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A Protocol for Genome-Wide Analysis of DNA Replication Timing in Intact Root Tips

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Plant Cell Division

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2382))

Abstract

DNA replication during S phase in eukaryotes is a highly regulated process that ensures the accurate transmission of genetic material to daughter cells during cell division. Replication follows a well-defined temporal program, which has been studied extensively in humans, Drosophila, and yeast, where it is clear that the replication process is both temporally and spatially ordered. The replication timing (RT) program is increasingly considered to be a functional readout of genomic features and chromatin organization. Although there is increasing evidence that plants display important differences in their DNA replication process compared to animals, RT programs in plants have not been extensively studied. To address this deficiency, we developed an improved protocol for the genome-wide RT analysis by sequencing newly replicated DNA (“Repli-seq”) and applied it to the characterization of RT in maize root tips. Our protocol uses 5-ethynyl-2′-deoxyuridine (EdU) to label replicating DNA in vivo in intact roots. Our protocol also eliminates the need for synchronization and frequently associated chemical perturbations as well as the need for cell cultures, which can accumulate genetic and epigenetic differences over time. EdU can be fluorescently labeled under mild conditions and does not degrade subnuclear structure, allowing for the differentiation of labeled and unlabeled nuclei by flow sorting, effectively eliminating contamination issues that can result from sorting on DNA content alone. We also developed an analysis pipeline for analyzing and classifying regions of replication and present it in a point-and-click application called Repliscan that eliminates the need for command line programming.

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Acknowledgments

This work was supported by grants from the NSF Plant Genome Research Program (NSF IOS-1025830 and IOS-2025811 to L.H.B.). Leigh Mickelson-Young and Emily E. Wear contributed equally to this work.

Author information

Authors and Affiliations

  1. Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA

    Leigh Mickelson-Young, Emily E. Wear, Linda Hanley-Bowdoin & William F. Thompson

  2. Texas Advanced Computing Center, University of Texas, Austin, TX, USA

    Jawon Song

  3. NVIDIA, Santa Clara, CA, USA

    Gregory J. Zynda

Authors
  1. Leigh Mickelson-Young
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  2. Emily E. Wear
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  3. Jawon Song
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  4. Gregory J. Zynda
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  5. Linda Hanley-Bowdoin
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  6. William F. Thompson
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Corresponding author

Correspondence to Leigh Mickelson-Young .

Editor information

Editors and Affiliations

  1. SiCE, Laboratoire Reproduction et Développement des Plantes, ENS Lyon, Lyon, France

    Marie-Cécile Caillaud

1 Electronic Supplementary Material

Fig. S1

Trim Galore! Parameter window. The default parameters are entered, as well the fastqc checkbox selected to generate the FASTQC report. Any known Illumina adapters and index sequences should be entered in the Adapter sequence to be trimmed textbox. Trim Galore! will auto-detect adapters if this parameter is left blank (PDF 409 kb)

Fig. S2

BWA-MEM parameters and output options windows. The BWA-MEM default parameters (a) and default output options (b) are auto-populated (PDF 365 kb)

Fig. S3

SAMtools view parameters window. (a) The SAMtools view app recommended parameters are selected. (b) The output from Plot distribution of MAPQ is a plot of the mapping quality (MAPQ) distribution found in the alignment file before filtering (PDF 292 kb)

Fig. S4

Picard MarkDuplicates parameter window. The recommended parameters for marking and removing duplicate read alignments (PDF 186 kb)

Fig. S5

SAMtools Flagstat output window. SAMtools Flagstat generates mapping statistics outputted as a nameable .txt file (PDF 136 kb)

Fig. S6

DeepTools multiBamSummary parameters window. (a) The recommended Correlation type is selected. A desired static bin size (bins in base pairs) should also be selected. (b) The Pearson correlation coefficients between individual biological replicates for early, middle, and late S samples represented as a heatmap with a hierarchical clustering dendrogram (PDF 226 kb)

Fig. S7

Repliscan default parameters. The Repliscan app default parameters are auto-populated. Select the plot checkbox to generate additional output files (see Table 2 and Subheading 3.11, step 9). (PDF 218 kb)

Fig. S8

Repliscan recommended parameters for maize B73 test dataset. For the maize test dataset, which has been filtered to remove alignments with MAPQ <10 and randomly downsampled, we recommend adjusting the Analysis bin size in base pairs as well as the remove parameter with the dependent parameter percentile cutoff. These setting adjustments are shown in the parameter window. See Notes 21 and 22 for a detailed description (PDF 220 kb)

Fig. S9

Repliscan input configuration example. (a) An input.txt configuration file is needed to assign individual files to analysis name labels. Individual files should be delimited by a tab. The name labels are used in output file naming and RT class segmentation naming. (b) Repliscan Inputs window requiring reference genome in Fasta format, a configuration file with a list of .bam files (input.txt), and a directory containing the .bam files (PDF 340 kb)

Fig. S10

Repliscan optional output example. Selecting the plot checkbox generates additional output files. (a) Included in these output files is a plot of the distribution of natural log transformed reads per bin in the G1 data and the selected cutoff (gray shaded area) from the remove and percentile cutoff parameters. For maize data that has been filtered to remove alignments with MAPQ <10, the distribution is negatively skewed (see Note 22). (b) An example of the .out file, which includes the parameter settings used and the auto-tuned RT class segmentation thresholds for individual chromosomes (see Subheading 3.11, step 8). The plots shown represent the files from the maize B73 test dataset with recommended parameters (Fig. S8) (PDF 520 kb)

Fig. S11

Analysis Name dropdown window example. Each app listed in the Repli-seq data analysis pipeline (Table 1) follows the same general organization. In the Analysis Name section, the specific name of the run and the location of the output folder can be modified (PDF 165 kb)

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Mickelson-Young, L., Wear, E.E., Song, J., Zynda, G.J., Hanley-Bowdoin, L., Thompson, W.F. (2022). A Protocol for Genome-Wide Analysis of DNA Replication Timing in Intact Root Tips. In: Caillaud, MC. (eds) Plant Cell Division. Methods in Molecular Biology, vol 2382. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1744-1_3

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  • DOI: https://doi.org/10.1007/978-1-0716-1744-1_3

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  • Publisher Name: Humana, New York, NY

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  • Online ISBN: 978-1-0716-1744-1

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