RBFOX splicing factors contribute to a broad but selective recapitulation of peripheral tissue splicing patterns in the thymus.

Thymic epithelial cells (TEC) control the selection of a T cell repertoire reactive to pathogens but tolerant of self. This process is known to involve the promiscuous expression of virtually the entire protein-coding gene repertoire, but the extent to which TEC recapitulate peripheral isoforms, and the mechanisms by which they do so, remain largely unknown. We performed the first assembly-based transcriptomic census of transcript structures and splicing factor (SF) expression in mouse medullary TEC (mTEC) and 21 peripheral tissues. Mature mTEC expressed 60.1% of all protein-coding transcripts, more than was detected in any of the peripheral tissues. However, for genes with tissue-restricted expression, mTEC produced fewer isoforms than did the relevant peripheral tissues. Analysis of exon inclusion revealed an absence of brain-specific microexons in mTEC. We did not find unusual numbers of novel transcripts in TEC, and we show that Aire, the facilitator of promiscuous gene expression, promotes the generation of long "classical" transcripts (with 5' and 3' UTRs) but has only a limited impact on alternative splicing in mTEC. Comprehensive assessment of SF expression in mTEC identified a small set of nonpromiscuously expressed SF genes, among which we confirmed RBFOX to be present with AIRE in mTEC nuclei. Using a conditional loss-of-function approach, we show that Rbfox2 promotes mTEC development and regulates the alternative splicing of promiscuously expressed genes. These data indicate that TEC recommission a small number of peripheral SFs, including members of the RBFOX family, to generate a broad but selective representation of the peripheral splice isoform repertoire.

A study of the peopling of Greenland using next generation sequencing of complete mitochondrial genomes.

OBJECTIVES: The Greenlandic population history is characterized by a number of migrations of people of various ethnicities. In this work, the analysis of the complete mtDNA genome aimed to contribute to the ongoing debate on the origin of current Greenlanders and, at the same time, to address the migration patterns in the Greenlandic population from a female inheritance demographic perspective. METHODS: We investigated the maternal genetic variation in the Greenlandic population by sequencing the whole mtDNA genome in 127 Greenlandic individuals using the Illumina MiSeq® platform. RESULTS: All Greenlandic individuals belonged to the Inuit mtDNA lineages A2a, A2b1, and D4b1a2a1. No European haplogroup was found. DISCUSSION: The mtDNA lineages seem to support the hypothesis that the Inuit in Greenland are descendants from the Thule migration. The results also reinforce the importance of isolation and genetic drift in shaping the genetic diversity in Greenlanders. Based on the mtDNA sequences, the Greenlandic Inuit are phylogenetically close to Siberian groups and Canadian Inuit.

Familial severe skeletal Class II malocclusion with gingival hyperplasia caused by a complex structural rearrangement at the KCNJ2-KCNJ16 locus.

The aim of this work was to identify the underlying genetic cause in a four-generation family segregating an unusual phenotype comprising a severe form of skeletal Class II malocclusion with gingival hyperplasia. SNP array identified a copy number gain on chromosome 1 (chr1); however, this chromosomal region did not segregate correctly in the extended family. Exome sequencing also failed to identify a candidate causative variant but highlighted co-segregating genetic markers on chr17 and chr19. Short- and long-read genome sequencing allowed us to pinpoint and characterize at nucleotide-level resolution a chromothripsis-like complex rearrangement (CR) inserted into the chr17 co-segregating region at the KCNJ2-SOX9 locus. The CR involved the gain of five different regions from chr1 that are shuffled, chained, and inserted as a single block (∼828 kb) at chr17q24.3. The inserted sequences contain craniofacial enhancers that are predicted to interact with KCNJ2/KCNJ16 through neo-topologically associating domain (TAD) formation to induce ectopic activation. Our findings suggest that the CR inserted at chr17q24.3 is the cause of the severe skeletal Class II malocclusion with gingival hyperplasia in this family and expands the panoply of phenotypes linked to variation at the KCNJ2-SOX9 locus. In addition, we highlight a previously overlooked potential role for misregulation of the KCNJ2/KCNJ16 genes in the pathomechanism of gingival hyperplasia associated with deletions and other rearrangements of the 17q24.2-q24.3 region (MIM 135400).

Facilitating accessible, rapid, and appropriate processing of ancient metagenomic data with AMDirT.

