WALTER: an easy way to online evaluate telomere lengths from terminal restriction fragment analysis

Lyčka, Martin; Peška, Vratislav; Demko, Martin; Kilar, Agata; Fajkus, Jiřı́; Fojtová, Miloslava

doi:10.1186/s12859-021-04064-0

Cited by 33 publications

(41 citation statements)

References 12 publications

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“…Telomere lengths and telomerase activities were then analysed in pptr and WT plants in parallel. While the mean telomere lengths, obtained using terminal restriction fragment length analysis followed by evaluation using WALTER tool ( 36 ), showed values of 1.4 and 1.5 kb in two independent WT plants, mean telomere lengths in three pptr lines revealed values of ca. 300 and 480 bp, respectively (Figure 8A , B), thus corresponding to ca.…”

Section: Resultsmentioning

confidence: 99%

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Evolution of plant telomerase RNAs: farther to the past, deeper to the roots

Fajkus

Kilar

Nelson

et al. 2021

Nucleic Acids Research

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The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats.

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Section: Resultsmentioning

confidence: 99%

“…Telomere signals were visualized using a PhosphoImager FLA-7000 (GE Healthcare). Telomere lengths were calculated by WALTER toolset v2.0 ( 36 ) using default setup with background correction.…”

Section: Methodsmentioning

confidence: 99%

Evolution of plant telomerase RNAs: farther to the past, deeper to the roots

Fajkus

Kilar

Nelson

et al. 2021

Nucleic Acids Research

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“…2B). Southern blot WALTER densitometry profiles (Lycka et al 2021) versus the nanopore data also showed similar profiles between Southern density distribution and the nanopore length distribution (Supplemental Fig. S3B), suggesting that nanopore sequencing can accurately measure telomere fragment length in a bulk population or single telomere population across different telomere length profiles.…”

Section: Telomere Length By Nanopore Sequencing Recapitulates Length Measurement By Southern Blotmentioning

confidence: 65%

“…S2A). We used WALTER , a tool for Southern blot densitometry, that generates summary length statistics (Lycka et al 2021), to compare Southern blot and nanopore telomere length profiles, and again saw comparable profiles (Supplemental Fig. S3A).…”

Section: Telomere Length By Nanopore Sequencing Recapitulates Length Measurement By Southern Blotmentioning

confidence: 94%

“…In ImageQuant, we used the 2-log ladder and the CEN4 band at 1.4kb as molecular markers to determine a linear range and the resulting .csv files were loaded into RStudio to generate telomere length plots. Second, PhotoShop .tif files were inverted and loaded into WALTER ScanToItensity and IntensityAnalyzer to remove background and generate intensity profiles of the telomere signal alone (Lycka et al 2021). The reported summary statistics were loaded into RStudio to produce boxplots of telomere length.…”

Section: Southern Blotting and Densitometrymentioning

confidence: 99%

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Chromosome specific telomere lengths and the minimal functional telomere revealed by nanopore sequencing

Sholes

Karimian

Gershman

et al. 2021

Preprint

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We developed a method to tag telomeres and measure telomere length by nanopore sequencing in the yeast S. cerevisiae. Nanopore allows long read sequencing through the telomere, subtelomere and into unique chromosomal sequence, enabling assignment of telomere length to a specific chromosome end. We observed chromosome end specific telomere lengths that were stable over 120 cell divisions. These stable chromosome specific telomere lengths may be explained by stochastic clonal variation or may represent a new biological mechanism that maintains equilibrium unique to each chromosomes end. We examined the role of RIF1 and TEL1 in telomere length regulation and found that TEL1 is epistatic to RIF1 at most telomeres, consistent with the literature. However, at telomeres that lack subtelomeric Y' sequences, tel1Δ rif1Δ double mutants had a very small, but significant, increase in telomere length compared to the tel1Δ single mutant, suggesting an influence of Y' elements on telomere length regulation. We sequenced telomeres in a telomerase-null mutant (est2Δ) and found the minimal telomere length to be around 75bp. In these est2Δ mutants there were many apparent telomere recombination events at individual telomeres before the generation of survivors, and these events were significantly reduced in est2Δ rad52Δ double mutants. The rate of telomere shortening in the absence of telomerase was similar across all chromosome ends at about 5 bp per generation. This new method gives quantitative, high resolution telomere length measurement at each individual chromosome end, suggests possible new biological mechanisms regulating telomere length, and provides capability to test new hypotheses.

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