Using short read sequencing to characterise balanced reciprocal translocations in pigs

Abstract

Background: A balanced constitutional reciprocal translocation (RT) is a mutual exchange of terminal segments of two non-homologous chromosomes without any loss or gain of DNA in germline cells. Carriers of balanced RTs are viable individuals with no apparent phenotypical consequences. These animals produce, however, unbalanced gametes and show therefore reduced fertility and offspring with congenital abnormalities. This cytogenetic abnormality is usually detected using chromosome staining techniques. The aim of this study was to test the possibilities of using paired end short read sequencing for detection of balanced RTs in boars and investigate their breakpoints and junctions. Results: Balanced RTs were recovered in a blinded analysis, using structural variant calling software DELLY, in 6 of the 7 carriers with 30 fold short read paired end sequencing. In 15 non-carriers we did not detect any RTs. Reducing the coverage to 20 fold, 15 fold and 10 fold showed that at least 20 fold coverage is required to obtain good results. One RT was not detected using the blind screening, however, a highly likely RT was discovered after unblinding. This RT was located in a repetitive region, showing the limitations of short read sequence data. The detailed analysis of the breakpoints and junctions suggested three junctions showing microhomology, three junctions with blunt-end ligation, and three micro-insertions at the breakpoint junctions. The RTs detected also showed to disrupt genes. Conclusions: We conclude that paired end short read sequence data can be used to detect and characterize balanced reciprocal translocations, if sequencing depth is at least 20 fold coverage. However, translocations in repetitive areas may require large fragments or even long read sequence data.

Classical, Molecular, and Genomic Cytogenetics of the Pig, a Clinical Perspective

“…The largest study of the applications of WGS into the study of porcine chromosome rearrangements performed short-read sequencing to seven carriers of karyotypically balanced rearrangements alongside 15 non-carriers [ 178 ]. Here, it was found that short-read sequencing was capable of accurately detecting the breakpoint junctions of six of seven carriers, with no false-positives detected.…”

“…Here, it was found that short-read sequencing was capable of accurately detecting the breakpoint junctions of six of seven carriers, with no false-positives detected. The main deficiency of short-read sequencing noted was that it was not capable of identifying breakpoint junctions occurring in repetitive sequences [ 178 ]. This study for the first time described the breakpoints of chromosome rearrangements in the pig genome, identifying several varieties of breakpoint signatures including microhomology, microinsertions, and blunt-end ligations also characteristic of human rearrangement breakpoint junctions [ 156 , 178 ].…”

“…The main deficiency of short-read sequencing noted was that it was not capable of identifying breakpoint junctions occurring in repetitive sequences [ 178 ]. This study for the first time described the breakpoints of chromosome rearrangements in the pig genome, identifying several varieties of breakpoint signatures including microhomology, microinsertions, and blunt-end ligations also characteristic of human rearrangement breakpoint junctions [ 156 , 178 ]. Genes disrupted by the breakpoint interrupting the gene sequence were also found at the sites of breakpoint junctions, with the heterozygous nature of the disruption suggested to be protective of any phenotypic effect associated with the rearrangement.…”

“…Genes disrupted by the breakpoint interrupting the gene sequence were also found at the sites of breakpoint junctions, with the heterozygous nature of the disruption suggested to be protective of any phenotypic effect associated with the rearrangement. The sequencing of these breakpoints therefore indicates that so-called balanced rearrangements may not be as balanced as once thought, with the presence of small deletions, insertions, and gene disruptions noted as occurring in the pig [ 178 ].…”

“…The factors influencing the formation of chromosome rearrangements in pigs are still largely unknown, despite the sequencing of a handful of rearrangement breakpoint junctions. No clear pattern presents itself, with breakpoints appearing with different breakpoint signatures and in a variety of chromosome regions and landscapes [ 148 , 156 , 178 ]. A study of mosaic rearrangement carriers indicated that relatives of mosaic carriers themselves carried mosaic rearrangements at 2.5× the frequency as control animals, indicating a possible genetic component to the formation of chromosome rearrangements [ 74 ].…”