Bioinformatic methods for detecting short tandem repeat expansions in short-read sequencing have identified new repeat expansions in humans, but require alignment information to identify repetitive motif enrichment at genomic locations. We present superSTR, an ultrafast method that does not require alignment. superSTR is used to process whole-genome and whole-exome sequencing data, and perform the first STR analysis of the UK Biobank, efficiently screening and identifying known and potential disease-associated STRs in the exomes of 49,953 biobank participants. We demonstrate the first bioinformatic screening of RNA sequencing data to detect repeat expansions in humans and mouse models of ataxia and dystrophy. Individual tandem repeats may vary in length and be subject to distinct selective pressures5. STR length is highly variable, and this has been attributed to events including strand-slippage during replication6, retrotransposition7, unequal crossing over in meiosis8, and issues in DNA repair9. STR length variation is captured but not routinely evaluated in short-read whole-genome sequencing data, even though bioinformatic methods exist to extract this length information.Short tandem repeats (STRs) are nucleotide sequences comprised of repetitive motifs 1–6 nucleotides (nt) in length found within the genomes of all living organisms. They number in the hundreds of thousands and make up 3–4% of a healthy human genome10. Germline REs play causal roles in over fifty neurogenetic and movement disorders, including Huntington’s disease, certain epilepsies, myotonic dystrophies, and many ataxias13. The presence of germline expansions associated with neurodegenerative disorders also appears to reduce overall risk of other diseases, including certain cancers14. Taken as a whole, these phenomena suggest complex, evolutionarily conserved mechanisms of action worthy of study for their role in human disease and biology.This STR length variation, particularly expansions of STRs , is associated with many phenomena and phenotypes. REs are known to be causally implicated in or associated with a wide range of human diseases. Somatic REs are a hallmark of hereditary non-polyposis colorectal cancers16. Investigation of STRs in individuals, particularly in the clinical context, are performed using laboratory-based methods such as repeat-primed polymerase chain reactions and Southern blotting targeting specific individual motifs at specific genomic loci17. These methods are unsuitable for screening of multiple repeats due to their low throughput. The development of long-read sequencing (LRS) has recently been shown to offer immense utility in analysis of STR loci18. LRS is particularly useful in uncovering the architecture and sizing of large and complex REs such as those associated with cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS)19 or benign adult familial myoclonic epilepsy (BAFME)20, but the relative expense and high volumes of data associated with obtaining equivalent sequencing depth across the genome offer significant barriers to its use in large-scale studies.Sites harbouring STRs within the genome (STR loci) have been identified and catalogued in genomic sequences using a set of well-established bioinformatic approaches28. These methods estimate the size of a putative RE at each potential STR locus, and if able to, proceed to test for statistical significance by case–control or outlier analysis24. Recent de novo methods perform RE discovery within alignments without prior knowledge of STR loci. These methods search for patterns of poor alignment to the reference genome characteristic of REs. A completely de novo strategy was used in discovery of the causal RE in CANVAS, a large RE not present in the reference genome29. Initial detection of this expansion involved identifying candidate genomic regions via linkage analysis30, followed by visual inspection of aligned reads in these areas. Such manual approaches are limited by the labour-intensiveness of the process and the number of samples needed for linkage analysis. The ExpansionHunter Denovo (EHDN) method automates de novo STR discovery by implementing heuristics on the mapping quality of alignments of reads to a reference31. EHDN was also used in the detection of CANVAS-associated REs32. STRetch is another de novo STR discovery method based on realignment of reads to a reference containing STR decoys, a computationally costly step that is complicated by the combinatoric nature of the number of STR contigs generated as decoys as the motif length analysed increases23.Short-read next-generation sequencing (NGS) based detection of REs via whole-exome and whole-genome sequencing  represents the bulk of sequencing data currently available. Analysis of expansion of repetitive elements in NGS data remains uncommon outside of research settings. A set of bioinformatic methods for detection of REs exist but are dependent upon alignment of repetitive regions to a reference. The first generation of bioinformatic RE detection methods for NGS data are reliant on catalogues of known, predefined STR loci identified in the reference genome35. Such methods have focused on substitution, insertion and deletion, and structural variation, but have not considered harmonization or optimisation for calling of RE status or size. Consistency in the choice of reference genome is also critical, both across reference builds (i.e. GRCh37 vs GRCh38) and within builds where variations on inclusion of alternative contigs and decoy sequences may impact calls. The recent release of a draft complete human genome generated by long-read sequencing36 is likely to result in the incorporation of low-complexity regions, including highly-repetitive centromeric and telomeric sequence, into the reference sequence. These changes and sources of variation in alignment have effects which become significant when considering the analysis of data at scale and across individuals and cohorts.Discovery of REs in all such methods relies entirely on information found in alignment to a reference genome. Important and extensive work on ensuring harmonization of alignment and variant calling methods across cohorts and sequencing centres has been implemented in genome and exome aggregation projects including gnomAD and the NCI Genomic Data Commons38. Both approaches use efficient algorithms based on string decompositions into unique, minimal representations, followed by steps that find maximal periodicities in the decomposed string40. TRhist was used in the detection of REs associated with familial adult myoclonic epilepsy20, neuronal intranuclear inclusion disease, and oculopharyngodistal myopathy41. These tools are conceptually similar to the previously published tools used in assembled genomes  but differ fundamentally when applied in this fashion, in that they process many millions of short genomic sequences rather than small numbers of much longer genomic scaffolds. Sequential processing of all reads causes extremely long processing times when analysing data from NGS experiments with moderate target depth, and no method exists for comparison of groups of samples using either repeat expansion detector.An alternative approach is to simply test all reads for repetition without alignment. Such an approach has been implemented in the TRhist utility, and may be implemented using alternative methods for processing of sequences in FASTA format, such as mreps40 used in mreps38 in individual samples within a cohort, then compares these samples to identify motifs that are associated with diseases or phenotypes of interest. Expansions of motifs and samples identified by superSTR may then be followed up with other methods. We demonstrate superSTR’s ability to identify samples and screen for motifs with REs in raw sequencing data from short-read WGS experiments, in biobank-scale analysis of WES, and for the first time in direct interrogation of repeat sequences in RNA-seq. The demonstration of superSTR on the UK Biobank identifies both known disease signals as well as novel signals which are poised for replication studies in disease specific cohorts.We present superSTR, the first method demonstrated to rapidly identify REs in population-scale cohorts without alignment in NGS experiments. superSTR uses a fast, compression-based estimator of the information complexity of individual reads to select and process only reads likely to harbour repeat expansions for processing using the linear-time maximal repetition detection algorithm44. It relies on repetitive sequence being more compressible than non-repetitive sequence44. We compute a compression ratio for each read in a NGS experiment, C, using the highly optimised zlib compression library43. A threshold imposed on this ratio is used to identify lower-complexity reads as demonstrated in simulated sequence data  cohort contains 120 samples with RE lengths experimentally validated by the Coriell Biobank and the laboratoriesp-values (p) < 0.05 in at least one disease group of individuals with RE in the Polaris RE cohort  motif analysis method. The DM1 samples identified by superSTR that were not identified by EHDN in analysis ranged from 65 repeats through to 500 repeats in size; the smallest size repeat identified as an outlier by EHDN was 500 repeats. Seven of 14 FRDA cases were called as AAG motif outliers, along with one carrier and a single individual who is likely a FRDA expansion carrier. 13 of 16 FXS cases were called as outliers for the CCG motif, along with 4 FXS premutation individuals, a non-affected relative of a FXS case, and a complex FXS case (NA07063). Full descriptions of outlier calls are contained in Supplementary Data TCF4 ), an intronic CAG expansion in the CA10 gene, and an apparent heterozygous expansion at the locus associated with spinocerebellar ataxia type 1 (SCA1) in ATXN1. The finding of a SCA1 expansion was not supported by confirmatory targeted-locus analysis using ExpansionHunter26.We did not identify individuals with the relatively smaller Huntington’s disease AGC expansion as outliers using either superSTR or the EHDN motif method. Ranking samples by AGC information score revealed several samples within the Diversity cohort with intermediate AGC scores, but no known AGC pathogenic repeat expansions. We performed follow-up EHDN locus analysis of these individuals, detecting CAG expansions in 47 as described in the Methods. We performed superSTR analysis on all individuals, and analysed differences in the information scores across all tri-, tetra- and pentamer motifs  within four-character UK Biobank ICD10 codes corresponding to diseases of the nervous system  and diseases of the eye and adnexa  between individuals with each diagnosis and the rest of the Biobank. p-values were corrected within each ICD code using Benjamini–Hochberg correction48 across the motifs detected in the set of individuals with each ICD10 code. Motifs were reported against an ICD code if they met a significance threshold of 0.05 after multiple-testing correction. Each sample with an ICD code identified as having a potentially significant associated motif was then processed with ExpansionHunter Denovo to localize expansions of the motif within that sample.We obtained and analysed 75nt paired-read WES data for 49,953 individuals with GP and hospital-level clinical records mapped to ICD10 coding from the UK Biobank’s FE WES datasetn = 13, p = 3.4 × 10−3) than those without, consistent with presence of DM1. Significant differences in AGC scores were also observed for individuals with hereditary corneal dystrophies , consistent with presence of FECD3. Follow-up analysis with EHDN localized these putative expansions to the expected DMPK and TCF4 genes for 77% and 67% of these samples respectively, along with several other loci . Among the genes to which EHDN mapped CAG repeats in individuals diagnosed with hereditary corneal dystrophies (H185) were genes with non-RE mutations linked to ocular disorders and dystrophies, including AGBL1 , PDK3 , POLG , and RAB28 .We identified 46 unique motifs achieving statistical significance. These were comprised of six trimers, 10 tetramers, and 30 pentamers in 60 disorders Table . The AGCn = 15, p = 3.8 × 10−2). Follow-up analysis of those individuals with EHDN localised AGG expansions to DCAF8L2 in 7 participants, FGFR2 in 2 participants, and EPHB3, ERICH6, CHD3, GDNF and WIZ in 1 participant each. Five of these genes have links to neuropathies. DCAF8  has been associated with giant axonal neuropathy 253, FGFR2 disruption leads to axonal neuropathy54, and GDNF reduces symptoms of neuropathy in mouse models55. The mouse ortholog of CHD3 participates in the nucleosome remodeling and deacetylase chromatin remodeling complex (NuRD), a complex required for peripheral nerve myelination which causes neuropathy in mouse models when disrupted56. EphB3, another murine ortholog of a screen result, is involved in adult axonal plasticity and recovery from injury57.We observed a statistically significant difference in the AGG motif for mononeuropathies of the upper limb . EHDN localized those reads to the Fragile X-associated FMR1 locus for 50% of these individuals. FXS premutation length expansions have previously been associated with migraine headache in family studies of fragile-X associated tremor ataxia syndrome and in relatives of individuals diagnosed with FXS58.We detected statistically significant differences in CCG motif information score for cluster headache syndrome sufferers  and cluster headache syndrome . Analysis with EHDN localized ACG repeats in the exomes of two of these individuals to the vicinity of exon 21 of CASZ1, which appears to be the main driver of significance in this group due to the rarity of this motif. CASZ1 is a zinc finger transcription factor expressed during brain development60 in which a single nucleotide polymorphism has previously been associated with migraine by meta-analysis and GWAS with odds ratio [1.06–1.17]61.The ACG motif is an exceptionally uncommon microsatellite in the human genome, appearing in only 16 genomic locations across the genome, of which only three appear in exonsATXN3. Incomplete penetrance is observed above 44 repeats, and pathogenicity occurs when > 52 repeats are present62. We analysed RNA-seq data of peripheral blood mononuclear cells (PBMCs) from 12 individuals with SCA3 and 12 age- and gender-matched control samples63. The AGC motif information score distribution was significantly greater than the control group  associated with an intronic heterozygous CTG expansion in DMPK. The pathogenic range is broad, starting at 50 repeats for mildly-affected individuals and can exceed 2,000 repeats in individuals with severe disease. DM1 expansions induce formation of nuclear RNA foci that can be visually detected by fluorescent in situ hybridization (RNA FISH)69.Myotonic dystrophy, type 1 (DM1) is caused by a CTG expansion in the 3’ untranslated region (UTR) of 71. The first study was performed in myotubes derived from induced pluripotent stem cells from a DM1 patient and an unaffected individual70. superSTR analysis was unable to detect differences between treatment and non-treatment groups  compared to the wild-type mice and HSALR mice treated with UGC-degrading Cas9 (n = 16). Outlier detection based on information scores correctly identified all four repeat-bearing HSALR mice as outliers with no false positives.We analysed data from two studies with RNA-seq of cells treated with CUG-degrading dCas924.RE detection methods rely on statistical analysis of their output. These analyses are performed in two ways: (1) case versus control analysis, where test statistics from individuals with possible REs are compared to a set of controls assumed to be free of pathogenic REs, or (2) outlier detection, where a set of samples of unascertained status undergo analysis to detect outliers. The latter study design assumes a heterogeneous cohort where only a small proportion of the cohort harbour a particular disease-causing RE, forming a potential group of outliers. If the outlier group is too large many of the statistical approaches will failNo statistical analysis methods are provided for TRhist; and as a result we were only able to evaluate its execution time, not its RE detection performance. We benchmarked TRhist, EHDN and superSTR using RE data from the Polaris dataset as described in the “In testing on WGS data, superSTR processed an average of 444 million paired reads per individual within 4.5 h (mean 258 min) using one CPU and less than 200 MB of RAM. TRhist execution times exceeded a cut-off at 36 h. EHDN completed analysis with mean execution time of 24 h 53 m and maximum time of 30 h 46 m. EHDN execution times were dominated by the alignment step, which required up to 15 CPUs and 30 GB of RAM at peak demand; EHDN consistently processed prepared alignments within an hour using a single CPU with peak memory demand of 2.5 GB of RAM.Both superSTR motif screening and EHDN case–control motif analysis identified statistically significant enrichment of the pathogenic ACG motifs in DM1, AAG motifs in FRDA, and CCG motifs in FXS. superSTR, but not EHDN, identified statistically significant enrichment of pathogenic AGC (CAG) motifs across the Huntington's disease (HD) cohort. At the individual sample level, superSTR outlier detection consistently outperformed EHDN motif-level outlier analysis in identifying pathogenic samples. EHDN showed a recall of 0.53 for DM1-affected individuals called as AGC motif outliers compared to superSTR’s recall of 1.0; 0.75 for FXS-affected individuals (outliers for CCG) compared to 0.81, and 0.29 for FRDA-affected individuals (AAG outliers) compared to 0.50. A lack of orthogonal validation across all STR loci in the Polaris data prevents calculation of statistics requiring evidence of negative status, such as precision or F1-score. Neither superSTR nor EHDN identified HD individuals as AGC motif outliers. A key difference between the two methods is that the primary purpose of EHDN is to generate results at the locus level, however superSTR can only be directly compared to EHDN’s motif-based analysis, which it outperforms. Thus, although the methods can be compared, they should be viewed as complementary. A detailed description of outlier calls for each sample is contained within the “superSTR and TRhist were computationally benchmarked on SCA3 RNA-seq samples described previously with an average library size of 15 million paired reads. EHDN does not support RNA-seq data. superSTR required mean execution time of 22 m using 1 CPU and 5 MB of RAM; TRhist required mean execution time of 4 h 34 m using 1 CPU and 2 GB of RAM. Detail of benchmarking and results is provided in “superSTR fundamentally differs from most existing bioinformatic STR detection methods in performing de novo searches for REs that requires neither alignment nor specification of the locations of putative repeats. superSTR furthermore sets up a statistical testing framework for motif screening and outlier detection, and provides an opportunity to detect REs in the many samples held in repositories such as dbGaP by enabling rapid screening and re-screening of such data where computational resources are scarce or where datasets are prohibitively large. These data sources contain immense potential for discovery of both known, but undetected, as well as novel REs, an area of intense research interest.The computational tool closest in capabilities to superSTR is the similarly alignment-free TRhist. superSTR substantially computationally outperforms that method by tenfold for execution time by exploiting inherent properties of genomic reads. TRhist, a prototype RE analysis tool, unlike superSTR, has no statistical analysis framework to evaluate signals, further preventing screening of biobank style cohorts.TCF4 locus. An appropriate selection of the outlier fraction is analysis-specific and should be determined by the user based on the rarity of the phenotype studied in the background population. For example, backgrounds containing individuals of unascertained status, as occurs comparing neurological disease cohorts to non-neurological disease cohorts, may require use of the prevalence of the disease in the population studied as outlier fractions; alternatively, more-stringent criteria may be merited when analysing backgrounds using true control samples).superSTR outperforms the motif-based analysis of the alignment-dependent EHDN. This is likely due to a number of factors, one of which is the ability of superSTR to make use of signal from reads that only contain partial repeats. As a result, superSTR can detect motif-level changes in shorter RE disorders such as Huntington's disease. EHDN's motif strategy analyses only pairs of reads that are mostly comprised of repeats with the same motif, whereas the locus-based analysis makes use of single reads comprised of repeats. Thus EHDN motif analysis cannot be expected to detect expansions shorter than approximately the fragment length (twice the read length plus the insert size). superSTR also significantly outperforms EHDN for motif-based outlier detection due to fundamental differences in how the threshold for calling outliers is estimated. superSTR defaults to using the conservative upper 95% CI estimate of the 95th quantile of the information score by the BCa bootstrap in only control samples with unascertained status. This behaviour is modifiable by the end-user through parameters. EHDN performs case resampling using both cases and controls using an assumption of a 5% outlier fraction. This assumption is violated in the Polaris cohort and may also be violated in other cohorts for particular REs where it routinely generates high rates of false negatives at loci where repeats are relatively common, such as the FECD3-linked 72, or in direct assay of potentially toxic repeat-containing RNA73. superSTR performs best where the repeat expansion is longer than the read length, and on motifs not common in the genome. superSTR can identify sufficiently large repeat expansions present in the allosome (such as the Fragile X repeat located in the FMR1 gene on the X-chromosome) but is not able to directly account for differences due to chromosome number due to the lack of alignment information. Alignment-based methods currently outperform superSTR when analysing allosomal expansions, and provide localisation and in some cases repeat size estimation, but their performance gains are balanced by the substantially increased computational cost associated with alignment and the significant multiple testing burden created in testing multiple loci per motif across large sets of diseases in cohorts such as the UK Biobank.We recommend use of superSTR in a complementary fashion to existing RE detection tools such as EHDN, EH and exSTRa. superSTR can be used to screen large cohorts for multiple phenotypes to identify outlier repeat motifs and enable any necessary realignment and follow-up with EHDN locus-based analysis for specific sets of individuals and phenotypes to genomically locate the source of the motif signal. It is also useful in studying hypotheses about RE disease that are not necessarily dependent on repeat localisation, such as proposed motif-phenotype correlation in DNA58, complicating matters further. There is also a suggestion of structure or underdiagnosis within the UK Biobank—FECD3 prevalence is estimated as 3–11% in Caucasians74, yet the prevalence of the ICD code representing all hereditary corneal dystrophies in the analysed cohort is 0.19%, despite the older age of participants in the UK Biobank relative to the general population.Our results in the UK Biobank whole exome sequencing motif screening recovers known RE-disease associations, identifying enrichment of pathogenic motifs in individuals with ICD codes for myotonic dystrophies and hereditary corneal dystrophies. They also identify samples with potentially novel REs localising to disease-linked genes where repetition has not been known to play a role. We caution that there are many significant sources of confounding within the UK Biobank, and that further work is needed to interpret the results presented here, especially since many participants have only partial clinical data. The use of ICD10 categories as labels remains challenging when investigating individuals with RE-associated disease where diseases grouped in a single category under an ICD code may have diverse causes. This may explain the weak p-value associated with the findings related to cluster headache. EHDN identifies a large number of participants with CCG expansions relative to the reference in FMR1, but prior reports of associations between FMR1 expansion and headache emphasise the role of repeat lengthWe also note that the UK Biobank WES data analysed here has undergone repeated revisions, with three separate releases of data aligned with different pipelines in response to issues identified in the alignment pipelines. superSTR reads only read sequence from the UK Biobank data, and discards alignment information, and our findings are unaffected by changes to these pipelines. Our findings in the UK Biobank require validation in more disease-specific cohorts and should be treated as leads for further investigation.75. Choice of RNA-seq library preparation method also appears to impact the viability of STR analysis. Ribosomal RNA depletion methods allow the capture of a wider array of features in RNA than poly-A captured RNA77 and may be necessary to study intronic RNA (e.g. in TCF4 expansions for FECD3). We observe some evidence of this complexity in DM1-derived myotubes, where the repeat is absent despite confirmation of its presence by RNA FISH imaging. These behaviours may require consideration given the emergent use of cell lines in investigating RE disorders and potential treatments. In summary, superSTR substantially broadens the ability to interrogate REs and will be a valuable tool for genomic discovery work.We demonstrate that superSTR can directly analyse repeats in RNA-seq data without alignment to reference transcriptomes or genomes. This allows the direct interrogation of transcripts for the presence of potentially toxic repeat-containing RNA, which may be missed in whole-exome sequencing due to involvement of RNA expressed from intronic or intergenic regions (e.g. in non-coding RNAs), or in the increased number of intergenic, non-expressed repetition detected in whole-genome sequencing. Prior studies have largely focused on detection of downstream transcriptional signatures of expansion rather than direct detection of expanded sequence itself. There are several potential complications to such analysis. REs contained in intronic and exonic regions will likely show tissue-specific patterns of expression similar to those of the genes in which they are contained. Intergenic REs may not be expressed at all, although recent work shows that transcription initiation at microsatellites is much more widespread than previously thought superSTR processes each read in a next-generation sequencing or RNA-seq experiment by compressing each read. Repetitive reads are more compressible than non-repetitive reads “”, and th43, set for the fastest possible compression speed. C is computed as the ratio of the size in bytes of the output data to the size in bytes of the input data (“C are read-length dependent and are supplied by the user from either pre-computed threshold tables (as used in this work) or user-run simulation. Reads passing this threshold are then analysed using the Kolpakov-Kucherov algorithm40 as implemented in a modified version of the mreps software with optimisation and modifications that enable its use in this context38. The repeat detection step used a resolution parameter of 1, corresponding to exact repeat detection, without limitations on the repeat or motif length, and no limit was imposed on the number of unique maximal repeats per read in the detection step. Each read identified as containing repetitive elements has their start and end location, motif, length and repeat purity recorded.The per-sample sequence processing component of superSTR .These summary vectors are then transformed into an information score, 78, with the alternative hypothesis that randomly selected samples of interest have larger information scores than samples from the background. We computed the p-value of test statistics against a sampling distribution computed by permutation testing of the labels of each sample using 10,000 permutations. p-values were calculated with approximation to the exact value79, using the arbitrary-precision mpmath library80 to perform the required Gauss–Legendre quadrature. P-values were then false discovery rate-corrected using the Benjamini–Hochberg procedure implemented in statsmodels81. Motifs were reported as significant if the corrected Mann–Whitney permutation p-value was less than 0.05 after false discovery rate correction.Motif screening is performed on labelled samples to determine whether the distribution of information scores is greater in a group of samples relative to a background, as would be expected if a repeat expansion was associated with the label. superSTR implements this with a one-sided Mann–Whitney U test in scipy82 as implemented in the arch package (v4.19)83, and values that exceed the upper 95% confidence interval of the estimate of this percentile are reported as outliers. superSTR also offers users the option to use a percentile bootstrap should the BCa-bootstrap fail .Outlier detection in superSTR is performed using a one-versus-many strategy. 95% confidence intervals for the 95th percentile of each motif’s information score are estimated in a background or control cohort using the bCa-bootstrapAll genomic and transcriptomic analysis described were conducted as case–control or outlier detection studies across all motifs detected with multiple-testing correction conducted using the Benjamini–Hochberg procedure to control for false-discovery rates.31. Both superSTR and EHDN (v.0.9.0) were run on all samples in the Illumina Polaris cohort.The Illumina Polaris cohort consists of 270 samples, comprised of 150 publicly available whole-genome sequences of individuals with unknown RE status (European Nucleotide Archive accession PRJEB20654), and a set of 120 whole-genome sequences from the Coriell Biobank with pathogenic REs of varying lengths, representing eight disorders associated with three motifs (European Genome-Phenome Archive accession EGAD00001003562). Pathogenic REs were characterised and validated by the submitting laboratories and Coriell Biobank in each sample. The latter data is available on application as detailed in the European Genome Archive. Samples for both cohorts were prepared with TruSeq PCR-free sample preparation, then sequenced by an Illumina HiSeq X with 150 × 150 nt paired read lengths to a target whole genome coverage depth of 30×. This dataset has been previously used to evaluate performance of RE detection methodsp-value in the superSTR case–control analysis.Whole-exome sequences of 49,953 individuals with unascertained RE status and available linked primary care records were obtained from the UK Biobank WES FE dataset in CRAM format . Read sequences were read directly from the UK Biobank-provided CRAM files for each individual by superSTR. These individuals were sequenced with the DT xGen Exome Research Panel v1.0, targeting a total 39 million base pairs of the genome . Samples were sequenced with 75 × 75 nt paired-end reads on the Illumina NovaSeq 6000 platform to a target 20× coverage depth. Primary care data containing readv2 and readv3 codes were converted to ICD-10 codes using the UK Biobank-supplied tables (UK Biobank Data Coding 19). A permissive mapping strategy was used in conversion where each read code was mapped to all possible ICD10 codes. Codes mapping to nervous system disease (G00–99) and diseases of the eye and adnexa (H00–H59) were tested for association with trimer, tetramer and pentamer motifs using superSTR’s case–control analysis mode. EHDN was then used to process all samples annotated with ICD10 codes meeting a significance threshold of 0.05 for a motif All transcriptomic data is sourced from published experiments that are publicly accessible within the NCBI Sequence Read Archive.63 (SRA accession SRP168964). Samples were depleted of ribosomal RNA, cDNA synthesised using the TruSeq Stranded Kit, then sequenced on an Illumina HiSeq X Ten instrument with 100nt long reads.RNA-seq data for SCA3 was obtained from a study describing the transcriptome of peripheral blood mononuclear cells in 12 SCA3-affected individuals with 12 age- and gender-matched controls from a study of patients at Xiangya Hospital in Changsha, China65 (SRA accession SRP186882). This study describes the transcriptome of the corneal endothelium of twelve individuals with FECD undergoing corneal transplantation, along with six control samples obtained from deceased individuals. Eight of these samples showed expansion in the TCF4 gene when assayed using a triplet-primed polymerase chain reaction. Samples were depleted of ribosomal RNA and sequenced with 2 × 125 cycles on an Illumina HiSeq 2500 instrument. Mean read lengths of the data deposited in the Sequence Read Archive for this experiment varied among samples from 132 to 124 nt. We trimmed reads in this experiment to a uniform 124nt length prior to processing with superSTR.RNA-seq data for FECD3 was obtained from a study conducted at the S. Fyodorov Eye Microsurgery Federal State Institution, Russia70. These cells have a CAG repeat length estimated by multiple orthogonal methods as being between 1793 and 2700 repeats in the DMPK gene84. This study used Illumina TruSeq PolyA library preparation, and samples were sequenced on an Illumina HiSeq 4000 instrument, with a uniform 99 × 99 nt read length.RNA-seq data for myotonic dystrophy, type 1 (DM1) was obtained from two studies of clearance of CTG-repeat RNA foci by RNA-targeted CRISPR-dCas9. The first study was conducted in human myoblasts derived from induced pluripotent stem cells that were in turn derived from a patient with DM1 (SRA accession SRP111361)LR mouse, which expresses 250 CTG repeats associated with the human skeletal actin promoter, and has 20–25 CTG repeats in the mouse Dmpk gene itself (SRA accession SRP266474)71. This study used Illumina TruSeq PolyA library preparation, and samples were sequenced on an Illumina HiSeq 4000 instrument. The SRA-deposited RNA-seq data for this experiment also varied in average spot length, with common read lengths in wild-type mice of 150 × 150 nt paired reads and 100 × 100 nt paired reads in the DM1-affected mice. We cropped reads in this experiment to a uniform 100 nt length prior to processing with superSTR. We assigned ‘expanded’ status to runs of samples from untreated HSALR mice in this experiment (8 runs) and ‘contracted’ status to treated HSALR mice and all WT mice in the experiment (12 runs).RNA-seq data for DM1 in a mouse model was obtained from a study of clearance of CTG-repeat RNA foci in skeletal muscle of HSAC ratio in classifying simulated repeat-containing reads against pseudorandom sequence drawn from four background nucleotide distributions. The uniform distribution, where each nucleotide is equiprobable, and distributions representing the genomic distributions of Homo sapiens, the GC-rich bacterium Streptomyces coelicolor, and the AT-rich intergenic regions of the protozoan Plasmodium falciparum. We randomly selected repeat motifs up to 15 nt with all possible repeat lengths up to the read length . Execution time was benchmarked on four WGS and four RNA-seq samples on the Walter and Eliza Hall Institute of Medical Research’s HPC system (Intel Xeon Sandy Bridge E5-2690 v4 CPUs with data stored on a Quantum Xcellis Storage Appliance). Accuracy was benchmarked using the Illumina Polaris dataset, testing disease groups within the Repeat Expansion cohort against the ‘control’ population of the Diversity cohort. Full details of benchmarking are contained in the “Supplementary Information 1.Supplementary Information 2.


