Downloaded 15 times
![11BIO-REFRESHER
Splice sites
v The spliceosome catalyzes the removal of introns and the ligation of
flanking exons.
v Splicing signals (from the point of view of an intron):
• One splice signal (site) on the 5’ end: usually GT (less common: GC)
• And a 3’ end splice site: usually AG
• Canonical splice sites look like this: …]5’-GT/AG-3’[…](https://image.slidesharecdn.com/wangglf7qtaei1ju6dik-signature-93f5ba2088ff1dbb4fd91778b23dfe7ca4d8b3123b2aa1ae2e11b5a824146952-poli-160204100646/85/Introduction-to-Apollo-for-i5k-11-320.jpg)
![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
annota'on
to
select
‘Set
3’
end’
or
‘Set
5’
end’
as
appropriate.
In
some
cases
all
the
data
may
disagree
with
the
annota'on,
in
other
cases
some
data
support
the
annota'on
and
some
of
the
data
support
one
or
more
alterna've
transcripts.
Try
to
annotate
as
many
alterna've
transcripts
as
are
well
supported
by
the
data.
43
MATCHING EXON BOUNDARY TO EVIDENCE
BECOMING ACQUAINTED WITH APOLLO SIMPLE CASES](https://image.slidesharecdn.com/wangglf7qtaei1ju6dik-signature-93f5ba2088ff1dbb4fd91778b23dfe7ca4d8b3123b2aa1ae2e11b5a824146952-poli-160204100646/85/Introduction-to-Apollo-for-i5k-43-320.jpg)
![Non-‐canonical
splices
are
indicated
by
an
orange
circle
with
a
white
exclama'on
point
inside,
placed
over
the
edge
of
the
offending
exon.
Canonical
splice
sites:
3’-‐…exon]GA
/
TG[exon…-‐5’
5’-‐…exon]GT
/
AG[exon…-‐3’
reverse
strand,
not
reverse-‐complemented:
forward
strand
45
SPLICE SITES
Zoom
to
review
non-‐canonical
splice
site
warnings.
Although
these
may
not
always
have
to
be
corrected
(e.g
GC
donor),
they
should
be
flagged
with
a
comment.
Exon/intron
splice
site
error
warning
Curated
model
BECOMING ACQUAINTED WITH APOLLO SIMPLE CASES](https://image.slidesharecdn.com/wangglf7qtaei1ju6dik-signature-93f5ba2088ff1dbb4fd91778b23dfe7ca4d8b3123b2aa1ae2e11b5a824146952-poli-160204100646/85/Introduction-to-Apollo-for-i5k-45-320.jpg)
![1. Two
exons
from
different
tracks
sharing
the
same
start/end
coordinates
display
a
red
bar
to
indicate
matching
edges.
2. Selec'ng
the
whole
annota'on
or
one
exon
at
a
'me,
use
this
edge-‐
matching
func'on
and
scroll
along
the
length
of
the
annota'on,
verifying
exon
boundaries
against
available
data.
Use
square
[
]
brackets
to
scroll
from
exon
to
exon.
User
curly
{
}
brackets
to
scroll
from
annota'on
to
annota'on.
3. Check
if
cDNA
/
RNAseq
reads
lack
one
or
more
of
the
annotated
exons
or
include
addi'onal
exons.
47
CHECKING EXON INTEGRITY
BECOMING ACQUAINTED WITH APOLLO SIMPLE CASES](https://image.slidesharecdn.com/wangglf7qtaei1ju6dik-signature-93f5ba2088ff1dbb4fd91778b23dfe7ca4d8b3123b2aa1ae2e11b5a824146952-poli-160204100646/85/Introduction-to-Apollo-for-i5k-47-320.jpg)
![• Check
‘Start’
and
‘Stop’
sites.
• Check
splice
sites:
most
splice
sites
display
these
residues
…]5’-‐GT/AG-‐3’[…
• Check
if
you
can
annotate
UTRs,
for
example
using
RNA-‐Seq
data:
– align
it
against
relevant
genes/gene
family
– blastp
against
NCBI’s
RefSeq
or
nr
• Check
for
gaps
in
the
genome.
• Addi'onal
func'onality
may
be
necessary:
– merging
2
gene
predic'ons
-‐
same
scaffold
– merging
2
gene
predic'ons
-‐
different
scaffolds
– spli`ng
a
gene
predic'on
– annota'ng
frameshias
– annota'ng
selenocysteines,
correc'ng
single-‐base
and
other
assembly
errors,
etc.
