Phylogeography of Dolomedes in southern New Zealand: A dissertation submitted in partial fulfilment of the requirements for the Degree of Bachelor of Science with Honours at Lincoln University

lntrogression is an important concept to consider when determining the taxonomy, and so the phylogeny, of any given species. It can have negative consequences on both populations and species as genotypic distinctiveness is removed, along with gene complexes that were co adapted to the respective environments through breeding (Rhymer and Simberloff 1996). However, it also can provide information on the direction of mitochondrial gene flow between species and the geography of introgression. We investigated the genetic structure of mitochondrial DNA (COi) and nuclear DNA (actin SC) for variations within and among populations of two nurseryweb spider species: Dolomedes aquaticus and D. minor. Specimens were collected from intermediately disturbed braided rivers located in the south of the South Island, New Zealand. The resulting sequences were compared against morphological characteristics to identify traits, both genetically and phenotypically, that indicate past occurrences of introgression. Successful amplification was gained from the majority of specimens for COi, but only from a third for actin SC. A comparison between the morphological examination and nuclear sequences were found to be in agreement for the identification of these specimens. Several D. aquaticus haplotypes supported introgression events from D. aquaticus to D. minor indicated in previous research, while an example of an additional introgression event was uncovered. No evidence was found for introgression from D. minor to D. aquaticus, indicating that the introgression is one directional and that isolation mechanisms that may be in place to prevent such an occurrence are more successful within one species. In addition, the distribution patterns of identical haplotypes were found to provide an indication for when and where introgression took place

Comprehensive Assessment of BARD1 Messenger Ribonucleic Acid Splicing With Implications for Variant Classification

Introduction: Case–control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 messenger RNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. Methods: Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast, and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants. Results: We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or reverse transcription PCR analysis performed for this study. Quantitative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5. Conclusions: No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/−1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate