Sex-ratio distorters enable pre-zygotic sexing for sex-specific transcriptomics in Anopheles gambiae embryos

Supplementary Files Supplementary Section 1. General Feature Format File of AgamP4.61_Embryo transcriptome annotation and InterproScan output of potential novel genes. File/s: -AgamP4.61_Embryo.gtf -NovelGenes_interproScan.xlsx Supplementary Section 2. DESeq2 counts generated from the short-read dataset across all conditions and timepoints and PCA output across all conditions and timepoints. File/s: -PCA_data.xlsx Supplementary Section 3. Differential transcript expression (DESeq2) across timepoints for each experimental condition. Data are not filtered for significance. File/s: -DTE_acrossTime_Males.xlsx -DTE_acrossTime_WT.xlsx -DTE_acrossTime_Females.xlsx Supplementary Section 4. Z-score values across all conditions and timepoints used to perform k-mean analysis and list of gene IDs used for GO enrichment analysis performed using ShinyGo(v0.8). File/s: -Cluster_analysis_z_scores.xlsx Supplementary Section 5. Conservation scores value obtained using An. gambiae conservation score tool. Pkl files containing per-base Cs values (3kb upstream, TSS,TED, 3kb downstream) for each transcript category (potential novel genes, potential novel transcript, 5000 random annotated transcripts) and for the embryonic clusters. -Plotting_cs_clusters (Python script to plot conservation score values across different embryonic clusters) -Plotting_cs_transcriptome_categories (Python script to plot conservation score values of transcript belonging to different annotation categories). Pkl files containing per-base Cs values: transcriptome_cs_cluster_0.pkltranscriptome_cs_cluster_1.pkltranscriptome_cs_cluster_2.pkltranscriptome_cs_cluster_3.pkltranscriptome_cs_cluster_4.pkltranscriptome_cs_cluster_5.pkltranscriptome_cs_cluster_6.pkltranscriptome_cs_potenial_novel_genes.pkltranscriptome_cs_potenial_novel_isoforms.pkltranscriptome_cs_real_matching_5000.pkl Supplementary Section 6. DenoiSex pipeline script and output. The folder contain the scripts to perform the sex bias correction on estimates counts generated using Kallisto (01_Bias_correction_analysis), the generation of reduced transcriptome (02_Generate_reduced_transcriptomes) and the estimation of Noise frool for expression analysis (03_Noise_floor_expression_analysis). The file 'DenoiSex_corrected_counts_booleans.xlsx' contains sex-corrected estimated transcript counts (generated using Kallisto) alongside with the original counts. The file also contains boolean values that indicating transcript above/below quantification noise treshold. File/s: -01_Bias_correction_analysis -02_Generate_reduced_transcriptomes -03_Noise_floor_expression_analysis -DenoiSex_corrected_counts_booleans.xlsx Supplementary Section 7. Differential transcripts expression across timepoints and between Male and Female embryo pools (DESeq2). Data are annotated based on AgamP4.61_Embryo transcriptome and are not filtered for significance. The file also incorporate information from the DenoiSex pipeline (Boolean value for quantificaiton noise tresholds). File/s: -DTE_acrossTime_Female.xlsx -DTE_acrossTime_Male.xlsx -DTE_acrossTime_WT.xlsx -DTE_Male_versus_Female.xlsx Supplementary Section 8. Differential splicing analysis output (splicing events) across timepoints for all the experimental conditions and between Male and Female embryo pools (SUPPA2). Transcript are annotated based on AgamP4.61_Embryo transcriptome and are filtered for significance as explained in the main text. File/s: -Differential_splicing_events_time_sex.xlsx Supplementary Section 9. The folder contains the output of a Motif enrichment analysis on splicing events during An. gambiae embryogenesis. Transcripts were selected based on SUPPA2 results used to identify differential splicing between male and female embryogenesis and across developmental stages. This file contains the output of SEA (https://doi.org/10.1101/2021.08.23.457422) performed on transcript regions involved in splicing across time and between sexes selected using MoSEA (https://github.com/comprna/MoSEA). The folder also contains the input sequences (fasta) for each splicing event category used as input for the SEA analysis. Control fasta sequences are also provided. File/s: -Plotting_SEA_output (script to plot merged SEA output files) -all_events_merged_sequences_Anopheles_sex (merged SEA sequences output files. differential splicing between sexes) -all_events_merged_sequences_Anopheles_time (merged SEA sequences output files, differential splicing across time) Sex: -SEA_output (contains folders for each SEA analysis on each splicing event category). -Male_versus_Female_a3_tot.fasta-Male_versus_Female_a5_tot.fasta-Male_versus_Female_af_tot.fasta-Male_versus_Female_al_tot.fasta-Male_versus_Female_control.fasta-Male_versus_Female_mx_tot.fasta-Male_versus_Female_ri_tot.fasta-Male_versus_Female_se_tot.fasta Time: -SEA_output (contains folders for each SEA analysis on each splicing event category). -splicing_WT_time_a3_tot.fasta-splicing_WT_time_a5_tot.fasta-splicing_WT_time_af_tot.fasta-splicing_WT_time_al_tot.fasta-splicing_WT_time_control.fasta-splicing_WT_time_mx_tot.fasta-splicing_WT_time_ri_tot.fasta-splicing_WT_time_se_tot.fasta Supplementary Section 10. Fle binding motif scan using FIMO. The folder contains FIMO output on differentially expressed transcripts and differential spliced transcripts between Male and Female embryos across development. -FIMO_Differential_splicing_Fle_motif (folder containing FIMO output)-FIMO_Differential_transcript_Fle_motif (folder containing FIMO output) Supplementary Section 11. List of Primers used for experimental validation of novel genes, sex-specific splicing and qPCR. -List_of_primers