Hidden assembly problems exposed

The human reference genome GRCh37 represents the highest quality mammalian genome assembly ever to be produced. It's played a major role in advancing both basic research and clinical research, and it continues to teach us that there's much we still don't know about human genomic biology. However, it is important to keep in mind that the assembly isn't perfect. While many users may be aware of some of the more visible issues with the assembly, such as gaps or missing genes, there are other assembly problems that may be less apparent. As noted in a previous GRC blog post, erroneous bases are one such problem. While the reference assembly is accurate to an error rate of ~1 in 100,000 bases, this still means that approximately 28,000 of the 2.85 billion bases are inaccurate. Using data from sources such as the 1000 genomes project, the GRC is working to address these sequencing errors for GRCh38, the next genome version that will be publicly released.

Component assembly problems represent another less-recognized source of error in the reference genome. The human reference genome is a clone-based assembly, sequenced using Sanger technology. Generating the consensus sequence for each of these genomic clone components involved their sub-cloning into plasmid vectors, sequencing and reassembly. During the course of the human genome project, many of the sequencing centers made note of problems encountered during the sequencing of these clones as annotations on the sequence records. Colloquially known as "black-tag" annotations, these represent regions of uncertainty due to force joins, unresolved tandem repeats, low quality or coverage sequence, and other biological and technical challenges. Although these annotations are part of public sequence records, they have historically been difficult to interpret in the context of the genome assembly because their coordinates were component, rather than scaffold or chromosome based.

The GRC has now mapped annotated clone assembly problems of assembly components to the coordinates of the top-level molecules in which they are found (scaffold or chromosome) in the human, mouse and zebrafish reference assemblies. These data are available in GFF3 and ASN.1 format on the GRC's public FTP site. These files allow users to see whether problems have been annotated at their coordinates of interest or if the locations of other genome features, such as segmental duplications or structural variation, fall in annotated problem regions and potentially represent false calls. Additionally, the GRC is cross-checking reports of genome problems with these coordinates in an effort to understand the underlying cause of these issues (Figure 1). It should be noted, however, that not all genomic components were equivalently annotated during the course of the human genome project. Thus, the absence of an annotation in these files does not mean that no problem was encountered during component sequencing, just that none was noted in its record. We encourage users to review the interpretations of their own assembly annotations in light of this data and we welcome your feedback!

Fig. 1

Figure 1. Graphical image of a region on GRCh37 chr. 15 (NC_000015.9; 99.63-99.65 Kb). The blue bar at top represents the component from which the chromosome sequence is derived at this location (AC036108.19 (RP11-6O2)), while an NCBI gene annotated in this vicinity is shown just below (green bar). At bottom, the small vertical black tick marks show the positions of clone assembly problems that were annotated on the component sequence. The GRC received two separate reports of assembly problems in this region (HG-971 and HG-352). Review shows that both correspond to the positions of annotations indicating problems in the assembly component. Both genome errors have been corrected by the addition of a new component to the assembly and will be released as FIX patches in GRCh37.p9.