DOI: 10.5061/dryad.50p47
title: Data from: High-throughput genotyping of green algal mutants reveals random distribution of mutagenic insertion sites and endonucleolytic cleavage of transforming DNA

description: A high-throughput genetic screening platform in a single-celled
 photosynthetic eukaryote would be a transformative addition to the plant
 biology toolbox. Here, we present ChlaMmeSeq (Chlamydomonas MmeI-based
 insertion site Sequencing), a tool for simultaneous mapping of tens of
 thousands of mutagenic insertion sites in the eukaryotic unicellular green
 alga Chlamydomonas reinhardtii. We first validated ChlaMmeSeq by in-depth
 characterization of individual insertion sites. We then applied ChlaMmeSeq
 to a mutant pool and mapped 11,478 insertions, covering 39% of annotated
 protein coding genes. We observe that insertions are distributed in a
 manner largely indistinguishable from random, indicating that mutants in
 nearly all genes can be obtained efficiently. The data reveal that
 sequence-specific endonucleolytic activities cleave the transforming DNA
 and allow us to propose a simple model to explain the origin of the poorly
 understood exogenous sequences that sometimes surround insertion sites.
 ChlaMmeSeq is quantitatively reproducible, enabling its use for pooled
 enrichment screens and for the generation of indexed mutant libraries.
 Additionally, ChlaMmeSeq allows genotyping of hits from Chlamydomonas
 screens on an unprecedented scale, opening the door to comprehensive
 identification of genes with roles in photosynthesis, algal lipid
 metabolism, the algal carbon-concentrating mechanism, phototaxis, the
 biogenesis and function of cilia, and other processes for which C.
 reinhardtii is a leading model system.
description: Supplemental Dataset 1:
 Mapped insertion sites of the individual mutants presented in Figure
 2.Supplemental_Dataset_1_mapping_sites_of_individual_mutants.xlsxSupplemental Dataset 2: Primer pairs used for the mutant insertion site characterization by PCR presented in Supplemental Figure 4.Supplemental_Dataset_2_primer_pairs_used_for_check-PCR.xlsxSupplemental Dataset 3: Oligonucleotide sequences used in this manuscript.Supplemental_Dataset_3_oligos_used_in_the_paper.xlsxSupplemental Dataset 4: Full list of hotspots and coldspots with FDR-adjusted p-values &lt;0.05.Supplemental_Dataset_4_hot-cold-spots.txtSupplemental Dataset 5: List of cassette fragmentation motifs and enrichment statistics.Supplemental_Dataset_5_cassette_fragmentation_motifs.txtSupplemental Dataset 6: All the programs written in our lab that were used for this project.Supplemental_Dataset_6_analysis_programs.tar.gzSupplemental Dataset 7: Insertion list for the dataset used for Figures 3 and 4, replicate a, raw.Supplemental_Dataset_7_insertion-list_Fig3-repl-a_raw.txtSupplemental Dataset 8: Insertion list for the dataset used for Figures 3 and 4, replicate b, raw.Supplemental_Dataset_8_insertion-list_Fig3-repl-b_raw.txtSupplemental Dataset 9: Insertion list for the dataset used for Figures 3 and 4, both replicates, filtered.Supplemental_Dataset_9_insertion-list_Fig3-both_filtered.txtSupplemental Dataset 10: Insertion list for the dataset used for Figure 5, 5' side, raw.Supplemental_Dataset_10_insertion-list_Fig5-5prime_raw.txtSupplemental Dataset 11: Insertion list for the dataset used for Figure 5, 3' side, raw.Supplemental_Dataset_11_insertion-list_Fig5-3prime_raw.txtREADME file for Supplemental Datasets 7-11