Montmorillonite clay modified by organosulfur surfactants possesses high cation exchange capacity (CEC) and adsorption capacity than their unmodified form (UM), therefore they may elevate the adverse impact of aflatoxin B1 (AFB1) on ruminal fermentation and methanogenesis. Chemical and mechanical modifications were used to innovate the organically modified nano montmorillonite (MNM). The UM was modified using sodium dodecyl sulfate (SDS) and grounded to obtain the nanoscale particle size form. The dose-response effects of the MNM supplementation to a basal diet contaminated or not with AFB1 (20 ppb) were evaluated in vitro using the gas production (GP) system. The following treatments were tested: control , UM diet [UM supplemented at 5000 mg /kg dry matter (DM)], and MNM diets at low (500 mg/ kg DM) and high doses (1000 mg/ kg DM).P < 0.05) on GP and pH values. Diets supplemented with the low dose of MNM either with or without AFB1 supplementation resulted in lower (P = 0.015) methane (CH4) production, ruminal pH (P = 0.002), and ammonia concentration (P = 0.002) compared to the control with AFB1. Neither the treatments nor the AFB1 addition affected the organic matter or natural detergent fiber degradability. Contamination of AFB1 reduced (P = 0.032) CH4 production, while increased (P < 0.05) the ruminal pH and ammonia concentrations. Quadratic increases (P = 0.012) in total short-chain fatty acids and propionate by MNM supplementations were observed.Results of the Fourier Transform Infra-Red Spectroscopy analysis showed shifts of bands of the OH-group occurred from lower frequencies to higher frequencies in MNM, also an extra band at the lower frequency range only appeared in MNM compared to UM. Increasing the dose of the MNM resulted in linear and quadratic decreasing effects (These results highlighted the positive effects of MNM on reducing the adverse effects of AFB1 contaminated diets with a recommended dose of 500 mg/ kg DM under the conditions of this study. This partly explains the reduction in ammonia by the low dose of MNM, but unexpectedly, the high supplementation level of MNM resulted in increases in ammonia and BCVFA concentrations. It seems that not only the MNM capture capacity for ammonia can be the sole indicator of rumen ammonia reduction. The balance between ammonia and BCVFA releases and uptakes by the specific rumen microbes may also affect their final concentrations [+) in the clay [The effects of the experimental clays and AFB1 additions on ruminal pH can be attributed to ammonia production. Decreases in the pH by UM were combined with decreases in ammonia concentration, while the pH increases observed in AFB1 contaminated diets were combined with ammonia increases. Ammonia is the end-product of ruminal protein fermentation, thus high ammonia concentration is an indicator of the high degradation of dietary protein . Howevertrations . It seemthe clay . These fThe results of this study are consistent with those of Khodabandehloo et al.  who obse4 with increases in propionate concentration either with or without AFB1 contamination compared to the control diet contaminated with AFB1. The consideration of MNM as a ruminal fermentation modifier was dose-dependent since the high MNM supplementation dose adversely affected the ruminal fermentation profile. It seems that modifications of the clays are a new potential approach as feed additives to elevate the adverse effects of mycotoxins while reducing the GHG emission from the livestock sector, but such in vitro experiments did not account for the absorption of AFB1 and/or its metabolites into the bloodstream after ingestion. Therefore the modified clays are required to assess in vivo (with various diet types) for recommendations and practical applications.The modified nano montmorillonite by SDS exhibited exceptional physicochemical properties compared to the unmodified clay (UM), like high cation exchange capacity which might improve its adsorption capacity. Diets contaminated with AFB1 adversely affected the ruminal fermentation process, while MNM can stimulate them. Supplementation of MNM at 500 mg/kg reduced CH


PrEPVacc is an international, multi-centre, double-blind vaccine study comparing experimental combination vaccine regimens including DNA/AIDSVAX BE and DNA/CN54gp140 with placebo control. Simultaneously, daily oral PrEP is compared for efficacy against daily Truvada in the context of the current PrEP availability situation at the study sites. An important clinical trial outcome is the accurate measurement of in vivo antibody titer induced through vaccination. Here we report the validation of two ELISAs for CN54gp140 and AIDSVAX BE at Uganda Virus Research Institute that demonstrates precision, specificity, and robustness for assessing the reciprocal antibody end point titer in human serum. This is a critical endpoint for determining whether vaccination can provide any protection against HIV in populations at risk of acquiring HIV. Despite the widespread rollout of antiretroviral therapy (ART), HIV remains a major global health challenge and continues to disproportionately affect those living in Sub-Saharan Africa with 20.6 million people living with the virus and 670,000 new infections in 2020 and accounting for approximately 54% of the total worldwide . VaccinaIt has still not yet proved possible to elicit broadly neutralizing antibodies (bnAbs) with a vaccine but the identification, characterization and production of bNAbs with increased breadth and potency has provided an opportunity to explore the potential of passively administered antibodies to reduce viremia or delay viral rebound following ART interruption in HIV-infected individuals –6. The aThe use of daily antiretrovirals (ARV) has been much more successful as a prophylactic intervention and has been recommended, among other prevention approaches, since 2015 by the WHO for populations with an HIV incidence above 3% . Furtherwww.prevacc.org). The selected antigens for PrEPVacc, AIDSVAX BE and CN54gp140, have been tested extensively in previous clinical trials with binding antibody titers determined by antigen-specific ELISA [PrEPVacc is an Africa-led Phase IIb trial assessing the safety and efficacy of two different vaccination regimens given with pre-exposure prophylaxis regimens in which a new form of oral PrEP (Descovy) taken daily is compared for efficacy against daily Truvada in the context of the current PrEP availability situation at the study sites . The protein was stored in aliquots at -80°C until use. The constituent proteins were mixed prior to use in accordance with instructions provided by the manufacturer (VaxGen).The assay was optimised using test serum samples from vaccinated volunteers within the HIV clinical trials Tamovac II (NCT01697007) and IDEA EV06 (NCT02376582). These samples had previously well characterised responses to AIDSVAX BE and CN54gp140 proteins and included negative (baseline) and positive responses of varying magnitude. Negative control serum samples were prepared as a pool derived from healthy, unvaccinated adults living in regionally relevant areas as well as 21 commercially procured serum samples. As no international standard preparation is available, a positive control sample for was prepared as a serum sample pool derived from individuals previously vaccinated with AIDSVAX or CN54gp140. All study participants and serum donors had given written informed consent for the use of their serum for vaccine research.To allow tracking and trending of assays over time, a high and low QC sample were prepared for both AIDSVAX BE and CN54 gp140 methodologies. These samples were derived from the positive control samples used for assay precision in which a dilution was selected to return a fixed and reproducible absorbance value of ~1.0 and ~0.3, respectively ), and gently blotted on clean blotting paper. Plates were then blocked with assay buffer (200 μL/well PBST supplemented with 1% Bovine Serum Albumin (1% BSA/PBST)) to saturate nonspecific binding sites, for 1 hour at 37°C. Plates were then washed four times with 0.05% PBST and gently blotted dry.All sample dilutions and controls were prepared in tissue-culture treated polystyrene, U-bottom plates using assay buffer (1% BSA/PBST). Sample dilutions for specificity testing were prepared at 1:100 dilutions. Standard titration curves were prepared as an 11-point dilution curve with 2-fold dilutions. QC standards were prepared as ready to use aliquots. All samples were plated in triplicate.Samples and controls, blank, were added (50 μL/well) and plates were incubated for 1 hour at 37°C. Plates were washed four times with 0.05% PBST and gently blotted before adding horseradish peroxidase-conjugated anti-human IgG goat antibody  diluted 1:10000 in assay buffer (1% BSA/PBST) for 1 hour at 37°C. After washing plates four times with 0.05% PBST and gently blotting, the substrate was added  1-Component Peroxidase Substrate). The TMB is oxidized by HRP using hydrogen peroxide, which will result in a blue coloured product. This reaction was then terminated, after 5 minutes protected from direct light, by the addition of a HCL-based stop solution (50 μL/well of TMB Stop Solution), which changed the color change from blue to yellow, due to pH changes. The optical density (OD) was determined by reading the assay plate at a wavelength of 450nm using a Biotek 800TS reader and Gen5 software and is directly correlated to the amount of target specific antibody binding in the serum.The AIDSVAX BE ELISA methodology was performed as previously described by Pitisuttithm et al . BrieflyPlates were washed manually four times (250 μL/well of