62 | 62
• Add:
– Important
project
informa'on
in
the
form
of
comments
– IDs
from
public
databases
e.g.
GenBank
(via
DBXRef),
gene
symbol(s),
common
name(s),
synonyms,
top
BLAST
hits,
orthologs
with
species
names,
and
everything
else
you
can
think
of,
because
you
are
the
expert.
– Comments
about
the
kinds
of
changes
you
made
to
the
gene
model
of
interest,
if
any.
– Any
appropriate
func'onal
assignments,
e.g.
via
BLAST,
RNA-‐Seq
data,
literature
searches,
etc.
CHECKLIST
for accuracy and integrity
MANUAL ANNOTATION CHECKLIST](https://image.slidesharecdn.com/wangglf7qtaei1ju6dik-signature-93f5ba2088ff1dbb4fd91778b23dfe7ca4d8b3123b2aa1ae2e11b5a824146952-poli-160204100646/85/Introduction-to-Apollo-for-i5k-62-320.jpg)
![Finished model
Example 94
Corroborate
integrity
and
accuracy
of
the
model:
-‐
Start
and
Stop
-‐
Exon
structure
and
splice
sites
…]5’-‐GT/AG-‐3’[…
-‐
Check
the
predicted
protein
product
vs.
NCBI
nr,
UniProt,
etc.](https://image.slidesharecdn.com/wangglf7qtaei1ju6dik-signature-93f5ba2088ff1dbb4fd91778b23dfe7ca4d8b3123b2aa1ae2e11b5a824146952-poli-160204100646/85/Introduction-to-Apollo-for-i5k-94-320.jpg)
Uploaded byMonica Munoz-Torres
Introduction to Apollo for i5k
The webinar focuses on Apollo, a collaborative genome annotation editing tool for the i5k research community aimed at improving genome curation. Participants will learn about the curation process from automated to manual annotation, focusing on how to accurately identify gene models using comparative analysis and experimental evidence. The session emphasizes the importance of collaborative efforts and training for effective genome annotation, allowing for better representation of biological functions and error elimination in automated analyses.
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Introduction to Apollo for i5k
- 1. Introduction to Apollo Collaborativegenome annotation editing A webinar for the i5K Research Community - Hemiptera Monica Munoz-Torres | @monimunozto Berkeley Bioinformatics Open-Source Projects (BBOP) Environmental Genomics & Systems Biology Division, Lawrence Berkeley National Laboratory i5k Pilot Project Species Calls | 9 February, 2016 http://GenomeArchitect.org
- 2. Outline • Today youwill discover effective ways to extract valuable information about a genome through curation efforts. Apollo Collabora've Cura'on and Interac've Analysis of Genomes
- 3. After this talkyou will... • Better understand ‘curation’ in the context of genome annotation: assembled genome à automated annotation à manual annotation • Become familiar with Apollo’s environment and functionality. • Learn to identify homologs of known genes of interest in your newly sequenced genome. • Learn how to corroborate and modify automatically annotated gene models using all available evidence in Apollo.
- 4. Experimental design, sampling. Comparativeanalyses Official / Merged Gene Set Manual Annotation Automated Annotation Sequencing Assembly Synthesis & dissemination. This is our focus.
- 5. We must careabout curation Marbach et al. 2011. Nature Methods | Shutterstock.com | Alexander Wild The gene set of an organism informs a variety of studies: • Characterization: Gene number, GC%, TEs, repeats. • Functional assignments. • Molecular evolution, sequence conservation. • Gene families. • Metabolic pathways. • What makes an organism what it is? What makes a bee a “bee”?
- 6. Genome Curation Identifies elementsthat best represent the underlying biology and eliminates elements that reflect systemic errors of automated analyses. Assigns function through comparative analysis of similar genome elements from closely related species using literature, databases, and experimental data. Apollo Gene Ontology Resources
- 7. A few thingsto remember when conducting manual annotation To remember… Biological concepts to be;er understand manual annota'on 7BIO-REFRESHER • KEEP A GLOSSARY HANDY from con$g to splice site • WHAT IS A GENE? defining your goal • TRANSCRIPTION mRNA in detail • TRANSLATION reading frames, etc. • GENOME CURATION steps involved
- 8. The gene: a“moving target” “The gene is a union of genomic sequences encoding a coherent set of potentially overlapping functional products.” Gerstein et al., 2007. Genome Res
- 9. 9 "Gene structure" byDaycd- Wikimedia Commons BIO-REFRESHER mRNA • Although of brief existence, understanding mRNAs is crucial, as they will become the center of your work.
