Ecological and population genomics of the wild yellow-necked mouse, Apodemus flavicollis

Species belonging to the genus Apodemus are one of the most common and broadly distributed small mammals in the Palearctic, making them ideal for studying ecological and evolutionary processes in natural systems. Although much research has focused on the ecology of Apodemus sp., relatively little is known about the evolutionary processes which govern their natural variation in the wild. This is, in part, due to the limitations of de novo genomic research in traditionally non-model organisms. However, recent advances in high-throughput library preparation techniques such as RADseq, have made non-model organisms more accessible for genome wide analyses. This thesis shows how a modified RADseq protocol (quaddRAD) can be used to conduct ecological and evolutionary genomics research in a large population of wild yellow-necked mice, Apodemus flavicollis, that is subject to highly seasonal conditions. I show how a high quality genomic dataset of 21,011 SNPs can be generated using quaddRAD, and discuss the rationale behind the methodology in detail. I then use the genotypes to construct a multi-generational pedigree, and describe the population’s demography, fitness and allele frequency dynamics over time. I find significant variation in the genetic contributions of mice to each generation, where by the end of the study, 53.6% of the sampled population was descended from a single individual. Contrary to the expected high levels of inbreeding and low genetic diversity in such a population, I find it is largely panmictic, which suggests a large degree of connectivity to nearby populations allowing genetic rescue through high levels of migration and gene flow. Finally, I show how heterothermic responses, which reduce an individuals energy budget by up to 65% under harsh conditions, are not only highly variable in the population, but also highly heritable. This suggests heterothermy could be subject to natural selection. I show that heterothermic responses form two distinct thermal strategies in the morphospace, which may be the result of different modes of selection acting on the population to maintain significant natural variation. This thesis shows the viability of quaddRAD for large scale genomics research in wild Apodemus sp., to cement their role as a model organism for ecological and evolutionary genomics research

Synchronous seasonality in the gut microbiota of wild mouse populations

The gut microbiome performs many important functions in mammalian hosts, with community composition shaping its functional role. However, the factors that drive individual microbiota variation in wild animals and to what extent these are predictable or idiosyncratic across populations remains poorly understood. Here, we use a multi-population dataset from a common rodent species (the wood mouse, Apodemus sylvaticus), to test whether a consistent “core” gut microbiota is identifiable in this species, and to what extent the predictors of microbiota variation are consistent across populations. Between 2014 and 2018 we used capture-mark-recapture and 16S rRNA profiling to intensively monitor two wild wood mouse populations and their gut microbiota, as well as characterising the microbiota from a laboratory-housed colony of the same species. Although the microbiota was broadly similar at high taxonomic levels, the two wild populations did not share a single bacterial amplicon sequence variant (ASV), despite being only 50km apart. Meanwhile, the laboratory-housed colony shared many ASVs with one of the wild populations from which it is thought to have been founded decades ago. Despite not sharing any ASVs, the two wild populations shared a phylogenetically more similar microbiota than either did with the colony, and the factors predicting compositional variation in each wild population were remarkably similar. We identified a strong and consistent pattern of seasonal microbiota restructuring that occurred at both sites, in all years, and within individual mice. While the microbiota was highly individualised, some seasonal convergence occurred in late winter/early spring. These findings reveal highly repeatable seasonal gut microbiota dynamics in multiple populations of this species, despite different taxa being involved. This provides a platform for future work to understand the drivers and functional implications of such predictable seasonal microbiome restructuring, including whether it might provide the host with adaptive seasonal phenotypic plasticity

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Identification and quantification of chimeric sequencing reads in a highly multiplexed RAD-seq protocol

Highly multiplexed approaches have become common in genomic studies. They have improved the cost-effectiveness of genotyping hundreds of individuals using combinatorially barcoded adapters. These strategies, however, can potentially misassigned reads to incorrect samples. Here, we used a modified quaddRAD protocol to analyse the occurrence of index hopping and PCR chimeras in a series of experiments with up to 100 multiplexed samples per sequencing lane (639 samples in total). We created two types of sequencing libraries: four libraries of type A, where PCRs were run on individual samples before multiplexing, and three libraries of type B, where PCRs were run on pooled samples. We used fixed pairs of inner barcodes to identify chimeric reads. Type B libraries show a higher percentage of misassigned reads (1.15%) than type A libraries (0.65%). We also quantify the commonly undetectable chimeric sequences that occur whenever multiplexed groups of samples with different outer barcodes are sequenced together on a single flow cell. Our results suggest that these types of chimeric sequences represent up to 1.56% and 1.29% of reads in type A and B libraries, respectively. We also show that increasing the number of mismatches allowed for barcode rescue to above 2 dramatically increases the number of recovered chimeric reads. We provide recommendations for developing highly multiplexed RAD-seq protocols and analysing the resulting data to minimize the generation of chimeric sequences, allowing their quantification and a finer control on the number of PCR cycles necessary to generate enough input DNA for library preparation.Funding Agencies|University of Huddersfield; Friedrich Miescher Laboratory of the Max Planck Society; Microsoft Azure</p

Molecular genetics of BCR-ABL1 negative myeloproliferative neoplasms in India

Introduction: Over the past decade, we have moved on from a predominantly morphological and clinical classification of myeloproliferative neoplasms (MPN) to a more evolved classification that accounts for the molecular heterogeneity that is unique to this subgroup of hematological malignancies. This usually incorporates mutations in Janus kinase 2 (JAK2), MPL, and calreticulin (CALR) genes. In this manuscript, we report the frequency of these mutations in a cohort of Indian patients at a tertiary cancer center. Materials and Methods: One hundred and thirty cases of MPN were included in this study. These cases were diagnosed and classified based on the World Health Organization 2008 criteria. JAK2 and MPL mutations were detected using high sensitivity allele-specific polymerase chain reaction using fluorescent labeled primers followed by capillary electrophoresis. A subset of JAK2 and CALR mutations were assessed using a fragment length assay. Results: Among the MPN, we had 20 cases of polycythemia vera (PV), 34 cases of essential thrombocythemia (ET), and 59 of myelofibrosis (MF). JAK2, MPL, and CALR mutations were mutually exclusive of each other. Seventeen cases were categorized as MPN unclassifiable (MPN-U). JAK2p.V617F and MPL mutations were present in 60% (78 of 130) and 5.3% (7 of 130) of all MPN. All the PV cases harbored the JAK2 p.V617F mutation. A total of 23.8% (31 of 130) of patients harbored CALR mutations. CALR exon 9 mutations were detected in 60.8% (14 of 23) and 50% (5 of 10) of JAK2 and MPL negative MF and ET cases, respectively. MPN-U cases included three JAK2 p.V617F positive, two MPL p.W515 L, and 12 CALR positive cases. Ten different types of CALR indels (8 deletions and 2 insertions) were detected of which Type I and Type II mutations were the most common, occurring at a frequency of 45.1% (14 of 31) and 22.5% (7 of 31), respectively. Discussion and Conclusion: We report frequencies of JAK2 p. V617F, MPL exon 10 and CALR mutations in 130 patients similar to those reported in western literature. These mutations carry not only diagnostic but also prognostic relevance

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AimThe SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery.MethodsThis was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin.ResultsOverall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P ConclusionOne in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease