
on
the
same
scaffold
– Merge
of
2
gene
predicRons
from
different
scaffolds
– Split
a
gene
predicRon
– Frameshies
– error
in
the
genome
assembly?
– Selenocysteine,
single-‐base
errors,
and
other
inconvenient
phenomena
54 | 54
7. Finalize
annotaRon
by
adding:
– Important
project
informaRon
in
the
form
of
canned
and/or
customized
comments
– IDs
from
GenBank
(via
DBXRef),
gene
symbol(s),
common
name(s),
synonyms,
top
BLAST
hits
(with
GenBank
IDs),
orthologs
with
species
names,
and
everything
else
you
can
think
of,
because
you
are
the
expert.
– Whether
your
model
replaces
one
or
more
models
from
the
official
gene
set
(so
it
can
be
deleted).
– The
kinds
of
changes
you
made
to
the
gene
model
of
interest,
if
any.
E.g.:
splits,
merges,
whether
the
5’
or
3’
ends
had
to
be
modified
to
include
‘Start’
or
‘Stop’
codons,
addiRonal
exons
had
to
be
added,
or
non-‐canonical
splice
sites
were
accepted.
– Any
funcRonal
assignments
that
you
think
are
of
interest
to
the
community
(e.g.
via
BLAST,
RNA-‐Seq
data,
literature
searches,
etc.)

FUTURE PLANS
interactive analysis and curation of variants
v InteracRve
exploraRon
of
VCF
files
(e.g.
from
GATK,
VAAST)
in
addiRon
to
BAM
and
GVF.
MulRple
tracks
in
one:
visualizaRon
of
geneRc
alteraRons
and
populaRon
frequency
of
variants.
v Clinical
applicaRons:
analysis
of
WEB APOLLO 55
1
1
2
Copy
Number
VariaRons
for
regulatory
effects;
overlaying
display
of
the
regulatory
domains.
Philips-‐Creminis
and
Corces.
2013.
Cell
50
(4):461-‐474
2
TADs:
topologically
associaRng
domains

FUTURE PLANS
educational tools
We
are
working
with
educators
to
make
Web
Apollo
part
of
their
curricula.
In
the
classroom.
Lecture
Series.
WEB APOLLO 56
At
the
lab.
Classroom
exercises:
from
genome
sequence
to
hypothesis.
CuraRon
group
dedicated
to
producing
educaRon
materials
for
non-‐model
organism
communiRes.
Our
team
provides
online
documentaRon,
hands-‐on
training,
and
rapid
response
to
users.

Exercises
Live
DemonstraRon
using
the
Apis
mellifera
genome.
57
1.
Evidence
in
support
of
protein
coding
gene
models.
1.1
Consensus
Gene
Sets:
Official
Gene
Set
v3.2
Official
Gene
Set
v1.0
1.2
Consensus
Gene
Sets
comparison:
OGSv3.2
genes
that
merge
OGSv1.0
and
RefSeq
genes
OGSv3.2
genes
that
split
OGSv1.0
and
RefSeq
genes
1.3
Protein
Coding
Gene
PredicJons
Supported
by
Biological
Evidence:
NCBI
Gnomon
Fgenesh++
with
RNASeq
training
data
Fgenesh++
without
RNASeq
training
data
NCBI
RefSeq
Protein
Coding
Genes
and
Low
Quality
Protein
Coding
Genes
1.4
Ab
ini&o
protein
coding
gene
predicJons:
Augustus
Set
12,
Augustus
Set
9,
Fgenesh,
GeneID,
N-‐SCAN,
SGP2
1.5
Transcript
Sequence
Alignment:
NCBI
ESTs,
Apis
cerana
RNA-‐Seq,
Forager
Bee
Brain
Illumina
ConRgs,
Nurse
Bee
Brain
Illumina
ConRgs,
Forager
RNA-‐Seq
reads,
Nurse
RNA-‐Seq
reads,
Abdomen
454
ConRgs,
Brain
and
Ovary
454
ConRgs,
Embryo
454
ConRgs,
Larvae
454
ConRgs,
Mixed
Antennae
454
ConRgs,
Ovary
454
ConRgs
Testes
454
ConRgs,
Forager
RNA-‐Seq
HeatMap,
Forager
RNA-‐Seq
XY
Plot,
Nurse
RNA-‐Seq
HeatMap,
Nurse
RNA-‐Seq
XY
Plot

Exercises
Live
DemonstraRon
using
the
Apis
mellifera
genome.
58
1.
Evidence
in
support
of
protein
coding
gene
models
(ConJnued).
1.6
Protein
homolog
alignment:
Acep_OGSv1.2
Aech_OGSv3.8
Cflo_OGSv3.3
Dmel_r5.42
Hsal_OGSv3.3
Lhum_OGSv1.2
Nvit_OGSv1.2
Nvit_OGSv2.0
Pbar_OGSv1.2
Sinv_OGSv2.2.3
Znev_OGSv2.1
Metazoa_Swissprot
2.
Evidence
in
support
of
non
protein
coding
gene
models
2.1
Non-‐protein
coding
gene
predicJons:
NCBI
RefSeq
Noncoding
RNA
NCBI
RefSeq
miRNA
2.2
Pseudogene
predicJons:
NCBI
RefSeq
Pseudogene

Web Apollo Workshop Instances
h/p://genomes.missouri.edu:8080/Amel_4.5_demo_1
h/p://genomes.missouri.edu:8080/Amel_4.5_demo_2
Workshop
DocumentaRon
at

FEDERATED ENVIRONMENT
other BBOP tools
BBOP Projects 60

• Berkeley
BioinformaJcs
Open-‐source
Projects
(BBOP),
Berkeley
Lab:
Web
Apollo
and
Gene
Ontology
teams.
Suzanna
E.
Lewis
(PI).
• §
ChrisRne
G.
Elsik
(PI).
University
of
Missouri.
• *
Ian
Holmes
(PI).
University
of
California
Berkeley.
• Arthropod
genomics
community:
i5K
Steering
Commi/ee,
Alexie
Papanicolaou
(CSIRO),
Monica
Poelchau
(USDA/NAL),
fringy
Richards
(HGSC-‐BCM),
BGI,
1KITE
h/p://www.1kite.org/,
and
the
Honey
Bee
Genome
Sequencing
ConsorRum.
• AgriPest
Base,
Hymenoptera
Genome
Database,
VectorBase,
FlyBase.
• Web
Apollo
is
supported
by
NIH
grants
5R01GM080203
from
NIGMS,
and
5R01HG004483
from
NHGRI,
and
by
the
Director,
Office
of
Science,
Office
of
Basic
Energy
Sciences,
of
the
U.S.
Department
of
Energy
under
Contract
No.
DE-‐
AC02-‐05CH11231.
• Insect
images
used
with
permission:
h/p://AlexanderWild.com
and
O.
Niehuis.
• For
your
a8enJon,
thank
you!
Thanks!
Web
Apollo
Nathan
Dunn
Colin
Diesh
§
Deepak
Unni
§
BBOP
Gene
Ontology
Chris
Mungall
Seth
Carbon
Heiko
Dietze
Alumni
Gregg
Helt
Ed
Lee
Rob
Buels*
Web
Apollo:
h/p://GenomeArchitect.org
GO:
h/p://GeneOntology.org
i5K:
h/p://arthropodgenomes.org/wiki/i5K
Thank you. 61

Web Apollo Workshop University of Exeter

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