- 10. 10BIO-REFRESHER Reading frames v Ineukaryotes, only one reading frame per section of DNA is biologically relevant at a time: it has the potential to be transcribed into RNA and translated into protein. This is called the OPEN READING FRAME (ORF) • ORF = Start signal + coding sequence (divisible by 3) + Stop signal
- 11. 11BIO-REFRESHER Splice sites v Thespliceosome catalyzes the removal of introns and the ligation of flanking exons. v Splicing signals (from the point of view of an intron): • One splice signal (site) on the 5’ end: usually GT (less common: GC) • And a 3’ end splice site: usually AG • Canonical splice sites look like this: …]5’-GT/AG-3’[…
- 12. 12BIO-REFRESHER Exons and Introns v Introns can interrupt the reading frame of a gene by inserting a sequence between two consecutive codons v Between the first and second nucleotide of a codon v Or between the second and third nucleotide of a codon "Exon and Intron classes”. Licensed under Fair use via Wikipedia
- 13.
- 14. 14GENE PREDICTION &ANNOTATION PREDICTION & ANNOTATION v Iden'fica'on and annota'on of genome features: • primarily focuses on protein-‐coding genes. • also iden'fies RNAs (tRNA, rRNA, long and small non-‐coding RNAs (ncRNA)), regulatory mo'fs, repe''ve elements, etc. • happens in 2 phases: 1. Computa'on phase 2. Annota'on phase
- 15. 15GENE PREDICTION &ANNOTATION COMPUTATION PHASE a. Experimental data are aligned to the genome: expressed sequence tags, RNA-‐sequencing reads, proteins (also from other species). b. Gene predic;ons are generated: -‐ ab ini$o: based on nucleo'de sequence and composi'on e.g. Augustus, GENSCAN, geneid, fgenesh, etc. -‐ evidence-‐driven: iden'fying also domains and mo'fs e.g. SGP2, JAMg, fgenesh++, etc. Result: the single most likely coding sequence, no UTRs, no isoforms. Yandell & Ence. Nature Rev 2012 doi:10.1038/nrg3174
- 16. 16GENE PREDICTION &ANNOTATION ANNOTATION PHASE Experimental data (evidence) and predic'ons are synthe'zed into gene annota'ons. Result: gene models that generally include UTRs, isoforms, evidence trails. Yandell & Ence. Nature Rev 2012 doi:10.1038/nrg3174 5’ UTR 3’ UTR
- 17. 17 In some cases algorithms and metrics used to generate consensus sets may actually reduce the accuracy of the gene’s representa'on. CONSENSUS GENE SETS Gene models may be organized into sets using: v combiners for automa'c integra'on of predicted sets e.g: GLEAN, EvidenceModeler or v tools packaged into pipelines e.g: MAKER, PASA, Gnomon, Ensembl, etc. GENE PREDICTION & ANNOTATION
- 18. ANNOTATION needs some refinement Noone is perfect, least of all automated annotation. 18 New technologies bring new challenges: • Assembly errors can cause fragmented annota'ons • Limited coverage makes precise iden'fica'on a difficult task Image: www.BroadInstitute.org
- 19. MANUAL ANNOTATION improving predictions Precise elucida;on of biological features encoded in the genome requires careful examina;on and review. Schiex et al. Nucleic Acids 2003 (31) 13: 3738-‐3741 Automated Predictions Experimental Evidence Manual Annotation – to the rescue. 19 cDNAs, HMM domain searches, RNAseq, genes from other species.
- 20. GENOME CURATION an inherentlycollaborative task GENE PREDICTION & ANNOTATION 20 So many sequences, not enough hands. Apis mellifera | Alexander Wild | www.alexanderwild.com
- 21. We have providedcontinuous training and support for hundreds of geographically dispersed scientists to conduct manual annotations efforts in order to recover coding sequences in agreement with all available biological evidence. 21 Lessons learned APOLLO • Collaborative work distills invaluable knowledge. • A little training goes a long way! Wet lab scientists can easily learn to maximize the generation of accurate, biologically supported gene models.
- 22.
