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Genomic analysis of the T-cell receptor (TCR) reveals the strength, breadth and clonal dynamics of the adaptive immune response to pathogens or cancer. The diversity of the TCR repertoire, however, means that sequencing is technically challenging, particularly for samples with low quality, degraded nucleic acids. Here, we have developed and validated FUME-TCRseq, a robust and sensitive RNA-based TCR sequencing methodology that is suitable for formalin-fixed paraffin-embedded samples and low amounts of input material. FUME-TCRseq incorporates unique molecular identifiers into each molecule of cDNA, allowing correction for sequencing errors and PCR bias. We used RNA extracted from colorectal and head and neck cancers to benchmark the accuracy and sensitivity of FUME-TCRseq against existing methods, and found excellent concordance between the datasets. Furthermore, FUME-TCRseq detected more clonotypes than a commercial RNA-based alternative, with shorter library preparation time and significantly lower cost. The high sensitivity and the ability to sequence RNA of poor quality and limited amount enables quantitative analysis of small numbers of cells from archival tissue sections, which is not possible with other methods. To demonstrate this we performed spatially-resolved FUME-TCRseq of colorectal cancers using macrodissected archival samples, revealing the shifting T-cell landscapes at the transition to an invasive phenotype, and between tumour subclones containing distinct driver alterations. In summary, FUME-TCRseq represents an accurate, sensitive and low-cost tool for the characterisation of T-cell repertoires, particularly in samples with low quality RNA that have not been accessible using existing methodology.

BackgroundRepeat expansion (RE) disorders affect ~1 in 3000 individuals and are clinically heterogeneous diseases caused by expansions of short tandem DNA repeats. Genetic testing is often locus-specific, resulting in under diagnosis of atypical clinical presentations, especially in paediatric patients without a prior positive family history. Whole genome sequencing (WGS) is emerging as a first-line test for rare genetic disorders, but until recently REs were thought to be undetectable by this approach.MethodsWGS pipelines for RE disorder detection were deployed by the 100,000 Genomes Project and Illumina Clinical Services Laboratory. Performance was retrospectively assessed across the 13 most common neurological RE loci using 793 samples with prior orthogonal testing (182 with expanded alleles and 611 with alleles within normal size) and prospectively interrogated in 13,331 patients with suspected genetic neurological disorders.FindingsWGS RE detection showed minimum 97·3% sensitivity and 99·6% specificity across all 13 disease-associated loci. Applying the pipeline to patients from the 100,000 Genomes Project identified pathogenic repeat expansions which were confirmed in 69 patients, including seven paediatric patients with no reported family history of RE disorders, with a 0.09% false positive rate.InterpretationWe show here for the first time that WGS enables the detection of causative repeat expansions with high sensitivity and specificity, and that it can be used to resolve previously undiagnosed neurological disorders. This includes children with no prior suspicion of a RE disorder. These findings are leading to diagnostic implementation of this analytical pipeline in the NHS Genomic Medicine Centres in England.FundingMedical Research Council, Department of Health and Social Care, National Health Service England, National Institute for Health Research, Illumina Inc

Aneuploidy, defined as the loss and gain of whole and part chromosomes, is a near-ubiquitous feature of cancer genomes, is prognostic, and likely an important determinant of cancer cell biology. In colorectal cancer (CRC), aneuploidy is found in virtually all tumours, including precursor adenomas. However, the temporal evolutionary dynamics that select for aneuploidy remain broadly uncharacterised. Here we perform genomic analysis of 755 samples from a total of 167 patients with colorectal-derived neoplastic lesions that cross-sectionally represent the distinct stages of tumour evolution, and longitudinally track individual tumours through metastasis and treatment. Precancer lesions (adenomas) exhibited low levels of aneuploidy but high intra-tumour heterogeneity, whereas cancers had high aneuploidy but low heterogeneity, indicating that progression is through a genetic bottleneck that suppresses diversity. Individual CRC glands from the same tumour have similar karyotypes, despite prior evidence of ongoing instability at the cell level. Pseudo-stable aneuploid genomes were observed in metastatic lesions sampled from liver and other organs, after chemo- or targeted therapies, and late recurrences detected many years after the diagnosis of a primary tumour. Modelling indicates that these data are consistent with the action of stabilising selection that ‘traps’ cancer cell genomes on a fitness peak defined by the specific pattern of aneuploidy. These data show that the initial progression of CRC requires the traversal of a rugged fitness landscape and subsequent genomic evolution, including metastatic dissemination and therapeutic resistance, is constrained by stabilising selection.