The Rationale for the GRC
The availability of a high quality human genome assembly has revolutionized
biomedical research. Genomics has now entered the realm of clinical genetics, with
many groups using either whole genome sequencing , or whole exome sequencing
 to identify variants
underlying diseases and informing treatment options . Advances in technology have
increased the number of sequenced human genomes; however, de novo assembly of next
generation sequencing reads is still problematic. The alignment of sequencing reads
from these new genomes to a high quality reference genome remains a critical aspect
of data interpretation .
While the human reference assembly is the highest quality mammalian assembly
available, it is not without shortcomings. The “finished” assembly  contained
over 300 gaps in the euchromatic portion of the genome, tiling path errors and
regions represented by uncommon alleles. Furthermore, assessment of genome-wide
variation revealed regions of the genome with complex, structurally diverse, allelic
representations – that were insufficiently represented in the reference
genome. Other analyses identified sequences that failed to align to the reference
assembly either because the reference assembly contained a valid deletion allele or
underrepresented multi-copy genes –. The Genome Reference Consortium (GRC)
was formed to
address these issues.
The GRC (the GRC consists of The Genome Institute at Washington University, The
Wellcome Trust Sanger Institute, The European Bioinformatics Institute, and The
National Center for Biotechnology Information) is an international consortium with
expertise in genome mapping, sequencing, and informatics. The goal of the GRC is to
provide high quality genome assemblies that will allow a user to place any sequence
greater than 500 bp into a chromosome context. While this report focuses largely on
recent GRC advances concerning the human reference assembly, the GRC is also
responsible for the mouse and zebrafish reference assemblies. Continued improvement
of the human reference assembly is critical as we move towards an era of clinical
and personal genomics. The reference genomes of mouse and zebrafish are similarly
critical in light of their importance as model organisms and the significant
investments made in creating community resources such as gene knockout
Two major problems faced the GRC at the outset of this project, the decentralized
nature of the Human Genome Project and the lack of a suitable data model for
representing complex genomes. Much of the data underlying curation decisions had not
been captured nor standardized. The human reference assembly had never been
submitted to the International Nucleotide Sequence Database Collaboration (INSDC)
 and thus
lacked stable, trackable sequence identifiers that could be accessed from any INSDC
Initial efforts at assembling the human genome were guided by the concept of “a
golden path” , a single clone tiling path that could be reduced to one
non-redundant haploid representation of the human genome. While this model fit well
with the prediction that single nucleotide variants (SNVs) would be the predominant
source of variation in the population, it is now clear that structural variation is
a much larger source of genomic diversity than previously recognized ,. Additionally,
this model did not deal robustly with sequences that were not part of chromosome
assemblies. These often represent sequences that cannot be easily ordered or
oriented on the chromosome assembly due to structural complexity but frequently
contain genes that may be of biological interest  or represent alternate
haplotypes of regions in the chromosome assembly ,. Earlier versions of the
reference genome assembly included some of these allelic variants (such as at the
MHC region) but the sequences themselves often were not used because they had no
relation to the chromosome sequence and could not be easily distinguished from
sequences reflecting biological or artificial duplication.
The GRC has addressed these problems by establishing common tools and standard
operating procedures (SOPs) so that the genome assembly is now constructed in a
regularized fashion. We have developed a single database to store all data
underlying the genome assembly. Finally, we have developed a system to track
individual regions that are under review. All of these data are made publicly
available through our Web site (http://genomereference.org/).
Additionally, the GRC has formalized an assembly model (Figure 1 and Box 1) that provides
for improved accounting
for all sequences, including those that are not part of chromosome assemblies, and
facilitates genome annotation by placing additional structure on those sequences.
Structurally complex regions can be represented by more than one tiling path; one
which will be integrated into the chromosome assembly while the others will be
instantiated as an independent sequence that, by alignment to the chromosome,
provides the chromosome context for the alternate allele.
Assembly representation for GRCh37.p3.