- 23. APOLLO: versatile genomeannotation editing • Apollo is a web-based genome annotation editor, integrated with JBrowse • Supports real time collaboration & generates analysis-ready data USER-CREATED ANNOTATIONS EVIDENCE TRACKS ANNOTATOR PANEL
- 24. BECOMING ACQUAINTED WITHAPOLLO 24 General process of curation 1. Select or find a region of interest, e.g. scaffold. 2. Select appropriate evidence tracks to review the gene model. 3. Determine whether a feature in an exis'ng evidence track will provide a reasonable gene model to start working. 4. If necessary, adjust the gene model. 5. Check your edited gene model for integrity and accuracy by comparing it with available homologs. 6. Comment and finish.
- 25. Apollo - versionat i5K Workspace@NAL 254. Becoming Acquainted with Web Apollo. 25 The Sequence Selec'on Window
- 26. Sort Apollo - versionat i5K Workspace@NAL 26 “Old Track Select Page” 4. Becoming Acquainted with Web Apollo. 26
- 27. 27 APOLLO annotation editing environment BECOMINGACQUAINTED WITH APOLLO Color by CDS frame, toggle strands, set color scheme and highlights. -‐ Upload evidence files (GFF3, BAM, BigWig), -‐ combina;on track -‐ sequence search track Query the genome using BLAT. Naviga'on and zoom. Search for a gene model or a scaffold. Get coordinates and “rubber band” selec'on for zooming. Login User-‐created annota'ons. New annotator panel. Evidence Tracks Stage and cell-‐type specific transcrip'on data. h;p://genomearchitect.org/web_apollo_user_guide
- 28. 28 | 28 BECOMINGACQUAINTED WITH APOLLO USER NAVIGATION Annotator panel. • Choose appropriate evidence from list of “Tracks” on annotator panel. • Select & drag elements from evidence track into the ‘User-‐created Annota$ons’ area. • Hovering over annota'on in progress brings up an informa'on pop-‐up. • Crea'ng a new annota'on
- 29. Adding a genemodel
- 30. Adding a genemodel
- 31. Adding a genemodel
- 32.
- 33. Editing functionality Example: Addingan exon supported by experimental data • RNAseq reads show evidence in support of a transcribed product that was not predicted. • Add exon by dragging up one of the RNAseq reads.
- 34. Editing functionality Example: Adjustingexon boundaries supported by experimental data
- 35.
- 36. 36 | 36 USERNAVIGATION BECOMING ACQUAINTED WITH APOLLO • ‘Zoom to base level’ reveals the DNA Track.
- 37. 37 | 37 USERNAVIGATION BECOMING ACQUAINTED WITH APOLLO • Color exons by CDS from the ‘View’ menu.
- 38. 38 | Zoom in/out with keyboard: shio + arrow keys up/down 38 USER NAVIGATION BECOMING ACQUAINTED WITH APOLLO • Toggle reference DNA sequence and transla;on frames in forward strand. Toggle models in either direc'on.
- 39.
- 40. “Simple case”: -‐ the predicted gene model is correct or nearly correct, and -‐ this model is supported by evidence that completely or mostly agrees with the predic'on. -‐ evidence that extends beyond the predicted model is assumed to be non-‐coding sequence. The following are simple modifica'ons. 40 ANNOTATING SIMPLE CASES BECOMING ACQUAINTED WITH APOLLO SIMPLE CASES
- 41. • A confirma'on box will warn you if the receiving transcript is not on the same strand as the feature where the new exon originated. • Check ‘Start’ and ‘Stop’ signals aoer each edit. 41 ADDING EXONS BECOMING ACQUAINTED WITH APOLLO SIMPLE CASES
- 42. If transcript alignment data are available & extend beyond your original annota'on, you may extend or add UTRs. 1. Right click at the exon edge and ‘Zoom to base level’. 2. Place the cursor over the edge of the exon un$l it becomes a black arrow then click and drag the edge of the exon to the new coordinate posi'on that includes the UTR. 42 ADDING UTRs To add a new spliced UTR to an exis'ng annota'on also follow the procedure for adding an exon. BECOMING ACQUAINTED WITH APOLLO SIMPLE CASES
- 43. 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 annota'on to select ‘Set 3’ end’ or ‘Set 5’ end’ as appropriate. In some cases all the data may disagree with the annota'on, in other cases some data support the annota'on and some of the data support one or more alterna've transcripts. Try to annotate as many alterna've transcripts as are well supported by the data. 43 MATCHING EXON BOUNDARY TO EVIDENCE BECOMING ACQUAINTED WITH APOLLO SIMPLE CASES
