Novel functional sequences uncovered through a bovine multiassembly graph

Abstract

Many genomic analyses start by aligning sequencing reads to a linear reference genome. However, linear reference genomes are imperfect, lacking millions of bases of unknown relevance and are unable to reflect the genetic diversity of populations. This makes reference-guided methods susceptible to reference-allele bias. To overcome such limitations, we build a pangenome from six reference-quality assemblies from taurine and indicine cattle as well as yak. The pangenome contains an additional 70,329,827 bases compared to the Bos taurus reference genome. Our multiassembly approach reveals 30 and 10.1 million bases private to yak and indicine cattle, respectively, and between 3.3 and 4.4 million bases unique to each taurine assembly. Utilizing transcriptomes from 56 cattle, we show that these nonreference sequences encode transcripts that hitherto remained undetected from the B. taurus reference genome. We uncover genes, primarily encoding proteins contributing to immune response and pathogen-mediated immunomodulation, differentially expressed between Mycobacterium bovis–infected and noninfected cattle that are also undetectable in the B. taurus reference genome. Using whole-genome sequencing data of cattle from five breeds, we show that reads which were previously misaligned against the Bos taurus reference genome now align accurately to the pangenome sequences. This enables us to discover 83,250 polymorphic sites that segregate within and between breeds of cattle and capture genetic differentiation across breeds. Our work makes a so-far unused source of variation amenable to genetic investigations and provides methods and a framework for establishing and exploiting a more diverse reference genome.

Structural variant-based pangenome construction has low sensitivity to variability of haplotype-resolved bovine assemblies

“…Sequencing technologies and assemblers evolve rapidly, and so even recently generated bovine assemblies, including the ones reported here, have been produced under non-uniform conditions (e.g., (Crysnanto et al, 2021; Talenti et al, 2021)). Given the differences we observed between HiFi- and ONT-based assemblies, especially in comparison to the CLR-based ARS-UCD1.2 reference, it was crucial to examine how pangenome construction responded to different assembly inputs.…”

“…Larger structural variants (SVs) and variation located in repetitive or challenging regions have rarely been studied across Bovinae due to the inherent limitations of short sequencing reads and incomplete reference genomes. Moreover, no reference assembly of a single individual can reflect the immense genomic diversity present in global breeds of domestic cattle (Crysnanto et al, 2019(Crysnanto et al, , 2021Crysnanto & Pausch, 2020;Talenti et al, 2021).…”

“…SVs may be involved in expression quantitative trait loci more often than previously estimated, and can impact gene expression more than shorter variants (Chiang et al, 2017). However, previous studies suffer from potential reference bias, because the use of a single taurine cattle reference assembly fails to reflect the immense genomic diversity present in global breeds of domestic cattle (Crysnanto et al, 2019(Crysnanto et al, , 2021Crysnanto & Pausch, 2020;Talenti et al, 2021). Pangenomes have long been proposed (Tettelin et al, 2005) as a way to better reflect variation present in a group of individuals (e.g., breed, species, clade, etc.).…”

Genome graphs detect human polymorphisms in active epigenomic state during influenza infection

“…We expect further explorations of other cell types to highlight additional MEIs that are not covered in the current set. In total, there may be many other functional sequences that are hidden by a haploid reference but that could be discovered through a pan-genomic reference [12].…”

Mining massive genomic data of two Swiss Braunvieh cattle populations reveals six novel candidate variants that impair reproductive success

“…This analysis is based on the latest reference sequence ARS-UCD1.2 of a Hereford cow, however, the reference sequence still spans many gaps and includes more than 2000 unplaced scaffolds that could potentially harbor important coding sequences [ 37 ]. Recently, it was shown that reference graphs based on sequence data of several breeds can include new functional sequences [ 97 ]. Another drawback of our approach is that we restricted our analysis to coding variants, while non-coding variants impairing the expression of a protein or more complex structural variants disturbing the function of genes might also be causative.…”