The document discusses the human reference assembly and some key points: 1. The current reference assembly (GRCh38) represents two haplotypes and includes alternate loci to represent structural variation. 2. The assembly is improved from previous versions through the inclusion of 178 regions with alternate loci representing 261 alternate loci and 96 patches of novel sequence totaling over 5 megabytes. 3. Relevant assembly data can be accessed from the Genome Reference Consortium website including sequence files, annotations, and reports on assembly regions like alternate loci and centromeres.

1. Understanding the reference assembly
Valerie Schneider
NCBI
26 October 2016
http://www.biorxiv.org/content/early/2016/08/30/072116

2. Dilthey et al.Paten et al.
Scientific Models

3. • Distinguishing features of the human reference
assembly
• Implications for genomic analyses and tools
• Where do you get assembly-relevant data?
Outline

5. Assembly Basics
Sanger-seq’d, clone based assembly BAC insert
BAC vector
Shotgun sequence clone
Assemble
GAPS
Finish
Minimal Tiling Path
Define switch points for adjacent components
(haploid mosaic)
Most contiguous
Highest sequence quality

6. Today’s reference assembly does not represent:
1.The most common allele
2.The longest allele
3.The ancestral allele
Assembly Basics
It represents the sequence available from the HGP

7. GRC Assembly Model
Sequences from haplotype 1
Sequences from haplotype 2
Old Assembly model: compress into a consensus
Current Assembly model: represent both haplotypes
many

8. Assembly (e.g. GRCh38)
Primary
Assembly
Unit
Non-nuclear
assembly unit
(e.g. MT)
PAR
Genomic
Region
(MHC)
Genomic
Region
(UGT2B17)
Genomic
Region
(MAPT)
Church et al., PLoS Biol. 2011 Jul;9(7):e1001091
GRC Assembly Model
ALT
2
ALT
3
ALT
4
ALT
5
ALT
6
ALT
7
ALT
1

9. The alignments of the alternate loci scaffolds to the
chromosomes are an integral part of the assembly
and can be downloaded from GenBank with the
assembly sequences

10. Assembly (e.g. GRCh38.p1)
Primary
Assembly
Unit
Non-nuclear
assembly unit
(e.g. MT)
ALT
1
ALT
2
ALT
3
ALT
4
ALT
5
ALT
6
ALT
7
PAR
Genomic
Region
(MHC)
Genomic
Region
(UGT2B17)
Genomic
Region
(MAPT)
Church et al., PLoS Biol. 2011 Jul;9(7):e1001091
Patches
Genomic
Region
(ABO)
Genomic
Region
(FOXO6)
Genomic
Region
(FCGBP)
GRC Assembly Model
Patches
FIX NOVEL
SCAFFOLD STATUS AT NEXT
MAJOR ASSEMBLY RELEASE
ALT
LOCI
--
(integrated)
Treat as:
Allelic
Treat as:
Preferred

11. 1q32 1q21 1p21
Dennis et al., 2012
GRC Assembly Model

12. GRC: Assembly Model
GRCh38
• 178 regions with alt loci: 2% of chromosome
sequence (61.9 Mb)
• 261 Alt Loci: 3.6 Mb novel sequence relative to
chromosomes

13. GRCh38.p9
• 96 Patches: >1 Mb novel sequence
• 48 FIX
• 48 NOVEL
GRC: Assembly Model

14. GRCh38: Alt Loci
Alignment Legend
no alignmentmismatchdeletion

15. chromosome
alt/patch
reads On-target alignment
Off-target alignments
(n=122,922)
GRCh38: Alt LociPLoS Biol. 2011 Jul;9(7):e1001091

16. Anatomy of an alt

17. Anatomy of an alt
AC012314.8
CU151838.1
ALT LOCI
AC012314.8
AC245052.3 CHR. 19
Due to anchor components, alternate loci contain some sequence
that is redundant to the primary assembly unit

18. GRCh38 Model Centromeres
Karen Miga (Kent Lab, UCSC)

19. GRCh38 Model Centromeres
WGS WGS WGS

20. GRCh38 Centromeres
Miga et al., Genome Res. 2014 Apr;24(4):697-707

21. GRCh38: Where’s the data?

22. GRCh38: Where’s the data?
GRCh38 Sequences for alignment pipelines

23. GRCh38: Where’s the data?

24. Assembly Sequence and Statistics Reports
GRCh38: Where’s the data?

25. GRCh38: Where’s the data?

26. GRCh38: Where’s the data?
Assembly Regions Report: Alts, Patches and Centromeres

27. GRCh38: Where’s the data?

29. GRCh38: Where’s the data?

30. GRCh38: Where’s the data?

31. Accessing the Datahttps://genomereference.org

32. Accessing the Datahttps://genomereference.org

33. Dumped daily
Frozen mappings to
prior assembly
versions in GFF3
Accessing the Datahttps://genomereference.org

34. Mapped to latest GRCh38 and GRCh37.p13
Accessing the Datahttps://genomereference.org

35. GRCh38 Collaborators
• NCBI RefSeq and gpipe annotation team
• Havana annotators
• Karen Miga
• David Schwartz
• Steve Goldstein
• Mario Caceres
• Giulio Genovese
• Jeff Kidd
• Peter Lansdorp
• Mark Hills
• David Page
• Jim Knight
• Stephan Schuster
• 1000 Genomes
GRC SAB
• Rick Myers
• Granger Sutton
• Evan Eichler
• Jim Kent
• Roderic Guigo
• Carol Bult
• Derek Stemple
• Jan Korbel
• Liz Worthey
• Matthew Hurles
• Richard Gibbs
GRC Creditshttps://genomereference.org

36. Alt Loci: Informatics Challenges

37. Masks and alt aware aligners reduce the incidence of
ambiguous alignments observed when aligning reads to
the full assembly
Mask1: mask chr for fix patches, scaffold for novel/alts. Mask2: mask only on scaffolds
Simulated Reads
GRCh38: Alt Loci

38. The Changing Reference

39. The Changing Reference

40. Dilthey et al.Paten et al.
The Changing Reference

41. • Distinguishing features of the human reference
assembly
• Implications for genomic analyses and tools
• Where do you get assembly-relevant data?
Outline