PBS supplemented with 0.05% Tween-20 (PBST)), and gently blotted on clean blotting paper. Plates were then blocked with assay buffer (200 μL/well PBST supplemented with 1% Bovine Serum Albumin (1% BSA/PBST)) to saturate nonspecific binding sites, for 1 hour at 37°C. Plates were then washed four times with 0.05% PBST and gently blotted dry.All sample dilutions and standards were prepared in tissue-culture treated polystyrene, U-bottom plates using assay buffer (1% BSA/PBST). Sample dilutions for specificity testing were prepared at 1:100 dilutions. Standard titration curves were prepared as an 11-point dilution curve with 2-fold dilutions. QC standards were prepared as ready to use aliquots. All samples were plated in triplicate.Samples and controls, blank, were added (50 μL/well) and plates were incubated for 1 hour at 37°C. Plates were washed four times with 0.05% PBST and gently blotted before adding horseradish peroxidase-conjugated anti-human IgG goat antibody  diluted 1:10000 in assay buffer (1% BSA/PBST) for 1 hour at 37°C. After washing plates four times with 0.05% PBST and gently blotting, the substrate was added  1-Component Peroxidase Substrate). The TMB is oxidized by HRP using hydrogen peroxide, which will result in a blue coloured product. This reaction was then terminated, after 5 minutes protected from direct light, by the addition of a HCL-based stop solution (50 μL/well of TMB Stop Solution), which changed the color change from blue to yellow, due to pH changes. The optical density (OD) was determined by reading the assay plate at a wavelength of 450nm using a Biotek 800TS reader and Gen5 software and is directly correlated to the amount of target specific antibody binding in the serum.Precision is defined as the closeness of agreement between independent test results obtained under stipulated conditions, called intra assay, inter assay and inter operator precision. To determine precision, a serum pool from vaccinated individuals was prepared spanning a dilution range of 1:100–1:102400. For intra assay precision, similar samples were measured at the same occasion in triplicate by a single operator on five separate plates; for inter assay precision, samples were measured in triplicate by a single operator on 5 separate occasions and for inter operator precision, samples were measured in triplicate by two operators on 5 separate occasions.The specificity of an analytical method is used to determine the capability of the method to assess the analyte in the presence of other components, such as are impurities, degradation products and/or sample matrix which may have an impact. Assay specificity was defined as the potential of the test to correctly assess the proportion of true test-negatives. It was used as a measurement of how well the assay performed in a group of known HIV-1 negative and non-vaccinated individuals. Specificity was assessed by testing 94 serum samples from individuals confirmed as HIV negative. The samples were assessed in triplicate at a 1:100 fixed dilution.Robustness is a measure of the assay’s capacity to remain unaffected by small but deliberate variations in method parameters, which indicated its reliability under anticipated conditions. Robustness evaluation was performed in two phases. Phase one evaluated manual plate washing against using an automated plate washer and then comparing readouts between two microplate readers BioTek 800TS and Tecan Infinite M200 Pro). Phase two evaluated varying the coating incubation times , plate blocking times , sample incubation times  and detection antibody incubation times . These are the key steps within the methodology at which variability can be introduced.The lower limit of detection (LLOD) is the lowest concentration of an analyte that can be detected but not necessarily quantified. The assay limit of detection (LOD) was evaluated by fitting four parameters logistic (4-PL) transformation to the data derived from Inter operator precision and determining the 99% confidence interval (99% CI) from the lower asymptote. For a standard curve, the LOD was the concentration corresponding to the interpolated intersection of the upper 99% CI of the lower asymptote with the 4-PL curve fit.The statistical cut-off of the ELISA was based on the upper limit of the 99.9% one-sided confidence interval of the specificity testing in HIV negative samples. The optical density (OD) corresponding to the dilution of each sample were averaged and the standard deviation was determined.All assays had to meet assay acceptance criteria to be considered a valid result  and plate readers (BioTek vs Tecan) was compared by means of a paired t-test with incubation parameter testing assessments using a one-sided Dunnett test .The quality control (QC)-specifications of each of the control samples were defined by computing standard deviation through all data. This data was plotted using Levey-Jennings plots to measure assay performance following the Westgard rules , 17. AddTo enable a single methodology to be developed for assessing responses to both CN54 and AIDSVAX BE the key steps identified for harmonization were assay incubation temperature, the preparation of QC samples and serum pools for titration curves. Precision was considered at two levels: repeatability (intra assay and inter assay) and intermediate (inter operator). To determine intermediate precision, positive serum pools derived from vaccinated subjects were titrated and tested by different operators on different days at two laboratories. Assay precision was determined using full data sets derived from Laboratory 2 (UVRI). The %CV for intra assay, inter assay and intermediate precision are shown in To assess non-specific background activity in human serum, 94 HIV-1 seronegative sera  were diluted at 1:100 and tested for non-specific background binding. Absorbance values (excluding samples that did not meet QC acceptance criteria) were used to calculate an ELISA specific positivity threshold to allow the distinction between CN54 and AIDSVAX BE negative and positive samples.Specificity evaluation showed an elevated background absorbance for AIDSVAX BE compared to CN54 and this translated to elevated assay cut-off using calculation  was determined by fitting four parameters logistic (4-PL) transformation to the data derived from Inter Operator Precision and determining the 99% confidence interval (99% CI) from the lower asymptote. This calculation recorded a value is 0.020 for CN54 ELISA and 0.014 for AIDSVAX BE ELISA .Six different parameters were assessed during robustness testing for each assay, split in to two phases. Phase 1 compared manual washing steps with the use of an automated plates washer and evaluated determining absorbance on two independent plate readers. Paired student t-tests were performed on the data sets along with an evaluation of the IC50 values for the different conditions. Comparison of manual and automated plate washing showed no difference between manual and automated plate washers p>0.05, .Phase 2 of robustness testing compared varying the incubation time for antigen coating (16–168 hours), varying the plate blocking times (60 minutes ±5 minutes), varying the sample incubation times (60 minutes ±5 minutes) and varying the detection antibody times (60 minutes ±5 minutes). No differences were observed for any of the conditions tested .The consistency of assay readouts were evaluated using a serum pool prepared from serum samples from volunteers who had received either CN54 or AIDSVAX BE vaccines within the EV06 and Tamovac II trials. Comparison of the samples indicated that although there was some cross reactivity between the CN54 High QC positive sample tested with the AIDSVAX BE ELISA there was no cross reactivity of AIDSVAX BE positive samples with the CN54 ELISA . NotwithDue to the observed cross-reactivity between the serum samples it was necessary to have two independent QC sample sets for the CN54 and AIDSVAX BE ELISA. These QC samples were used to track assay consistency over time and were interpreted using Levey-Jennings plots and Westgard rules. Evaluation of the data obtained during assay optimization and validation did not show any breaches of the Westgard rules , 19 thatELISA has been the preferred methodology for measuring the levels of binding antibody responses within many of the late-stage HIV efficacy studies to date including RV144  and HIVIThe results of the validation demonstrated that the harmonized ELISA methodology is specific, precise, and capable for repeatable titration of antibodies specific for CN54gp140 and AIDSVAX BE HIV envelope proteins with average precision values ranging from 6.2% to 13%. These values are consistent with established criteria for ELISA assays within the field, although there are no values specified within official guidance.Determination of the assay-cut off was a key objective for the validation as this is the threshold for determining positivity in a clinical sample. This value was calculated by assessed by screening normal human serum samples from USA, Tanzania and Uganda and resulted in the determination of statistical cut-off of 0.106 and 0.259 for CN54 and AIDSVAX BE respectively. To facilitate a more stringent positivity threshold, the final cut-off used for clinical immunogenicity evaluation will be fixed at an absorbance value of 0.200 and 0.500 was applied for the CN54 and AIDSVAX BE ELISA methodologies.Assessment of the sample origins revealed that there was no difference in values for all samples of different origins when tested using AIDSVAX BE methodology Click here for additional data file.S2 Data(DOCX)Click here for additional data file.