Background: Access to sample-level metadata is important when selecting public metagenomic sequencing datasets for reuse in new biological analyses. The Standards, Precautions, and Advances in Ancient Metagenomics community (SPAAM, https://spaam-community.org) has previously published AncientMetagenomeDir, a collection of curated and standardised sample metadata tables for metagenomic and microbial genome datasets generated from ancient samples. However, while sample-level information is useful for identifying relevant samples for inclusion in new projects, Next Generation Sequencing (NGS) library construction and sequencing metadata are also essential for appropriately reprocessing ancient metagenomic data. Currently, recovering information for downloading and preparing such data is difficult when laboratory and bioinformatic metadata is heterogeneously recorded in prose-based publications. Methods: Through a series of community-based hackathon events, AncientMetagenomeDir was updated to provide standardised library-level metadata of existing and new ancient metagenomic samples. In tandem, the companion tool 'AMDirT' was developed to facilitate rapid data filtering and downloading of ancient metagenomic data, as well as improving automated metadata curation and validation for AncientMetagenomeDir. Results: AncientMetagenomeDir was extended to include standardised metadata of over 6000 ancient metagenomic libraries. The companion tool 'AMDirT' provides both graphical- and command-line interface based access to such metadata for users from a wide range of computational backgrounds. We also report on errors with metadata reporting that appear to commonly occur during data upload and provide suggestions on how to improve the quality of data sharing by the community. Conclusions: Together, both standardised metadata reporting and tooling will help towards easier incorporation and reuse of public ancient metagenomic datasets into future analyses.

Multiple Breath Washout for Early Assessment of Pulmonary Complications in Patients With Primary Antibody Deficiencies: An Observational Study in Pediatric Age.

Background: In primary antibody deficiencies (PADs), pulmonary complications are the main cause of morbidity, despite immunoglobulin substitutive therapy, antibiotic treatment of exacerbations, and respiratory physiotherapy. Current Italian recommendations for surveillance of PADs respiratory complications include an annual assessment of spirometry and execution of chest high-resolution computed tomography (HRCT) every 4 years. Objective: This study aimed to evaluate the effectiveness of the lung clearance index (LCI) as an early marker of lung damage in patients with PADs. LCI is measured by multiple breath washout (MBW), a non-invasive and highly specific test widely used in patients with cystic fibrosis (CF). Methods: Pediatric patients with PADs (n = 17, 10 male, 7 female, and age range 5-15 years) underwent baseline assessment of lung involvement with chest HRCT, spirometry, and multiple breath nitrogen washout. Among them, 13 patients were followed up to repeat HRCT after 4 years, while performing pulmonary function tests annually. Their baseline and follow-up LCI and forced expiratory volume at 1 s (FEV1) values were compared, taking HRCT as the gold standard, using logistic regression analysis. Results: Lung clearance index [odds ratio (OR) 2.3 (confidence interval (CI) 0.1-52) at baseline, OR 3.9 (CI 0.2-191) at follow-up] has a stronger discriminating power between altered and normal HRCT rather than FEV1 [OR 0.6 (CI 0.2-2) at baseline, OR 1.6 (CI 0.1-13.6) at follow-up]. Conclusion: Within the context of a limited sample size, LCI seems to be more predictive of HRCT alterations than FEV1 and more sensitive than HRCT in detecting non-uniform ventilation in the absence of bronchiectasis. A study of a larger cohort of pediatric patients followed longitudinally in adulthood is needed to challenge these findings.

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma.

Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies PromethION platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150 bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from specific chromosomes of the long-read data achieved good specificity and sensitivity. However, results of somatic SNV calling highlight the need for the development of specialised joint calling algorithms. We find the comparative genome-wide performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6 Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans.

Accurate targeted long-read DNA methylation and hydroxymethylation sequencing with TAPS.

We present long-read Tet-assisted pyridine borane sequencing (lrTAPS) for targeted base-resolution sequencing of DNA methylation and hydroxymethylation in regions up to 10 kb from nanogram-level input. Compatible with both Oxford Nanopore and PacBio Single-Molecule Real-Time (SMRT) sequencing, lrTAPS detects methylation with accuracy comparable to short-read Illumina sequencing but with long-range epigenetic phasing. We applied lrTAPS to sequence difficult-to-map regions in mouse embryonic stem cells and to identify distinct methylation events in the integrated hepatitis B virus genome.