- 44. Non-‐canonical splice sites flags. Double click: selec'on of feature and sub-‐features Evidence Tracks Area ‘User-‐created Annota$ons’ Track Edge-‐matching Apollo’s edi'ng logic (brain): § selects longest ORF as CDS § flags non-‐canonical splice sites 44 ORFs AND SPLICE SITES BECOMING ACQUAINTED WITH APOLLO SIMPLE CASES
- 45. Non-‐canonical splices are indicated by an orange circle with a white exclama'on point inside, placed over the edge of the offending exon. Canonical splice sites: 3’-‐…exon]GA / TG[exon…-‐5’ 5’-‐…exon]GT / AG[exon…-‐3’ reverse strand, not reverse-‐complemented: forward strand 45 SPLICE SITES Zoom to review non-‐canonical splice site warnings. Although these may not always have to be corrected (e.g GC donor), they should be flagged with a comment. Exon/intron splice site error warning Curated model BECOMING ACQUAINTED WITH APOLLO SIMPLE CASES
- 46. Apollo calculates the longest possible open reading frame (ORF) that includes canonical ‘Start’ and ‘Stop’ signals within the predicted exons. If ‘Start’ appears to be incorrect, modify it by selec'ng an in-‐frame ‘Start’ codon further up or downstream, depending on evidence (proteins, RNAseq). It may be present outside the predicted gene model, within a region supported by another evidence track. In very rare cases, the actual ‘Start’ codon may be non-‐canonical (non-‐ATG). 46 ‘Start’ AND ‘Stop’ SITES BECOMING ACQUAINTED WITH APOLLO SIMPLE CASES
- 47. 1. Two exons from different tracks sharing the same start/end coordinates display a red bar to indicate matching edges. 2. Selec'ng the whole annota'on or one exon at a 'me, use this edge-‐ matching func'on and scroll along the length of the annota'on, verifying exon boundaries against available data. Use square [ ] brackets to scroll from exon to exon. User curly { } brackets to scroll from annota'on to annota'on. 3. Check if cDNA / RNAseq reads lack one or more of the annotated exons or include addi'onal exons. 47 CHECKING EXON INTEGRITY BECOMING ACQUAINTED WITH APOLLO SIMPLE CASES
- 48.
- 49. Evidence may support joining two or more different gene models. Warning: protein alignments may have incorrect splice sites and lack non-‐conserved regions! 1. In ‘User-‐created Annota<ons’ area shio-‐click to select an intron from each gene model and right click to select the ‘Merge’ op'on from the menu. 2. Drag suppor'ng evidence tracks over the candidate models to corroborate overlap, or review edge matching and coverage across models. 3. Check the resul'ng transla'on by querying a protein database e.g. UniProt, NCBI nr. Add comments to record that this annota'on is the result of a merge. 49 Red lines around exons: ‘edge-‐matching’ allows annotators to confirm whether the evidence is in agreement without examining each exon at the base level. COMPLEX CASES merge two gene predictions on the same scaffold BECOMING ACQUAINTED WITH APOLLO COMPLEX CASES
- 50. One or more splits may be recommended when: -‐ different segments of the predicted protein align to two or more different gene families -‐ predicted protein doesn’t align to known proteins over its en're length -‐ Transcript data may support a split, but first verify whether they are alterna've transcripts. 50 COMPLEX CASES split a gene prediction BECOMING ACQUAINTED WITH APOLLO COMPLEX CASES
- 51. DNA Track ‘User-‐created Annota;ons’ Track 51 COMPLEX CASES annotate frameshifts and correct single-base errors Always remember: when annota'ng gene models using Apollo, you are looking at a ‘frozen’ version of the genome assembly and you will not be able to modify the assembly itself. BECOMING ACQUAINTED WITH APOLLO COMPLEX CASES
- 52. 52 COMPLEX CASES correcting selenocysteinecontaining proteins BECOMING ACQUAINTED WITH APOLLO COMPLEX CASES
- 53. 53 COMPLEX CASES correcting selenocysteinecontaining proteins BECOMING ACQUAINTED WITH APOLLO COMPLEX CASES