PAEP) gene isoforms and relative exon usage in endometrial glands. We report a previously unannotated endometrial gland cilia transcript GALNT11 and its susceptibility to exon skipping. Key endometrial receptivity gene transcripts are also reported to change in endometrial glands of women with recurrent pregnancy loss. The endometrial gland cilia and PAEP targets identified in this study could be used to identify a perturbed endometrium, isolate causes of recurrent pregnancy loss and develop targeted therapies in personalised medicine.Endometrial glands are essential for fertility, consisting of ciliated and secretory cells that facilitate a suitable uterine environment for embryo implantation. This study sought to determine whether an endometrial gland-specific transcriptome and splicing profile are altered in women with recurrent pregnancy loss. Our data provide a comprehensive catalogue of cilia and progestagen-associated endometrial protein (n = 5) vs a control group (n = 5). We identify target genes altered in women with recurrent pregnancy loss. Endometrial gland markers could be used to identify inappropriate endometrial receptivity.Successful embryo implantation is a trade-off between the lining of the womb which receives an implanting embryo, termed the endometrium, and a good quality embryo. For days 21–24 of the menstrual cycle, the endometrium undergoes changes into a receptive state in which it can receive an implanting embryo. Inappropriate endometrial receptivity is thought to underlie recurrent pregnancy loss. Improving pregnancy success in women with recurrent pregnancy loss requires an increased understanding of the endometrium at the molecular level. Genes contain the instructions for the cell and which genes are turned on or off determine how well it can do its role. We sought to determine a gene expression pattern of human endometrial glands in women with recurrent pregnancy loss ( A less-receptive endometrium is implicated in subfertility and recurrent implantation failure, as implantation fails in over 50% of IVF patients despite the selection of good-quality embryos. Whereas an over-receptive endometrium may underlie recurrent pregnancy loss by allowing the implantation of low-quality embryos which increases the risk of pregnancy loss (The endometrium is the lining of the womb in which the embryo implants at the start of pregnancy. The regenerated endometrium becomes receptive to implantation in the period following ovulation termed the implantation window (7–10 days after the luteinising hormone (LH) surge; LH+7–10). Inappropriate receptivity is thought to underlie reproductive failure in women with recurrent pregnancy loss ≥3 miscarriages), subfertility (>12 months of inability to conceive) and recurrent implantation failure   were recruited from fertile women who elected to donate eggs having met the criteria for egg donation, while an exclusion criteria for control participants was a history of pregnancy loss. Recurrent pregnancy loss participants (n = 5) had a history of three or more first trimester losses .Library preparation was performed using the TruSeq Stranded mRNA Library Prep kit (Illumina). The final library was quantified by a Roche KAPA library quantification kit (Illumina) and by the Agilent 2100 Bioanalyser. Paired-end RNA sequencing (2 × 150 bp) was carried out on an Illumina NextSeq 550.2 including a gland. This sub-block was mounted onto an aluminium pin with conductive glue and sputter coated with gold/palladium. The endometrial gland was imaged by Gatan 3View inside an FEI Quanta 250 FEGSEM microscope at 3.0 kV accelerating voltage and a vacuum of 40 Pa  were mapped to pathways using the publicly available software Toppgene . A B&H FDR was used to correct findings N > 10%, reads where >50% of read has Qscore ≤ 5). Paired FASTQ files were aligned to GRCh38 human genome reference using GENCODE v29 gene annotations ; 3.2.2 in the STAR manual). The two-pass alignment method was used, with soft clipping activated.Raw FASTQ reads underwent adapter trimming and quality filtering  files sorted by chromosomal coordinates assessed for saturation of known splice junctions were calculated using RSeqQC v3.0.1 ' v4.0.2 (rMATS turbo) was used to statistically measure differences in splicing between replicates of WT and mutant sequence (PAEP) and SYT13 involved in transport vesicle docking to the plasma membrane. Significantly enriched biological processes in the glandular epithelium from women with recurrent pregnancy loss included metal ion homeostasis and isoprenoid catabolic processes . The recurrent pregnancy loss group is a heterogeneous group. The two most significantly enriched biological processes in the glandular epithelium of women with recurrent pregnancy loss include tissue morphogenesis and positive regulation of cell differentiation . A previously unannotated GALNT11 transcript was reported (GALNT11-211); although GALNT11 is expressed at low levels, there was a significant splicing difference between the numbers of reads spanning the exons  and were enriched in the cilium and microtubule skeleton cellular components . Specifihe exons . GALNT11CTN1-219 . GAS5 anEighty-three gene transcripts were significantly changed in intron retention (<0.05 FDR) and were enriched in gene pathways from biological processes termed: RNA helicase activity, catalytic activity acting on RNA, long chain dehydrogenase activity and positive regulation of projection organisation . Eighty-PAEP gene transcripts were upregulated in endometrial glands from women with recurrent pregnancy loss compared to controls. These included PAEP-206, PAEP-201, PAEP-208, PAEP-209, PAEP-205, PAEP-207 and PAEP-202  demonstrated cryptic splice site usage in PAEP  and other antisense oligonucleotides (ASOs) are becoming increasingly used for the treatment of a range of genetic conditions and cancers, and so identification of RNA transcripts which can be targeted with ASOs and SSOs is a very promising avenue for treatment . Equipment in the Biomedical Imaging Unit was supported by MR/L012626/1 Southampton Imaging under MRC UKRMP.Data discussed in this study have been deposited on the NCBI’s Gene Expression Omnibus .Informed written consent was given by all participants.Participant information was anonymised.J P and J S performed sample collection and laboratory analysis. G W, J S and J P performed analysis of transcriptomic data. All authors contributed interpretation all data and the writing of the study. J C, Y C and R L initiated, designed and obtained funding for the study.


