Servant, N. | bioRxiv Science

bioRxiv2026

An alignment-last approach enables rapid transcriptomic biomarker discovery in large cohorts

Canonical transcriptomic analysis requires committing from the outset to a reference genome or transcriptome, which imposes a predefined feature set, usually annotated genes or isoforms. Alignment and annotation dilute the signal through feature-level aggregation, discard any sequence absent from the reference, and require reprocessing the entire dataset for each new question (mutations, fusions, transposable elements). Here, we introduce the alignment-last paradigm, in which the read becomes the unit of comparison across samples, and alignment is deferred to annotate only the relevant sequences. Querying the merome, a reference-free cohort k-mer index, with just a handful of reads (about 0.01% of a sample's) reveals the cohort's transcriptomic structure in bulk and single-cell data. At single-cell resolution, these reads outperform genes for cell classification and rediscover, without supervision, a transposable-element signature (VL30) of exhausted T cells. Finally, unsupervised read-level differential analysis recovers established lncRNA biomarkers; uncovers new prognostic transposable-element reads in adrenocortical carcinoma and sarcomas; and extracts signals even from reads that fail to align.

bioinformatics↗

bioRxiv2026

X-inactivation escapee domains are CTCF-cohesin independent chromatin compartments

X-chromosome inactivation involves chromosome-wide gene silencing accompanied by extensive chromatin changes, as well the loss of topologically associating domains. Yet discrete regions of the inactive X chromosome retain activity within localised 3D domains, which contain active genes that variably escape from X inactivation. The transcription factor and architectural protein CTCF has been proposed to be implicated in escape by insulating escape domains or sustaining their topology via cohesin-mediated loop extrusion. Here, we test the role of CTCF and cohesin in escape using acute degron-mediated depletion of CTCF and RAD21 in neural progenitor cells with established escape profiles. Although CTCF occupancy correlates with escape status on the inactive X chromosome, its removal - together with loss of loop extrusion - does not disrupt escapee gene expression, or domain organization, nor does it result in spreading of silencing or activation of genes in cis. Rather, we show that facultative escape regions are self-sustaining compartments of active chromatin enriched in H3K27 acetylation and depleted in H3K27 methylation, with the magnitude of compartment strength scaling up with the degree of transcriptional activity on the inactive X chromosome. These active escapee compartments are propagated independently of CTCF and RAD21-dependent 3D architecture. Our findings identify chromatin compartmentalization as the primary feature of facultative escapee domains.

genetics↗