- 54. 1. Apollo allows annotators to make single base modifica'ons or frameshios that are reflected in the sequence and structure of any transcripts overlapping the modifica'on. These manipula'ons do NOT change the underlying genomic sequence. 2. If you determine that you need to make one of these changes, zoom in to the nucleo'de level and right click over a single nucleo'de on the genomic sequence to access a menu that provides op'ons for crea'ng inser'ons, dele'ons or subs'tu'ons. 3. The ‘Create Genomic Inser<on’ feature will require you to enter the necessary string of nucleo'de residues that will be inserted to the right of the cursor’s current loca'on. The ‘Create Genomic Dele<on’ op'on will require you to enter the length of the dele'on, star'ng with the nucleo'de where the cursor is posi'oned. The ‘Create Genomic Subs<tu<on’ feature asks for the string of nucleo'de residues that will replace the ones on the DNA track. 4. Once you have entered the modifica'ons, Apollo will recalculate the corrected transcript and protein sequences, which will appear when you use the right-‐click menu ‘Get Sequence’ op'on. Since the underlying genomic sequence is reflected in all annota'ons that include the modified region you should alert the curators of your organisms database using the ‘Comments’ sec'on 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’ op'on from the menu. 54 COMPLEX CASES annotating frameshifts and correcting single-base errors & selenocysteines BECOMING ACQUAINTED WITH APOLLO COMPLEX CASES
- 55. 55 | 55 USERNAVIGATION BECOMING ACQUAINTED WITH APOLLO • Information Editor
- 56. 56 The Annota'on Informa;on Editor 56 USER NAVIGATION BECOMING ACQUAINTED WITH APOLLO
- 57. 57 The Annota'on Informa;on Editor • Add PubMed IDs • Include GO terms as appropriate from any of the three ontologies • Write comments sta'ng how you have validated each model. 57 USER NAVIGATION BECOMING ACQUAINTED WITH APOLLO
- 58. 58 | 58 USERNAVIGATION BECOMING ACQUAINTED WITH APOLLO • Keeping track of each edit
- 59. 59 Annota'ons, annota'on edits, and History: stored in a centralized database. 59 USER NAVIGATION BECOMING ACQUAINTED WITH APOLLO
- 60. Follow the checklist un'l you are happy with the annota'on! And remember to… – comment to validate your annota'on, even if you made no changes to an exis'ng model. Think of comments as your vote of confidence. – or add a comment to inform the community of unresolved issues you think this model may have. 60 | 60 Always Remember: Apollo cura'on is a community effort so please use comments to communicate the reasons for your annota'on. Your comments will be visible to everyone. COMPLETING THE ANNOTATION BECOMING ACQUAINTED WITH APOLLO
- 61.
- 62. • Check ‘Start’ and ‘Stop’ sites. • Check splice sites: most splice sites display these residues …]5’-‐GT/AG-‐3’[… • Check if you can annotate UTRs, for example using RNA-‐Seq data: – align it against relevant genes/gene family – blastp against NCBI’s RefSeq or nr • Check for gaps in the genome. • Addi'onal func'onality may be necessary: – merging 2 gene predic'ons -‐ same scaffold – merging 2 gene predic'ons -‐ different scaffolds – spli`ng a gene predic'on – annota'ng frameshias – annota'ng selenocysteines, correc'ng single-‐base and other assembly errors, etc. 62 | 62 • Add: – Important project informa'on in the form of comments – IDs from public databases e.g. GenBank (via DBXRef), gene symbol(s), common name(s), synonyms, top BLAST hits, orthologs with species names, and everything else you can think of, because you are the expert. – Comments about the kinds of changes you made to the gene model of interest, if any. – Any appropriate func'onal assignments, e.g. via BLAST, RNA-‐Seq data, literature searches, etc. CHECKLIST for accuracy and integrity MANUAL ANNOTATION CHECKLIST
- 63.
- 64. 64i5K Workspace@NAL The collaborativecuration process at i5k 1. A computa'onally predicted consensus gene set has been generated using mul'ple lines of evidence; e.g. HVIT_v0.5.3-‐Models 2. i5K Projects will integrate consensus computa'onal predic'ons with manual annota'ons to produce an updated Official Gene Set (OGS): Achtung! • If it’s not on either track, it won’t make the OGS! • If it’s there and it shouldn’t, it will s'll make the OGS!
