1.
RefSeq curation and annotation of the
reference human genome GRCh38
Kim D. Pruitt
National Center for Biotechnology Information
National Library of Medicine
National Institutes of Health
www.ncbi.nlm.nih.gov/refseq/

2.
RefSeq Background
• RefSeq provides -
• Human genome annotation
• Known transcripts & proteins (manually curated)
• Model transcripts & proteins (annotation pipeline)
• Collaborations -
• Genome Reference Consortium (GRC)
• HUGO Gene Nomenclature Committee (HGNC)
• Consensus CDS (CCDS) Collaboration (HAVANA curators)
• RefSeqGene/Locus Reference Genomic (LRG)/LSDB
RefSeq: www.ncbi.nlm.nih.gov/refseq/ Gene: www.ncbi.nlm.nih.gov/gene/
An NCBI project to provide reference sequence
standards that incorporate current knowledge.
Archaea – Bacteria – Eukaryotes - Virus

3.
Curation support of genic regions of
the reference human assembly
• RefSeqGene and LRG collaboration
• Genomic and cDNA standards for clinical reporting
• Report potential issues to the GRC
• Consensus CDS collaboration
• Stabilized human CDS annotation
• Report potential issues to the GRC
• RefSeq
• Curation of genes, transcript & protein records
• Report potential issues to the GRC
• Review GRC patch updates for gene annotation impact

4.
Genome annotation leverages
curation + computation
Genes:
• Type, location, length
Sequence:
• Accuracy, length
• Alternate splice products
• Functional annotation
Align curated RefSeqs
Align transcripts, proteins
Align RNA-Seq
Filter best alignments
Build model RefSeqs
Assign accessions, GeneID
Evidence-based genome
annotation pipeline
Manual Curation
Sequence - Literature
Transcripts Proteins
Known RefSeqs 50,540 39,363
Model RefSeqs 112,735 60,599
Annotated Genes Count
Protein-coding 20,576
Non-coding 18,037
Pseudogene 12,474

5.
Transition from GRCh37 to GRCh38
• Identify gene/sequence differences vs. GRCh38
• Automatic update at synonymous mismatches
• Curation review of remainder
• >5,100 Known RefSeq transcripts updated since October 2013
• 47,031 Known RefSeqs identical to genome
• 2,916 intentionally retain a mismatch or indel
• ~600 pending
• ~132 genes merged
0 200 400 600 800 1000 1200
2013 Q1
2013 Q3
2014 Q1
2014 Q3
2015 Q1
2015 Q3
Number of updates
* GRCh38 12/24/2013
*

6.
Updating RefSeq to match GRCh38
• Post GRCh38 review:
• NM_173477 updated to match genome (NM_173477.4)
• Model RefSeq XM_005257026.1 promoted to Known RefSeq
GRCh38
GRCh37
alignment
alignment

7.
RefSeq curation & genome
maintenance
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GRCh38
GRCh37
GRCh37 Issue:
SCX duplication
MROH1 split
GRCh38 update:
Gap closed
MROH1 complete
One SCX gene
gap

8.
RefSeq curation & genome
maintenance
• POLR2A (GeneID:5430) NM_000937.4 has a 2 nt deletion
vs. GRCh38
• This maintains the correct reading frame
GRCh38
alignment

9.
RefSeq curation & genome
maintenance
• RefSeq reported this sequence issue to the GRC

10.
GRCh38 ALT LOCI and PATCHES
Pre-Patch & ALT review
Polymorphic pseudogenes
Haplotype & CNV variation
ALT-specific RefSeq records
Curator-stored placement data
Evidence-based genome
annotation pipeline
Manual Curation
Assembly-ALT alignments
Alignment quality reports
Subsequent genome
annotation build corrects
the annotation
Interim alignment updates

11.
Polymorphic pseudogenes
• RefSeq provides different transcripts to represent the protein-
coding gene versus the pseudogene
• Curators store assembly placement information (chromosome
versus ALT) in a local database
• This is used by annotation pipeline to ensure correct annotation
Assembly Unit GSTT1 GSTT2 GSTT2B GSTTP1 GSTTP2
GRCh38 chr22 null pseudo coding pseudo null
ALT_REF_LOCI_1 coding coding coding pseudo pseudo
An example – GSTT cluster on chromosome 22:

12.
GSTT* variation, chromosome 22
• Copy number variation of glutathione-S-transferase theta genes
is associated with digestive track cancers and more
• Accurate gene annotation is important to downstream users
GRCh38 chr22
GRCh38 ALT
pseudogene
chr22 = null allelecoding allele
ulcerative colitis - laryngeal cancer - esophageal cancer - colorectal cancer

13.
GSTT2 polymorphism
AT splice donor Premature
stop codon
GT splice donor Stop codon
GRCh38 chr22
GRCh38 ALT

14.
GRCh38 chr22 GSTT2 pseudogene
GRCh38 chr22

15.
Data access
• Genes:
• <…ncbi root url…>/gene/
• ftp://ftp.ncbi.nlm.nih.gov/gene/
• NCBI YouTube ‘Download genomic sequence for a gene’
• https://www.youtube.com/watch?v=RHz2nZbzjpA
• RefSeq transcripts and proteins:
• Links from NCBI Gene
• Nucleotide/protein query:
• human[organism] + use facets to specify RefSeq and molecule type
• ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/
• NCBI Genome Annotation
• Links from NCBI Assembly or Genome resources
• <ncbi>/assembly/ or <ncbi>/genome/

16.
Data access to annotated genome
Gene
Assembly details

17.
Genome FTP formats
• FASTA
• genome, transcripts, proteins
• GenBank file format
• – genome transcripts, proteins
• GFF genome annotation
• Feature table
• features and locations in
tabular format
• AGP, Assembly details & statistics
• Repeat masker results
• Md5checksums
• Documentation
• README files
• <ncbi>/genome/doc/ftpfaq/

18.
Acknowledgements
RefSeq Curators
Annotation pipeline
Paul Kitts
Terence Murphy
Francoise Thibaud-Nissen
Eric Cox
Catherine Farrell
Tamara Goldfarb
Tripti Gupta
Vinita Joardar
Vamsi Kodali
Kelly McGarvey
Mike Murphy
Nuala O'Leary
Shashi Pujar
Bhanu Rajput
Sanjida Rangwala
Lillian Riddick
Dave Webb
Matt Wright
Susan Hiatt
www.ncbi.nlm.nih.gov/refseq/
Collaborators
Elspeth Bruford (HGNC)
Jen Harrow (HAVANNA)
Locus-Specific Databases
Expert databases
Individual scientists

19.
NCBI Posters & Booth 2405