The completion of the complete “telomere-telomere” (T2T) human genome last year highlighted that genome sequences previously thought to be “complete” were in fact not complete at all.

Moreover, many recent genomes have been sequenced using short read sequencing technologies that fragment the DNA into short segments, typically 150–300 bp long, and then compared to a reference sequence. While fast, accurate and relatively economical short read methodologies typically miss large portions of the genome, about 10% overall. Missing segments include high G/C regions and repetitive sequences including segmental duplications, simple repeats and transposable elements (TEs).

TEs are repetitive sequences that move to other locations in the genome, and the mobility of these sequences contributes greatly to the variability of the genome. Repetitive sequences often underlie the formation of structural variants (SVs), genomic differences resulting from duplications, insertions, deletions, and inversions. SVs are often overlooked when using short read sequencing (particularly those mediated by repeats), but they may play an important role in genome dysregulation and disease.

Researchers have turned to long-read sequencing for a more complete analysis of genomes, as these technologies allow much longer segments of DNA to be sequenced and can accurately display a more complete picture of the genome. Recent advances have increased the accuracy and utility of long reads, allowing researchers to explore previously undiscovered genomic features, and not just in humans.

Jackson Laboratory (JAX) and University of Connecticut Health Center Associate Professor Christine Beck, Ph.D., led a team that examined the genomes of another known species, the mouse, and revealed the details of 20 different inbred strains that will be critical to inform research in genetics and genomics. on mice move forward.

Structural differences between strains of mice

Mice have their own reference genome, known as GRCm39, based on the sequence C57BL/6J, a strain of the Mus musculus domesticus subspecies. But many widely used strains of laboratory mice are also descended from two other subspecies, Mus musculus castaneus and Mus musculus musculus, and there are many genetic differences between different inbred strains.

For work presented in “Resolving Structural Variations in Different Mouse Genomes Reveals Chromatin Remodeling by Transposable Elements” published in Cellular genomics, Dr. Beck selected a wide range of widely used strains, including seven founding parents of genetically diverse panels of Collaborative Cross (CC) and Diversity Outbred (DO) mice, six resulting CC strains with abnormalities of unknown genetic origin, and seven other frequently used strains. with different genetic backgrounds.

Ardian Ferrage, PhD student and lead author of the study, then assembled the genomes of these 20 mice and used these sequences to identify the SVs present in the animals that distinguished their genomes from that of the C57BL/6J reference. Using PAV, a program developed by Beck’s lab associate Dr. Peter Audano, Ardian showed that SVs are predominant in mouse genomes and contribute significantly to genomic variability. In fact, SVs contain almost five times more affected bases than previously published single nucleotide variants from various mouse genomes.

They also found much greater SV diversity between mouse genomes than between human genomes, suggesting that a single reference mouse genome is not suitable for mapping genomic data between mouse strains. Importantly, long read sequencing is vital to capture this variation. In 18 strains of mice, the research team found an additional 213,688 insertions, 64,277 deletions, and 97 inversions in long reads compared to short reads.

Mobile elements and consequences of structural variations

While only small amounts of TEs are still able to mobilize in human genomes, they are more mobile in mice. Because of this, Beck and her team focused on mobile element variants (TEVs), which they found to make up nearly 40% of all SVs, with the majority (60%) being inserts. There are several types of TEVs, known as short and long interspersed nuclear elements (SINE and LINE), which are predictably characterized by their size. LINEs were almost twice as common as SINEs in mouse genomes, from 47% to 24%.

Because of their size, LINEs also account for almost half of the variable sequence content in mouse genomes, compared to only 24% of non-TEV SV and 2.1% of SINE. Various endogenous retroviral sequences generated the remaining 28% of TEVs. Retroviruses are RNA viruses whose genomes are reverse-transcribed into DNA, which is then inserted into the genome. While many modern retroviruses are associated with diseases such as AIDS and cancer, normal mammalian genomes contain large amounts of DNA derived from thousands of years of retroviruses known as endogenous retroviruses or ERVs, which help drive genomic variation in mice.

So what are the possible implications of all this genomic variation and activity? The researchers looked at SV in the context of known genomic features and predicted the severity of the consequences. Of the recently discovered SVs in gene sequences, the vast majority (94,863) were in introns, sequences that are spliced ​​from pre-mRNA so they do not change protein structure; 1469 were in untranslated segments (UTRs) at both ends of the gene; and 510 in actual protein coding sequences.

They also identified a previously undetected insertion of a retroviral element into a specific gene, Mutyh, a DNA repair gene associated with a known mutational signature in certain strains of mice. The underlying variant was unknown, but the team found that the insertion was associated with a significant reduction in Mutyh gene expression. The discovery shows that unknown SVs can alter important regions of the genome and reside in genes associated with traits related to health and function, including disease.

Finally, in collaboration with researcher Jax Dr. Laura Reinholdt, team investigated the effect of TE on embryonic stem cell differences. TEs contribute to genome diversity, and their variations can change important aspects of gene expression between strains. Indeed, the study found more than 22,000 TEVs associated with significant changes in the availability of stem cell chromatin, a key regulator of gene expression, in embryonic stem cells from 10 genetically distinct mouse strains.

Focusing again on a specific example, they examined a strain-specific (CAST/EiJ) intron insertion in the Slc47a2 gene, which was accompanied by a chromatin availability signal unique to the strain. They found increased levels of Slc47a2 expression compared to strains without an insert, with a strain-specific transcript and a possible pluripotency factor binding region, indicating an important role for TEV in early development.

A better understanding

Given the importance of the mouse as a model for mammalian genetics and human disease, it is necessary to fully understand the functional implications of genomic variation. Comprehensive detection and characterization of SV between the genomes of mouse strains is an important part of this understanding, and the results and data obtained by Dr. Beck and her collaborators have taken an important step forward in this area.

The authors have created a sequence resolution SV resource, a mouse embryonic stem cell expression resource, and chromatin availability data for the research community that may aid further research into mouse evolution and the genomics underlying traits of interest.