- 65. 65 The ‘Replace Models’rules 65 BECOMING ACQUAINTED WITH APOLLO http://tinyurl.com/apollo-i5k-replace
- 66. 66i5K Workspace@NAL 3. In some cases algorithms and metrics used to generate consensus sets may actually reduce the accuracy of the gene’s representa'on. Use your judgment, try choosing a different model to begin the annota'on. 4. Isoforms: drag original and alterna'vely spliced form to ‘User-‐created Annota<ons’ area. 5. If an annota'on needs to be removed from the consensus set, drag it to the ‘User-‐created Annota<ons’ area and label as ‘Delete’ on the Informa$on Editor. 6. Overlapping interests? Collaborate to reach agreement. 7. Follow guidelines for i5K Pilot Species Projects, at h;p://goo.gl/LRu1VY The collaborative curation process at i5k
- 67.
- 68. What’s new?... findinginspiration in PubMed. Example 68 “Molecular analysis of bed bug populations from across the USA and Europe found that >80% and >95% of the respective populations contained V419L and/ or L925I mutations in the voltage-gated sodium channel gene, indicating widespread distribution of target-site-based pyrethroid resistance.” Homalodisca vitripennis | Alexander Wild | www.alexanderwild.comHalyomorpha halys | Fondazione Edmund Mach - Italy Now for our species of interest. . .
- 69. Example Example 69 Cura'on example using the Hyalella azteca genome (amphipod crustacean).
- 70. What do weknow about this genome? • Currently publicly available data at NCBI: • >37,000 nucleo'de seqsà scaffolds, mitochondrial genes • 344 amino acid seqsà mitochondrion • 47 ESTs • 0 conserved domains iden'fied • 0 “gene” entries submi;ed • Data at i5K Workspace@NAL (annota'on hosted at USDA) -‐ 10,832 scaffolds: 23,288 transcripts: 12,906 proteins Example 70
- 71. PubMed Search: what’snew? Example 71
- 72. PubMed Search: what’snew? Example 72 “Ten popula'ons (3 cultures, 7 from California water bodies) differed by at least 550-‐fold in sensi;vity to pyrethroids.” “By sequencing the primary pyrethroid target site, the voltage-‐gated sodium channel (vgsc), we show that point muta'ons and their spread in natural popula'ons were responsible for differences in pyrethroid sensi'vity.” “The finding that a non-‐target aqua'c species has acquired resistance to pes'cides used only on terrestrial pests is troubling evidence of the impact of chronic pes;cide transport from land-‐based applica'ons into aqua'c systems.”
- 73. How many sequencesare there, publicly available, for our gene of interest? Example 73 • Para, (voltage-‐gated sodium channel alpha subunit; Nasonia vitripennis). • NaCP60E (Sodium channel protein 60 E; D. melanogaster). – MF: voltage-‐gated ca'on channel ac'vity (IDA, GO:0022843). – BP: olfactory behavior (IMP, GO: 0042048), sodium ion transmembrane transport (ISS,GO:0035725). – CC: voltage-‐gated sodium channel complex (IEA, GO:0001518). And what do we know about them?
- 74. Retrieving sequences fora sequence similarity search. Example 74 >vgsc-‐Segment3-‐DomainII RVFKLAKSWPTLNLLISIMGKTVGALGNLTFVLCIIIFIFAVMGMQLFGKNYTEKVTKFKWSQDG QMPRWNFVDFFHSFMIVFRVLCGEWIESMWDCMYVGDFSCVPFFLATVVIGNLVVSFMHR
- 75. BLAT search input Example75 >vgsc-‐Segment3-‐DomainII RVFKLAKSWPTLNLLISIMGKTVGALGNLTFVLCIIIFIFAVMGMQLFGKNYTEKVTKFKWSQDG QMPRWNFVDFFHSFMIVFRVLCGEWIESMWDCMYVGDFSCVPFFLATVVIGNLVVSFMHR
- 76. BLAT search results Example76 • High-‐scoring segment pairs (hsp) are listed in tabulated format. • Clicking on one line of results sends you to those coordinates.
- 77. BLAST at i5K heps://i5k.nal.usda.gov/blast Example 77 >vgsc-‐Segment3-‐DomainII RVFKLAKSWPTLNLLISIMGKTVGALGNLTFVLCIIIFIFAVMGMQLFGKNYTEKVTKFKWSQDG QMPRWNFVDFFHSFMIVFRVLCGEWIESMWDCMYVGDFSCVPFFLATVVIGNLVVSFMHR
- 78. BLAST at i5K heps://i5k.nal.usda.gov/blast Example 78
- 79. BLAST at i5K:hsps in “BLAST+ Results” track Example 79
- 80. Creating a newgene model: drag and drop Example 80 • Apollo automa'cally calculates longest ORF. • In this case, ORF includes the high-‐scoring segment pairs (hsp), marked here in blue. • Note that gene is transcribed from reverse strand.