The top panel shows an ideogram representation of the human genome. The
primary assembly unit contains sequences for the non-redundant haploid
assembly; this includes the scaffolds that make up the chromosome sequence
as well as unplaced and unlocalized scaffolds that are thought to represent
novel sequence (not shown in this picture). Alternate loci and patches are
placed in separate assembly units to facilitate annotation. Note the seven
alternate scaffolds in the MHC region are all placed in different assembly
units, as they all represent different representations of the same
sequences. Other alternate loci can be added to these assembly units at the
next major release if they don’t overlap the existing alternates. All
patches are placed in the PATCHES assembly unit and minor releases are
cumulative such that the latest minor release will contain all patches. The
red triangle, yellow circles, and blue circles represent regions that
contain additional sequences that are not given actual chromosome
coordinates, but rather are given a chromosome context via alignment to the
primary assembly. The red triangles represent regions’ alternate loci;
these are sequences that provide an additional tiling path to the one given
in the chromosome representation and are essential for representing
structurally complex loci. The circles represent patch sequences; these are
minor updates made to the assembly outside of the major build cycle. Yellow
circles represent “fix” patches: regions of the chromosome
assembly that will change with the next major assembly update. Blue circles
represent “novel” patches: these are sequences that represent
new alternate loci in the next major assembly update. Unlocalized and
unplaced sequences are not represented in this figure. Sequences within the
assembly are placed within containers known as assembly units. Note: a
region can point to more than one type of extra chromosomal sequence; for
example, a region could point to an alternate locus and to a fix or novel
Box 1. Assembly Definitions
AGP: A file used to describe the instructions for building a contig,
scaffold, or chromosome sequence. This file specifies the order, orientation,
and switch points for each component.
Alternate Locus: A sequence that provides an alternate
representation of a locus found in a largely haploid assembly. These sequences
don’t represent a complete chromosome sequence, although there is no hard
limit on the size of the alternate locus; currently these are less than 5
Assembly: A set of sequences (chromosomes, unlocalized, unplaced,
and alternate loci) used to represent an organism’s genome.
Assembly Unit: Collections of sequences used to define discrete
parts of an assembly.
Component: The basic genomic level sequence used to construct the
genome; typically these are clone sequences, Whole Genome Shotgun sequences, or
PCR fragments. These sequences must be submitted to GenBank/EMBL/DDBJ.
Contig: A contiguous sequence generated from determining the
non-redundant path along an ordered set of component sequences. A contig should
contain no gaps.
Patch: A genome patch is a scaffold sequence that is part of a minor
genome release. These sequences either correct errors in the assembly (a FIX
patch) or add additional alternate loci (a NOVEL patch). These sequences allow
us to update the assembly information without disrupting the chromosome
coordinate system. FIX patches will be removed at the next major assembly
release, as the changes will be rolled into the new assembly. NOVEL patches will
be moved from the PATCHES assembly unit to a proper assembly unit.
Primary Assembly Unit: Represents the collection of sequences that,
when combined, represent a non-redundant haploid genome.
Scaffold: An ordered and oriented set of contigs. A scaffold will
contain gaps, but there is typically some evidence to support the contig order,
orientation, and gap size estimates.
TPF: Tiling Path File; this provides the order of the component
sequences that are used to build a higher order sequence (contig, scaffold, or
Switch Point: The base at which the contig sequence stops being
generated from one component sequence and switches to using the next component
sequence. There must be at least one switch point between adjacent component
sequences in a contig.
Unlocalized sequence: A sequence found in an assembly that is
associated with a specific chromosome, but that cannot be ordered or oriented on
Unplaced sequence: A sequence found in an assembly that is not
associated with any chromosome.
We have also introduced the concept of a “minor” assembly update, in the
form of genome patches. This mechanism provides users with timely access to genome
improvements without inducing frequent changes to the coordinate system upon which
assembly annotations are based. Because genome patches take the same form as
alternate loci the two forms of data can be similarly managed.
The release cycle for major assembly updates will not occur on a fixed schedule. In
order to minimize the need for frequent re-annotation, major assembly updates will
occur infrequently when we have produced at least 100 fix patches or affected
>1% of the euchromatic sequence. The GRC will announce planned updates on
their Web site at least 6 months in advance of any major assembly release.
Additional, detailed information regarding major releases will be publicly announced
via the Web site as data freeze dates approach. Minor assembly updates will be made
Assembly Quality and Improvement
We have produced a major release of the human reference assembly, GRCh37, which was
submitted in June of 2009 to the INSDC (GCA_000001405.1), and four minor assembly
updates, with the last patch, GRCh37.p4 (GCA_000002405.5), released in April 2011.
Detailed information concerning genome assembly construction is on our Web site
The top part of Figure 2 shows
the distribution of issue types that were resolved for these assembly releases. Some
assembly updates are relatively minor, involving the correction of a single
nucleotide discrepancy in the assembly (e.g., HG-445; http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/issue_detail.cgi?id=HG-445)
while others involved multiple components and required generation of new,
region-specific tiling paths (e.g., HG-2; http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/issue_detail.cgi?id=HG-2).
(Figure 2) .
Distribution of issues addressed and an example region.
(Top Panel) Issues for GRCh37, GRCh37.p1, and GRCh37.p2, broken down by type.
