Short-range, orientation-reversing template-switching events occur at a high frequency in the human and yeast genomes

Abstract

20The identification of structural variations in genomes using next-generation sequencing approaches 21 greatly facilitates the study of genetic and genomic diseases. The data generated using these 22 approaches also provide interesting new means to examine DNA repair, recombination, and 23 replication to better understand sources of genomic instability. To better utilize this data, we 24 developed SCARR (Systematic Combination of Alignments to Recreate Rearrangements) to identify 25 DNA rearrangements, and used it to examine the occurrence of orientation-reversing events in human 26 and budding yeast genomes. SCARR exceeds the sensitivity of previous genome sequencing 27 approaches, and identifies rearrangements genome-wide with base-pair resolution, which helps 28 provide insights into the mechanisms involved in their formation. We find that short-range orientation-29 reversing events occur at high rates in both human and yeast genomes. We quantified these 30 rearrangements in yeast strains lacking various DNA repair factors, and propose that these short-31 range events often occur through template-switching events within a replication fork. We hypothesize 32 that this mechanism may act as an error-prone alternative to fork reversal to restart stalled replication 33 forks. 343 57 mitochondria using next-generation sequencing [11]. This type of approach revealed that these 58 events in organelles are also favored by sequence similarities as short as 5 bases, and may even 59 occur in the absence of homology. The circumstances leading to their formation, as well as their 60 possible consequences, remain poorly understood due to the challenges involved in their detection by 61 traditional biochemical methods [12]. 62The rapidly expanding repositories of next-generation sequencing data have opened several new 63 avenues for understanding genomic instability including its roles in evolution and genomic diseases. A 64 landmark study in the field identified tens of thousands of SVs within the human genome [2], which 65 included deletions, duplications, insertions, as well as a small number of inversions. New 66 computational approaches to detect and investigate SVs continue to be developed to improve 67 detection rates and focus on specific types of SVs, such as the harder to detect inversions [13][14][15]. 68Nevertheless, complex SVs such as those that result from FoSTeS and other template-switching 69 events often remain undetected by current next-generation sequencing analysis pipelines. 70To avoid a bias for simple SVs, we developed SCARR (Systematic Combination of Alignments to 71 Recreate Rearrangements), which uses no a priori assumptions about the relative positions of DNA 72 sequences around a breakpoint. SCARR instead iterates through all possible genome-wide alignment 73 combinations to find the best match. Using this approach, it is possible to identify deletions, tandem 74 duplications and breakpoints resulting from template-switching events at base-pair resolution with 75 reasonable accuracy. Consequently...

Short-range template switching in great ape genomes explored using pair hidden Markov models

“…While a process of template switching can occur locally as a mechanism to bypass DNA lesions during replication, it is traditionally considered an error-free pathway mediated by proliferating cell nuclear antigen polyubiquitination, in which replication proceeds in the same direction as the nascent strand following the formation of a hemicatenane structure with the newly synthesised sister chromatid [ 10 – 12 ]. However, error-prone reverse-oriented template switching has now been observed in multiple eukaryotes including humans [ 13 , 14 ], and has been shown to leave a footprint of clustered mutagenesis in the human genome [ 15 ]. Despite our understanding of these individual mechanisms, computationally capturing their mutational footprints in an evolutionary context remains difficult, especially when focusing on local mutational mechanisms, which may present as a plausible cluster of accumulated substitutions and indels within a sequence alignment.…”

Short-range template switching in great ape genomes explored using a pair hidden Markov model

“…While a process of template switching can occur locally as a mechanism to bypass DNA lesions during replication, it is traditionally considered an error-free pathway mediated by proliferating cell nuclear antigen polyubiquitination, in which replication proceeds in the same direction as the nascent strand following the formation of a hemicatenane structure with the newly synthesised sister chromatid (10)(11)(12). However, error-prone reverse-oriented template switching has now been observed in multiple eukaryotes including humans (13,14), and has been shown to leave a footprint of clustered mutagenesis in the human genome (15). Despite our understanding of these individual mechanisms, computationally capturing their mutational footprints in an evolutionary context remains difficult, especially when focusing on local mutational mechanisms, which may present as a plausible cluster of accumulated substitutions and indels within a sequence alignment.…”