- 81.
- 82.
- 83. Also, flanking sequences(other gene models) vs. NCBI nr Example 83 In this case, two gene models upstream, at 5’ end. BLAST hsps
- 84. Review alignments Example 84 HaztTmpM006234 HaztTmpM006233 HaztTmpM006232
- 85. Hypothesis for vgscgene model Example 85
- 86. Editing: merge thethree models Example 86 Merge by dropping an exon or gene model onto another. Merge by selec'ng two exons (holding down “Shio”) and using the right click menu. or…
- 87. Result of mergingthe gene models: Example 87
- 88. Editing: correct offendingsplice site Example 88 Modify exon / intron boundary: -‐ Drag the end of the exon to the nearest canonical splice site. or -‐ Use right-‐click menu.
- 89. Editing: set translationstart Example 89
- 90. Editing: delete exonnot supported by evidence Example 90 Delete first exon from HaztTmpM006233
- 91. Editing: add anexon supported by RNAseq Example 91 • RNAseq reads show evidence in support of transcribed product, which was not predicted. • Add exon at coordinates 97946-‐98012 by dragging up one of the RNAseq reads.
- 92. Editing: adjust offendingsplice site using evidence Example 92
- 93. Editing: adjust otherboundaries supported by evidence Example 93
- 94. Finished model Example 94 Corroborate integrity and accuracy of the model: -‐ Start and Stop -‐ Exon structure and splice sites …]5’-‐GT/AG-‐3’[… -‐ Check the predicted protein product vs. NCBI nr, UniProt, etc.
- 95. Information Editor • DBXRefs: e.g. NP_001128389.1, N. vitripennis, RefSeq • PubMed iden'fier: PMID: 24065824 • Gene Ontology IDs: GO:0022843, GO: 0042048, GO:0035725, GO:0001518. • Comments • Name, Symbol • Approve / Delete radio bu;on Example 95 Comments (if applicable)
- 96.
- 97. PUBLIC DEMO 97 |97 APOLLO ON THE WEB instructions At i5K 1. Register for access to Apollo at the i5K Workspace@NAL at h;ps://i5k.nal.usda.gov/web-‐apollo-‐registra'on 2. Contact the coordinator for each species community to receive more informa'on about how to contribute. Contact info is available on each organism’s page.
- 98. PUBLIC DEMO 98 |98 APOLLO ON THE WEB instructions Public Honey bee demo available at: h;p://GenomeArchitect.org/WebApolloDemo Username: demo@demo.com Password: demo
- 99. APOLLO demonstration PUBLIC DEMO 99 Demonstra'on video is available at h;ps://youtu.be/VgPtAP_fvxY
- 100. OUTLINE Apollo Collabora've Cura'on and Interac've Analysis of Genomes 100OUTLINE • BIO-‐REFRESHER biological concepts for cura'on • ANNOTATION automa'c predic'ons • MANUAL ANNOTATION necessary, collabora've • APOLLO advancing collabora've cura'on • EXAMPLE demos
- 101. Apollo Development Nathan Dunn EricYao Christine Elsik’s Lab, University of Missouri Suzi Lewis Principal Investigator BBOP Moni Munoz-Torres Colin DieshDeepak Unni JBrowse. Ian Holmes’ Lab University of California, Berkeley
- 102. • Berkeley BioinformaticsOpen-source Projects (BBOP), Berkeley Lab: Apollo and Gene Ontology teams. Suzanna E. Lewis (PI). • § Christine G. Elsik (PI). University of Missouri. • * Ian Holmes (PI). University of California Berkeley. • Arthropod genomics community & i5K Steering Committee. • Stephen Ficklin, GenSAS, Washington State University • Apollo is supported by NIH grants 5R01GM080203 from NIGMS, and 5R01HG004483 from NHGRI. Also supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231 • For your attention, thank you! Apollo Nathan Dunn Colin Diesh § Deepak Unni § Gene Ontology Chris Mungall Seth Carbon Heiko Dietze BBOP Learn more about Apollo at http://GenomeArchitect.org Thank you! NAL at USDA Monica Poelchau Mei-Ju Chen Christopher Childers Gary Moore HGSC at BCM fringy Richards Kim Worley JBrowse Eric Yao *