The incidence of non-alcoholic fatty liver disease (NAFLD)-associated hepatocellular carcinoma (HCC) is increasing worldwide, but the steps in precancerous hepatocytes which lead to HCC driver mutations are not well understood. Here we provide evidence that metabolically driven histone hyperacetylation in steatotic hepatocytes can increase DNA damage to initiate carcinogenesis.Global epigenetic state was assessed in liver samples from high-fat diet or high-fructose diet rodent models, as well as in cultured immortalized human hepatocytes (IHH cells). The mechanisms linking steatosis, histone acetylation and DNA damage were investigated by computational metabolic modelling as well as through manipulation of IHH cells with metabolic and epigenetic inhibitors. Chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) and transcriptome (RNA-seq) analyses were performed on IHH cells. Mutation locations and patterns were compared between the IHH cell model and genome sequence data from preneoplastic fatty liver samples from patients with alcohol-related liver disease and NAFLD.Genome-wide histone acetylation was increased in steatotic livers of rodents fed high-fructose or high-fat diet. In vitro, steatosis relaxed chromatin and increased DNA damage marker γH2AX, which was reversed by inhibiting acetyl-CoA production. Steatosis-associated acetylation and γH2AX were enriched at gene clusters in telomere-proximal regions which contained HCC tumour suppressors in hepatocytes and human fatty livers. Regions of metabolically driven epigenetic change also had increased levels of DNA mutation in non-cancerous tissue from NAFLD and alcohol-related liver disease patients. Finally, genome-scale network modelling indicated that redox balance could be a key contributor to this mechanism.Abnormal histone hyperacetylation facilitates DNA damage in steatotic hepatocytes and is a potential initiating event in hepatocellular carcinogenesis.The online version contains supplementary material available at 10.1186/s13073-022-01071-5. Telomerase (TERT) gene which is present in around 60% of cancers and is the earliest detectable of all the known mutations  an an22] anWe assessed whether the global chromatin changes are associated with DNA damage by performing immunofluorescence for ɣH2AX, a well-established marker of DNA damage , 36 whicTo investigate whether the DNA damage in vitro was dependent on histone acetylation, we inhibited the major biochemical pathways which produce acetyl-CoA, the substrate for histone acetylation. We found that the steatosis-associated ɣH2AX was reversed by a 4-h pharmacological inhibition of any of the major sources of acetyl-CoA  in the patients; similarly, 17 out of 21 genes at the chromosome 5 cluster had identified mutations . However, closer examination of the carcinoma risk factors in the TCGA patient history showed no statistically significant difference in the frequency of mutations within these 2 clusters between a group of alcoholic liver disease plus NAFLD cases  and a viral hepatitis group . Nonetheless, the combination of the cluster-wide epigenetic changes and the mutations in multiple genes within these clusters supports the possibility that large chromosomal regions have increased mutation risk from metabolism-associated epigenetic change in steatosis, which may overlap with mutagenic processes in viral hepatitis. Furthermore, the observation that the TERT gene, which is the most commonly mutated gene in HCC, had high histone acetylation and ɣH2AX levels in steatotic cells suggested that the mechanism could contribute to carcinogenesis.We next sought to identify which regions of the genome experience steatosis-associated increased ɣH2AX as that would identify which genes and biological processes could be disrupted by DNA damage. We performed chromatin immunoprecipitation with anti-H4K16ac and anti-ɣH2AX antibodies and next-generation sequencing on the enriched DNA from control and steatotic IHH cells. The ɣH2AX peak regions were compared between the control and steatotic cells. Seventy-seven of the 100 regions with biggest peak increases in steatosis compared to control cells were clustered on 7 chromosomes . These clusters displayed colocalization of H4K16 acetylation and ɣH2AX peaks  at oleic acid peak regions compared to control peaks, we re-examined the respective ChIP-seq peak regions in our sequenced whole genomes from human donors. While we did not observe any SVs at the control peak regions, we observed a deletion that affects tylation . It is ae in HCC . TherefoFinally, we examined the mutational signatures that are present in the human liver biopsy WGS datasets at the regions which correspond to the ɣH2AX peak regions. This indicated that the proportion of SNVs which are attributable to ROS-associated mutational processes are more pronounced in NAFLD clones in the steatotic ɣH2AX peak regions than control ɣH2AX peak regions , lipid storage (PLIN2), lipotoxicity (ATF4) and oxidative stress responses  , and the computationally predicted increase in aldehyde dehydrogenase (ALD5A1) and decrease in retinol dehydrogenase were also observed.Gene expression analysis identified 1769 upregulated and 2081 downregulated transcripts in oleic acid-supplemented IHH cells compared to those grown in control media  Fig. C. Genes JUN was the most significantly downregulated transcript which may indicate that garcinol reduces cell proliferation or increases apoptosis. Additionally, there was no indication that β-oxidation (CPT1A), lipotoxicity (ATF4), acetyl-CoA biogenesis  nor oxidative stress responses  were altered by garcinol although a slight decrease in a marker for lipid storage was seen (PLIN2) . There did not appear to be any overall cluster-specific transcriptional effect on protein-coding genes, with all clusters having a mix of significantly up- and downregulated genes. However, the RNA-seq also revealed that oleic acid-treated cells had a greater percentage of lincRNA or antisense transcripts with significantly increased expression , than were significantly decreased  which was a divergence from the relative percentage of all combined transcript types . Closer examination of the ChIP-seq enriched gene clusters also revealed that the highest upregulated transcript in each cluster was a non-coding RNA. The effects of garcinol treatment may mirror this, as the most significantly downregulated transcripts in 3 of the 5 clusters were non-coding RNAs .In this study, we provide evidence that energy metabolism can direct epigenetic change and DNA damage across the genome, including to the earliest mutated gene in HCC. While acetylation changes and HDAC expression changes are known in HCC  and NAFLTERT, may be predisposed for sensitivity [Overall, our data support the concept that increased acetyl-CoA flux in steatotic hepatocytes leads to increased histone acetylation at certain genomic regions thus increasing their sensitivity to the prevailing oxidative stress. We observed that many of the genomic regions which had the highest increases in ɣH2AX and H4K16ac in steatosis also had relatively high levels (compared to the rest of the genome) in cells grown in control media. This suggests that these regions, such as the telomere-proximal cluster which contains sitivity . There isitivity , but thesitivity . DespiteThe computational modelling of the metabolic changes induced by the different inhibitors of acetyl-CoA production suggests that reduced oxidative stress is responsible for the ɣH2AX decreases. This is supported by the presence of ROS-associated mutational signatures of the SNVs that are found at the genomic regions in fatty livers which correspond to the oleic acid-induced ɣH2AX peaks. NAD/NADP-consuming enzymes in folate and pyruvate metabolism were predicted to be altered, and dysregulation of either process has been shown to influence oxidative stress in liver , 49. TheTERT gene (Fig. The RNA-seq comparison of control media and oleic acid supplementation in IHH cells suggested that the increase in histone acetylation and ɣH2AX are linked to increased oxidative stress due to lipotoxicity and/or increased ROS generation from mitochondrial fat metabolism. These data also highlighted the possibility that oleic acid stimulated cell proliferation may contribute to increases in genome-wide acetylation. Garcinol treatment provided a variety of mechanistic insights as it reverses histone acetylation and ɣH2AX at the ene Fig. Aiii but ene Fig. B. The trThe RNA-seq data also revealed that an in vitro transcriptomic consequence of epigenome change in steatosis is increased transcription from lowly expressed non-coding genes. This fits with the transcriptionally permissive nature of histone hyperacetylation which may stimulate transcription from cryptic promoters. It will be important to identify whether these transcripts contribute to pathogenic changes in hepatocytes either by themselves, perhaps by promoting structural and base pair-level mutability, or by modulating the expression of protein-coding genes.The worldwide rates of HCC are increasing, and there is a switch from the incidence being driven by a relatively small population with viral hepatitis who have a high risk for HCC, to a large population with NAFLD who have a relatively low HCC risk . TherefoBesides HCC, future work will aim to determine whether regionally increased histone acetylation can progress the severity of NAFLD to NASH and cirrhosis through dysregulation of genes involved in inflammation and fibrosis. Finally, several drugs undergoing preclinical and clinical trials for reducing liver steatosis and fibrosis in NAFLD will likely impact acetyl-CoA metabolism  and/or hTERT gene is particularly affected indicates that this mechanism may be of particular importance for understanding the preneoplastic stages of HCC. To our knowledge, this is the first example of histone hyperacetylation being an initiating event in carcinogenesis. Future work can aim to manipulate this mechanism for reduction of HCC risk in patients with fatty liver disease.Our investigations reveal a new mechanism in which macronutrient-sourced metabolites induce transcriptional changes and promote mutations across the genome, including at oncogenes. The observation that the Additional file 1. Liver Cohort Baseline Clinical Data.Additional file 2. Supplementary Figures S1-S4.Additional file 3. ChIPseq significant histone peaks.Additional file 4. RNAseq DEG and GSEA.