a.i.r. (artificial intelligence rna-seq) software  (Sequentia Biotech)

Journal: Nature

Article Title: Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs

doi: 10.1038/s41586-024-07472-3

Figure Lengend Snippet: a , Experimental design. CD, chow diet. b , Glucose tolerance of unexposed male offspring (F 1 ) of HFD-exposed bucks. Top: AUC ipGTT . Bottom: frequency distribution analysis to identify tolerant and intolerant animals. n = 60 male mice across 4 cohorts with 5 litters each and 3 males per litter (eLFD and eHFD bucks); n = 10–15 (sLFD and sHFD bucks) including 1 cohort with 5 litters and 3 males per litter. c , d , Glucose tolerance ( c ; mean ± s.e.m.) and insulin sensitivity ( d ; mean ± s.e.m.) of male offspring (F 1 ) of HFD-exposed bucks. Data represent a re-phenotyping of the animals in c carried out 8 weeks after the first phenotyping. Significance calculated by a two-way ( c ; n in graph) or one-way ( d ; n as in c ) analysis of variance (ANOVA; *** P < 10 −4 ). ITT, insulin tolerance test. e , f , PCA plot ( e ) and functional enrichment analysis (KEGG (Kyoto Encyclopedia of Genes and Genomes); f ) of peripheral tissue RNA-seq data from HFDt and HFDi F 1 animals. eWAT, epididymal white adipose tissue; metab., metabolism; AA, amino acid; TH, thyroid hormone; OXPHOS, oxidative phosphorylation; FA, fatty acid; TCA, tricarboxylic acid. g , Left: overlap between genes differentially expressed in tissues from HFDi mice and their human orthologues associated with childhood obesity. Right: functional enrichment analysis (KEGG) of the overlapping genes ( n = 693) pre-classified as protective and risk genes for childhood obesity on the basis of the β -score for BMI-SDS. DEGs, differentially expressed genes; Hs , Homo sapiens ; FDR, false discovery rate; NSCLC, non-small cell lung cancer; CML, chronic myeloid leukaemia; NAFLD, non-alcoholic fatty liver disease. h , Scatter plot of children’s body weight trajectories as a function of paternal BMI at conception in families with mothers who were lean (red line; r = 0.2611; P value < 10 −4 ) or overweight (blue line; r = 0.3467; P value < 10 −4 ) at conception. Significant association calculated by linear regression analysis. i , Insulin sensitivity, measured as ISI Matsuda (top) or homeostatic model assessment for insulin resistance (HOMA-IR; bottom) indices in children as a function of parental weight status at conception. n lean–lean = 106; overweight–lean = 184; lean–overweight = 114; overweight–overweight = 415. Data represented as mean ± s.e.m. Significance calculated by two-way ANOVA (details in the graph).

Article Snippet: Reads mapping and differential expression analysis were carried out using the A.I.R. (Artificial Intelligence RNA-Seq) software from Sequentia Biotech with the following pipeline: BBDuk (reads trimming; BBDUkguide ), STAR (reads mapping to the mouse genome GRCm38 (ENSEMBL); https://github.com/alexdobin/STAR ), featureCounts (gene expression quantification; https://subread.sourceforge.net/featureCounts.html ) and NOISeq (statistical analysis of differentially expressed genes; http://bioinfo.cipf.es/noiseq/doku.php ).

Techniques: Functional Assay, RNA Sequencing Assay

Journal: Nature

Article Title: Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs

doi: 10.1038/s41586-024-07472-3

Figure Lengend Snippet: ( a ) Representative H&E (top) and TRA98 (bottom) staining in testes from LFD (left) or HFD (right) fed mice. N = 6-7 mice/group independently stained and analysed ( b ) Quantification of the testis tubule diameter (average of the horizontal and vertical diameters) from H&E-stained testes (N = 60–70 seminiferous tubules – 10/mouse/group – significance calculated with Mann-Whitney test to compare ranks). ( c ) Motility of cauda spermatozoa from LFD and HFD-fed mice (N = 7 – significance calculated by two-tailed t-test). ( d - e ) First cleavage (d) and positive IVF (% of fertilised oocytes that complete pre-implantation development - e) rates in embryos generated via IVF with sperm from LFD or HFD-fed mice (N = 4 IVF each done with a pool of spermatozoa from 10 mice – significance calculated by two-tailed t-test). ( f ) PCA representation of the RNA-seq analysis of Round Spermatids (RS) isolated from testes of mice fed with LFD or HFD (f). ( g - h ) Scatter plot (g) and heatmap (h) representation of differentially expressed genes in round spermatids. ( i - j ) UMAP representation (i) and cluster annotation (j - based on publicly available datasets - GSE112393 ) of single-cell RNA-seq analysis of testes from LFD and HFD-fed mice (n = 3). ( k ) Representative marker genes for the different germ cell populations.

Article Snippet: Reads mapping and differential expression analysis were carried out using the A.I.R. (Artificial Intelligence RNA-Seq) software from Sequentia Biotech with the following pipeline: BBDuk (reads trimming; BBDUkguide ), STAR (reads mapping to the mouse genome GRCm38 (ENSEMBL); https://github.com/alexdobin/STAR ), featureCounts (gene expression quantification; https://subread.sourceforge.net/featureCounts.html ) and NOISeq (statistical analysis of differentially expressed genes; http://bioinfo.cipf.es/noiseq/doku.php ).

