Short template switch events explain mutation clusters in the human genome

Löytynoja, Ari; Goldman, Nick

doi:10.1101/gr.214973.116

Cited by 31 publications

(77 citation statements)

References 56 publications

(80 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Evidence of further evolution of the wsp cluster was detected on 28-d. RSCV-40, in addition to having the wspA Δ285-298 driver mutation, had 3 separate mutations in wspD which led to an early stop codon. This unusual mutation cluster is consistent with error-prone translesion synthesis or DNA template switching facilitated by microhomology 28 . However, as the mutations occur at the wspD 3’ end, the WspD N-terminus may still be expressed and functional (Fig S3).…”

Section: Resultssupporting

confidence: 64%

ThePseudomonas aeruginosaWsp pathway undergoes positive evolutionary selection during chronic infection

Gloag

Marshall

Snyder

et al. 2018

Preprint

View full text Add to dashboard Cite

Running title: Evolution of P. aeruginosa RSCVs in a chronic infection model 22Author Contributions: ESG and CWM performed the experimental work. JSH 23 performed the colony PCR. SBC infected and sampled the porcine burn wounds. DS 24 generated the sequence library. MW and GRL quantified the strain frequency in the 25 wounds. ESG, CWM, MW, VSC, and DJW conceptualized the project and wrote the 26 manuscript. 27Chronic infections are those that persist despite extensive treatment. These 55 persistent infections are often attributed to difficult to eradicate biofilms, which are 56 communities of adhered microorganisms encased in an extracellular polymeric 57 substance (EPS) 1,2 . Complicating chronic infections is the high likelihood that 58 bacterial populations adaptively evolve, producing persistent phenotypes with 59 increased fitness. 60 61One of the most understood bacterial adaptive responses to chronic infection is that 62of Pseudomonas aeruginosa to the cystic fibrosis (CF) lung 3 . CF patients exhibit 63 mucus accumulation, where P. aeruginosa biofilms commonly colonize the mucus 64 lining and establish persistent pulmonary infections 4-6 . Evolved phenotypic variants 65 of this organism are routinely isolated from CF patient sputum samples, and several 66 of these variants are often associated with worsening patient prognosis 7 . Of 67 particular interest are the rugose small-colony variants (RSCVs), which are isolated 68 from up to 50% of P. aeruginosa-positive CF sputum samples 8,9 . When isolated, 69 their frequencies range drastically between 0.1 − 100% 9,10 . In contrast there is little-70 to-no reports of the frequency of P. aeruginosa adapted variants from other chronic 71 infections, such as chronic wound infections. 72 73 Common to RSCVs are mutations in pathways that lead to elevated cyclic 74 diguanylate monophosphate (c-di-GMP) 7 . C-di-GMP is a messenger molecule that 75 signals the transition from planktonic to biofilm lifestyle in many bacteria 11 . In P. 76 aeruginosa, increased c-di-GMP, among many responses, leads to overproduction of 77 exopolysaccharides, Psl and Pel, and matrix proteins 12,13 . As a result, RSCVs have 78 hyper-biofilm phenotypes 13,14 , increased tolerance to antimicrobials 10 , and enhanced 79 resistance to immunity 15,16 . P. aeruginosa RSCVs also evolve from in vitro grown 80 biofilms 17,18 , suggesting that there is strong selection for ecological diversification in 81 both in vivo and in vitro biofilms. 82 defined by a matte, rugose colony morphology that stained intensely by the two dyes, 132 indicating exopolysaccharide overproduction, were isolated from all three timepoints 133 (Fig 2A). Two RSCV sub-populations were observed; one that had a pink, rugose 134 phenotype and a second that had an orange, textured phenotype (Fig 2A). 135 136The RSCV abundance in the wounds was quantified by expressing their frequency 137 as a percentage of the total P. aeruginosa burden. The RSCV frequency was low in 138

show abstract

Section: Resultssupporting

confidence: 64%

ThePseudomonas aeruginosaWsp pathway undergoes positive evolutionary selection during chronic infection

Gloag

Marshall

Snyder

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…In some cases, two such orientation reversal events are paired to return the DNA strand to its original orientation. A similar mechanism was recently proposed to explain mutation clusters observed between chimpanzee and human reference genomes, as well as between the assembled genomes of individual humans [33]. The rate at which we identified short-range orientation reversal events in healthy human tissue further supports the idea that they play a role in both the evolution of genomes and the appearance of genetic diversity within species.…”

Section: Discussionsupporting

confidence: 86%

“…Since HR is impaired in BRCA2-deficient cells, fork restart through U-turns could explain a reduced sensitivity to fork stalling in the absence of fork reversal. The rate at which we observe short-range orientation-reversing template-switching events in healthy tissues and the proposed evolutionary model also suggest that the SVs resulting from U-turns can occur with little to no deleterious effects [33].…”

Section: Discussionmentioning

confidence: 99%

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

Tremblay‐Belzile

Giorgio

Loubert‐Hudon

et al. 2020

Preprint

View full text Add to dashboard Cite

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...

show abstract

“…Correspondingly, template switching during CRISPR/Cas9-mediated HDR in HEK293T cells has been recently demonstrated by Paix et al (2017) . We hypothesized that the presence of microhomologies in the target sequence may explain why certain targets are prone to template switching with subsequent erroneous repair, as previously seen in genomic rearrangement events ( Löytynoja and Goldman, 2017 ) ( Fig. 6 C).…”

Section: Discussionmentioning

confidence: 81%

CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments

Boel

Saffel

Steyaert

et al. 2018

Disease Models &Amp; Mechanisms

124

View full text Add to dashboard Cite

Targeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome. Our results suggest that HDR rates are strongly determined by repair-template composition, with the most influential factor being homology-arm length. However, we found that repair using ssODNs does not only lead to precise sequence replacement but also induces integration of repair-template fragments at the Cas9 cut site. We observed that error-free repair occurs at a relatively constant rate of 1-4% when using different repair templates, which was sufficient for transmission of point mutations to the F1 generation. On the other hand, erroneous repair mainly accounts for the variability in repair rate between the different repair templates. To further improve error-free HDR rates, elucidating the mechanism behind this erroneous repair is essential. We show that the error-prone nature of ssODN-mediated repair, believed to act via synthesis-dependent strand annealing (SDSA), is most likely due to DNA synthesis errors. In conclusion, caution is warranted when using ssODNs for the generation of knock-in models or for therapeutic applications. We recommend the application of in-depth NGS analysis to examine both the efficiency and error-free nature of HDR events.

show abstract

Short template switch events explain mutation clusters in the human genome

Cited by 31 publications

References 56 publications

ThePseudomonas aeruginosaWsp pathway undergoes positive evolutionary selection during chronic infection

ThePseudomonas aeruginosaWsp pathway undergoes positive evolutionary selection during chronic infection

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

CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments

Contact Info

Product

Resources

About