1. The NCBI Annotation Pipeline
6/13/2018
The NCBI Annotation Pipeline
Genome Assembly
Annotation
Automated
Manual
Section 1-Sequence Characteristics
- number of basepairs
- characteristics of fragments
- contamination
NCBI Genome Resources

2. Assembly (sequence chromosome)
Remove contaminants
Bin by chromosome arms
Sequence Layout
Sequence Building
Place on chromosomes
NCBI Genome Resources

3. Sequence Assembly - a modified greedy approach
6/13/2018
Sequence Assembly - a modified greedy approach
Sequence Layout
Curated Finished Regions
Curated assembly instructions
MegaBLAST hits
Consider TPF clone order
BAC chromosome assignment
annotation
STS markers
personal communication
Remove conflicting overlaps, redundant BACs
BAC Sequence
Fragments
Assemble
Order
NCBI Contig
Sequence Building
Consider fragment:fragment sequence overlaps for each BAC pair in layout
Meld overlapping sequence
Order and Orient (o+o ):
alignments (mRNA, EST)
BAC annotation
paired plasmid reads
113,310 Melds
NCBI Genome Resources

4. Genome Build Process Freeze Contig Build & Release Exclude Problem
dbSNP
STS
Clones
Collaboration
Curation
GenomeScan
GenBank
LocusLink
RefSeq
Update:
Links
gi’s
Prepare for release
LocusLink
Annotation
Contig Build
&
Release
Assembly
Resource
Updates
Freeze
Input Data:
Sequences
Curated NTs
TPF
BLAST hits
Public Release
Sequences (contig mRNA protein)
Exclude
Problem
accessions
Analysis & Review
Corrections for next build
Map Viewer
FTP
BLAST
Input Resources
NCBI Genome Resources

5. What is being annotated?
6/13/2018
What is being annotated?
Feature Method
Genes: By alignment, by prediction
Markers:
By ePCR
Variation:
By alignment
Clones/Cytogenetic location:
By alignment (BAC ends)
Phenotype (MIM):
Via Gene identification, associated markers
Cytogenetic Position:
By annotated BAC-END sequenced clones
By FISH-mapped clones used in assembly
Section 1-Sequence Characteristics
- number of basepairs
- characteristics of fragments
- contamination
NCBI Genome Resources

6. Genome Annotation: Genes
6/13/2018
Genome Annotation: Genes
Automated:
By mRNA alignment (RefSeq, GenBank)
protein coding and transcribed pseudogenes
By EST alignments
GenomeScan predictions
Products:
Sequences - RefSeq RNAs and proteins
Resources - MapViewer, FTP, BLAST
Section 1-Sequence Characteristics
- number of basepairs
- characteristics of fragments
- contamination
NCBI Genome Resources

7. RefSeq: a reagent for Contig Annotation
Potential Problems:
Gene Families
Partial
Chimeric
Intron read-through
Linker
Vector
Wrong organism
genome
RefSeq mRNAs
GenBank mRNAs
RefSeq Advantages:
Separate Gene Families
Not Partial
Means to correct
problem sequences
ESTs
TBLASTN
RPSBLAST
RefSeq process results
in excluding problem
GenBank sequences
from annotation pipeline
GenomeScan
NCBI Genome Resources

8. NCBI Reference Sequences (RefSeq)
Genome Oriented Resource
A sequence for each macromolecule Central Dogma: Chromosome, mRNA, preprotein, mature protein
Linked on a residue by residue basis
Objectively non-redundant and comprehensive
Curated Resource
Authoritative source by genome
Derivative of GenBank but corrected, merged, extended
Publicly distributed, Entrez Genomes Web site
Reagents for Genome Annotation and Analysis
Substrate for Functional Genomics
NCBI Genome Resources

9. Reference Sequences
Goal: One sequence entry for each naturally occurring DNA, RNA and protein molecule
Key:
curated
calculated
NC_000000
NM_000000
NP_000000
XM_000000
XP_000000
chromosome
NT_000000
contig
mRNA
predicted mRNA
protein
predicted protein
NG_000000
gene
Multiple products for one gene are instantiated as separate RefSeqs with the same LocusID.
NCBI Genome Resources

10. RefSeq: Scope
The NCBI Reference Sequence (RefSeq) project provides non-redundant sequence data including bacterial and viral genomes, mitochondrion, chromosomes, constructed genomic contigs, transcripts, and proteins.
NCBI Genome Resources

11. Process Flow Technology: Sybase relational databases
Support multiple users
Instant updates in-house
Daily updates public web site
C, C++, Perl, stored procedures, triggers
In-house processing
External Collaboration
LocusLink
Public Release:
LocusLink Web Site
Annotation Data
Automated
BLAST
Analysis
RefTrack
Provisional & Predicted Records
(Transcripts & Proteins)
Status
QBLAST
Update
Reviewed Records
(Genomic Regions,Transcripts, Proteins)
Manual Curation
Accessible in:
BLAST
BLink
Entrez
FTP
LocusLink
RefTrack Tracking Database
. Decisions
. Status
. Accessions
. History
Genome
Annotation
Pipeline
NCBI Genome Resources

12. Manual Curation Process
RefSeq infrastructure: supporting analysis
Manual Curation Process
Tools:
User Input
Genome Analysis
Database QC checks
Assignment
Disease Genes
Gene Families
Gene Clusters
Identified Problems
Analysis:
Precomputed BLAST
Vector
ESTs
GenBank ‘nr’
HTG
Contigs
blastx
blastp
Spidey alignments
Map Viewer
Evidence Viewer
Review
Alternate Splicing
Gene Families
Review
Sequence
Database data
Literature
Database
Editing
Sequence
Editing
Sequence changes
Remove contamination
Correct errors
Extend UTRs
Splice Variants
Annotation
Annotation:
Sequin
Ingenue
Final Check
Quality Control
Store Data & QC:
RefSeq validator
Public
Public
NCBI Genome Resources

13. New Type of RefSeq: Genomic Regions
Why?
Correct Assembly through Duplications, Paralogous Gene Clusters
Optimize Annotation in Gene Clusters
Goals:
1. Establish genomic reference sequence
2. Provide annotation [genes, pseudogenes, variation]
3. Provide corresponding mRNA, protein RefSeq
Used in:
Genome Assembly & Annotation Pipeline
NCBI Genome Resources

14. Gene Annotation Automated Annotation: Manual Annotation:
6/13/2018
Gene Annotation
Automated Annotation:
By mRNA alignment (RefSeq, GenBank)
By EST alignments
GenomeScan predictions
Initial RefSeq record (provisional, predicted status)
LocusLink – maintaining associations; connections to function
Manual Annotation:
RefSeq – genomic regions, mRNA,protein (reviewed status)
Local regions of Genome Assembly – provide curated assembly instructions (join a->b; don’t join a->c)
Section 1-Sequence Characteristics
- number of basepairs
- characteristics of fragments
- contamination
NCBI Genome Resources

15. Products of annotation
6/13/2018
Products of annotation
RefSeqs (transcripts, proteins)
Gene id (LocusID)
features in chromosome coordinates
features in contig (NT accession)
coordinates
Available in:
Map Viewer
Graphical display
Tabular display
Sequence downloads
FTP
RefSeqs (contigs, transcripts, proteins)
Mapping Data
LocusLink & Other resources
Section 1-Sequence Characteristics
- number of basepairs
- characteristics of fragments
- contamination
NCBI Genome Resources

16. Map Viewer: Query support
NCBI Genome Resources

17. Map Viewer: Tabular report
NCBI Genome Resources

18. Genes in regions of conserved synteny
Anchored by human gene order
Anchored by mouse gene order
NCBI Genome Resources

19. Query by sequence: Review the alignment
6/13/2018
Query by sequence: Review the alignment
A click away:
Alignments (BLAST hit)
Gene Description (LocusLink)
Report of all features in the region
Contig sequence
Sequence in the region
other mRNAs aligning in the region
Define your own gene model based on alignments in the region
Section 1-Sequence Characteristics
- number of basepairs
- characteristics of fragments
- contamination
NCBI Genome Resources

20. Quality Control - Genome review
6/13/2018
Quality Control - Genome review
Is the sequence correct?
Is the feature correctly placed?
Is there a feature that should be placed?
Are the attributes of the feature correct?
Approaches:
In-house analysis & review (manual curation)
Shared information (UCSC/Ensembl)
Solicited review by experts in local regions
Section 1-Sequence Characteristics
- number of basepairs
- characteristics of fragments
- contamination
NCBI Genome Resources

21. Acknowledgments http://www.ncbi.nlm.nih.gov/genome/guide/human.html
Genome Build Team:
Richa Agarwala
Hsiu-Chuan Chen
Slava Chetvernin
Deanna Church
Olga Ermolaeva
Renata Geer
Wratko Hlavina
Wonhee Jang
Jonathan Kans
Ken Katz
Paul Kitts
David Lipman
Adam Lowe
Donna Maglott
Jim Ostell
Kim Pruitt
Sergey Resenchuk
Victor Sapojnikov
Greg Schuler
Steve Sherry
Andrei Shkeda
Tatiana Tatusova
Lukas Wagner
Sarah Wheelan
Acknowledgments
RefSeq Curator Staff
BLAST Team
Entrez Team
NCBI Service Desk Staff
Collaborators:
Human Gene Nomenclature Committee
OMIM Staff
The Jackson Laboratory
Rat Genome Database