Techniques: Staining, MANN-WHITNEY, Two Tailed Test, Generated, RNA Sequencing Assay, Isolation, Marker

Journal: Nature

Article Title: Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs

doi: 10.1038/s41586-024-07472-3

Figure Lengend Snippet: a , Distribution of sncRNA biotypes in cauda spermatozoa from LFD- and HFD-fed mice. n = 3 samples per diet. lincRNA, long intergenic non-coding RNA; miRNA, microRNA. b , Biotype-specific differential expression analysis ( P adj < 0.05; log 2 [fold change (FC)] > |1|) of cauda spermatozoa sncRNAs. c , Histogram (bars at a bin centre of 0.5 with the overlapping non-linear fit curve) showing the distribution of the log 2 [FC(HFD versus LFD)] in nuclear- (n) and mitochondrial- (mt) derived tsRNAs (left) and rsRNAs (right). d , Volcano plot representation of differentially expressed mt-tsRNAs (differential expression calculated by EdgeR; significance defined as P adj < 0.05). e , Fragmentation pattern of mt-tRNAs in cauda spermatozoa from LFD- and HFD-fed mice. Significance tested by a two-tailed t -test, HFD versus LFD (mean ± s.e.m.; n = 3; * P < 0.05). f , Pearson-based correlation analysis of individual sncRNA biotype expression in human sperm with BMI (two-tailed P value with 95% confidence interval). g , Differentially expressed tsRNAs in human sperm from lean and overweight donors calculated by DESeq2-based continuous differential expression analysis ( n = 18 donors). VST, variance-stabilizing transformation. h , Heat map representation of mature mt-tRNA levels in cauda spermatozoa from LFD- and HFD-fed mice. i , Relative abundance of the indicated sncRNA biotypes in epididymosomes (Epi) or spermatozoa (Sp) isolated from the caput, corpus and cauda epididymis. Data reanalysed from refs. , . j , Uniform manifold approximation and projection (UMAP) representation of mtDNA transcription during spermatogenesis from testis single-cell RNA-seq data.

Article Snippet: Reads mapping and differential expression analysis were carried out using the A.I.R. (Artificial Intelligence RNA-Seq) software from Sequentia Biotech with the following pipeline: BBDuk (reads trimming; BBDUkguide ), STAR (reads mapping to the mouse genome GRCm38 (ENSEMBL); https://github.com/alexdobin/STAR ), featureCounts (gene expression quantification; https://subread.sourceforge.net/featureCounts.html ) and NOISeq (statistical analysis of differentially expressed genes; http://bioinfo.cipf.es/noiseq/doku.php ).

Techniques: Expressing, Derivative Assay, Two Tailed Test, Transformation Assay, Isolation, RNA Sequencing Assay

Journal: Nature

Article Title: Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs

doi: 10.1038/s41586-024-07472-3

Figure Lengend Snippet: a , MA plot representation of differentially expressed genes in HFD_A versus LFD embryos. b , Gene Ontology-based functional enrichment analysis of differentially expressed genes in HFD_A versus LFD embryos (see for details). c , d , Heat map representation of the relative expression (HFD versus LFD) of OXPHOS genes in early two-cell embryos ( c ) and in a publicly available dataset of single-embryo RNA-seq analysis of mouse pre-implantation development ( d ). TE_blast, trophectoderm blastocyst; ICM_blast, inner cell mass blastocyst. e , PCA-based representation of the developmental timing in HFD and LFD embryos laid over the analysis of mouse pre-implantation development of ref. . f , RNA-seq-based quantification of the expression of genes important for mitochondrial function across adult tissues and germ cells of male mice exposed to 2 weeks of HFD as well as early embryos and adult tissues from unexposed male offspring of HFD-fed fathers. 2CEs, two-cell embryos; TF, transcription factor; ROS, reactive oxygen species; gastro, gastrocnemius. g , Pearson-based correlation matrix of IMPC-derived metabolic phenotypes in WT offspring of parents heterozygous (het.) for IMPC-selected genes (see for details). h , Bar plot representation of the relative (WT versus control) glucose intolerance (measured as AUC ipGTT ) in WT offspring of fathers heterozygous for genes important for mitochondrial structure and function. Black arrows indicate genes for which cryopreserved heterozygous sperm samples were analysed. i , Distribution of sncRNA biotypes in cauda spermatozoa from the indicated mutant mice ( n = 10 mice per gene). j , Heat map representation of the relative abundance of 5′ n-tsRNAs and 5′ mt-tsRNAs in mutant spermatozoa. Control, LFD and HFD samples are cryopreserved and resequenced to serve as reference and technical controls.

Article Snippet: Reads mapping and differential expression analysis were carried out using the A.I.R. (Artificial Intelligence RNA-Seq) software from Sequentia Biotech with the following pipeline: BBDuk (reads trimming; BBDUkguide ), STAR (reads mapping to the mouse genome GRCm38 (ENSEMBL); https://github.com/alexdobin/STAR ), featureCounts (gene expression quantification; https://subread.sourceforge.net/featureCounts.html ) and NOISeq (statistical analysis of differentially expressed genes; http://bioinfo.cipf.es/noiseq/doku.php ).

Techniques: Functional Assay, Expressing, RNA Sequencing Assay, Derivative Assay, Control, Mutagenesis