Issue types are: Clone Problem: The issue is contained within a single
clone. This may be a single nucleotide difference or a clone mis-assembly.
Path Problem: There is evidence that the tiling path within a given region
is incorrect and we will need to update the path. GRC Housekeeping: Changes
use to help regularize the tiling path. Missing Sequence: Sequence that we
can’t yet place on the assembly. Mapping studies are ongoing to help
place these sequences. Variation: There is evidence to suggest that complex
variation is complicating a region and an alternate allele may need to be
produced. Gap: The issue concerns filling a gap. Unknown: Issue is still
under investigation for classification. (Bottom Panel) Details for issue
HG-2, a Path Problem. The representation in NCBI36 was a mixed haplotype.
The tiling paths for NCBI36 and GRCh37 are shown. Blue clones are anchor
clones that are in NCBI36, the GRCh37 chr4 path, and the GRCh37 alternate
locus path. Red clones represent the UGT2B17 insertion path and dark gray
clones represent the UGT2B17 deletion path. The light gray clone was not
used in NCBI36, but was used in GRCh37 to complete the alternate locus.
While the model changes described above facilitated our assembly management and
reporting, we also wished to investigate whether these updates would allow for
improved genome analysis. To investigate this, we first tried to recover sequence
identified as novel in a personal genome, theYH1 human assembly . Roughly 25%
could be placed in a chromosome context using GRCh37.p2 (see supplemental table 1
and supplemental figure 1 at http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/supplement/).
remaining sequences are being investigated to determine if they warrant inclusion
a future assembly release.
We also wished to investigate the impact on alignment of next generation sequencing
reads. We selected two samples from the 1,000 Genomes project , NA12156 and NA12878,
accessions ERX000125 and ERX000080, respectively) and aligned their reads to GRCh37,
with and without the alternate loci. We demonstrated that removal of the alternate
loci leads to misalignment of approximately two-thirds of the alternate-locus
specific reads (see supplemental table 2, supplemental figure 2 at http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/supplement/).
data clearly demonstrate that that inclusion of alternate representations for
genomic loci can improve alignment quality and thus avoid spurious variation
We envision the high quality reference assemblies generated by the GRC having a
long-term role in biomedical research because they most accurately capture all forms
of human genetic variation and facilitate investigation of human disease in model
organisms. With this in mind, we have built a reference assembly infrastructure to
support transparent curation and assembly production. We have also updated the
assembly model so that it better represents our current understanding of genome
structure and diversity. We will use this model to encompass new discoveries and
ultimately capture all significant variations in the human population structure as
discovered through projects such as 1,000 genomes. Additionally, we wish to engage
the research and clinical communities to identify regions that require targeted
effort and to incorporate information from groups performing detailed work on
specific loci. The GRC can only be truly successful with community input. Users can
report problems directly to the GRC via our Web page (http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/ReportAnIssue.shtml).
It is difficult to overstate the importance of the human reference assembly, even
the age of personal genomics. Given current sequencing and assembly technology,
there is a clear need for a high quality reference that can represent structural
diversity across all populations. Providing a representation of this diversity is
critical for next generation sequence analysis. Even using an assembly with only
three regions with alternative alleles, we show improved alignment quality and by
extension variation calling, which is the primary product of personal genomics. More
genomic alignment tools that can take the alternate representations into account
need to be developed.
Understanding how genotype influences phenotype necessitates an accurate and complete
picture of all loci in multiple populations. For many genomic regions, this can be
denoted by a sequence with annotated SNPs and small indels, but other loci will
require multiple sequence instances for complete representation. Some human loci,
such as the 1q21 region, which remains misassembled in GRCh37.p2, are sufficiently
complex that significant effort is needed to obtain even one correct sequence for
the region. Additional work is required to sort out the haplotypes segregating among
various populations, many of which contribute to phenotypes associated with multiple
developmental disorders .
While assemblies using next generation sequencing are beginning to approach the
quality of long-read Whole Genome Shotgun assemblies , they continue to fail in
complex regions. While it is likely that sequencing and assembly technology will
improve such that de novo assembly of individual genomes will approach the quality
of the human reference, it is not clear when this will happen. However, even when
this is a common occurrence, we see a role for the GRC in integrating the data from
thousands of human genomes to produce a “gold-standard” reference
assembly. We anticipate a continued need for a high quality reference assembly that
will allow any human sequence to be placed into a chromosome context quickly and
easily. As we march down the path of personal genomics it is critical that we devote
resources to the current reference assembly in order to support clinical
applications. As we continue to understand how genotype influences phenotype, the
best possible reference assembly available must be made available to the research