Newborn Genetic Screening-Still a Role for Sanger Sequencing in the Era of NGS.

In the Norwegian newborn screening (NBS) program, genetic testing has been implemented as a second or third tier method for the majority of NBS disorders, significantly increasing positive predictive value (PPV). DNA is extracted from dried blood spot (DBS) filter cards. For monogenic disorders caused by variants in one single gene or a few genes only, Sanger sequencing has been shown to be the most time- and cost-efficient method to use. Here, we present the Sanger sequencing method, including primer sequences and the genetic test algorithms, currently used in the Norwegian newborn screening program.

Performance of Expanded Newborn Screening in Norway Supported by Post-Analytical Bioinformatics Tools and Rapid Second-Tier DNA Analyses.

In 2012, the Norwegian newborn screening program (NBS) was expanded (eNBS) from screening for two diseases to that for 23 diseases (20 inborn errors of metabolism, IEMs) and again in 2018, to include a total of 25 conditions (21 IEMs). Between 1 March 2012 and 29 February 2020, 461,369 newborns were screened for 20 IEMs in addition to phenylketonuria (PKU). Excluding PKU, there were 75 true-positive (TP) (1:6151) and 107 (1:4311) false-positive IEM cases. Twenty-one percent of the TP cases were symptomatic at the time of the NBS results, but in two-thirds, the screening result directed the exact diagnosis. Eighty-two percent of the TP cases had good health outcomes, evaluated in 2020. The yearly positive predictive value was increased from 26% to 54% by the use of the Region 4 Stork post-analytical interpretive tool (R4S)/Collaborative Laboratory Integrated Reports 2.0 (CLIR), second-tier biochemical testing and genetic confirmation using DNA extracted from the original dried blood spots. The incidence of IEMs increased by 46% after eNBS was introduced, predominantly due to the finding of attenuated phenotypes. The next step is defining which newborns would truly benefit from screening at the milder end of the disease spectrum. This will require coordinated international collaboration, including proper case definitions and outcome studies.

Second-Tier Next Generation Sequencing Integrated in Nationwide Newborn Screening Provides Rapid Molecular Diagnostics of Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) and other T cell lymphopenias can be detected during newborn screening (NBS) by measuring T cell receptor excision circles (TRECs) in dried blood spot (DBS) DNA. Second tier next generation sequencing (NGS) with an amplicon based targeted gene panel using the same DBS DNA was introduced as part of our prospective pilot research project in 2015. With written parental consent, 21 000 newborns were TREC-tested in the pilot. Three newborns were identified with SCID, and disease-causing variants in IL2RG, RAG2, and RMRP were confirmed by NGS on the initial DBS DNA. The molecular findings directed follow-up and therapy: the IL2RG-SCID underwent early hematopoietic stem cell transplantation (HSCT) without any complications; the leaky RAG2-SCID received prophylactic antibiotics, antifungals, and immunoglobulin infusions, and underwent HSCT at 1 year of age. The child with RMRP-SCID had complete Hirschsprung disease and died at 1 month of age. Since January 2018, all newborns in Norway have been offered NBS for SCID using 1st tier TRECs and 2nd tier gene panel NGS on DBS DNA. During the first 20 months of nationwide SCID screening an additional 88 000 newborns were TREC tested, and four new SCID cases were identified. Disease-causing variants in DCLRE1C, JAK3, NBN, and IL2RG were molecularly confirmed on day 8, 15, 8 and 6, respectively after birth, using the initial NBS blood spot. Targeted gene panel NGS integrated into the NBS algorithm rapidly delineated the specific molecular diagnoses and provided information useful for management, targeted therapy and follow-up i.e., X rays and CT scans were avoided in the radiosensitive SCID. Second tier targeted NGS on the same DBS DNA as the TREC test provided instant confirmation or exclusion of SCID, and made it possible to use a less stringent TREC cut-off value. This allowed for the detection of leaky SCIDs, and simultaneously reduced the number of control samples, recalls and false positives. Mothers were instructed to stop breastfeeding until maternal cytomegalovirus (CMV) status was determined. Our limited data suggest that shorter time-interval from birth to intervention, may prevent breast milk transmitted CMV infection in classical SCID.

Ecological speciation by temporal isolation in a population of the stonefly Leuctra hippopus (Plecoptera, Leuctridae).

Stream dwelling invertebrates are ideal candidates for the study of ecological speciation as they are often adapted to particular environmental conditions within a stream and inhabit only certain reaches of a drainage basin, separated by unsuitable habitat. We studied an atypical population of the stonefly Leuctra hippopus at a site in central Norway, the Isterfoss rapids, in relation to three nearby and two remote conspecific populations. Adults of this population emerge about a month earlier than those of nearby populations, live on large boulders emerging from the rapids, and are short-lived. This population also has distinct morphological features and was studied earlier during the period 1975-1990. We reassessed morphological distinctness with new measurements and added several analyses of genetic distinctness based on mitochondrial and nuclear sequence markers, as well as AFLP fingerprinting and SNPs mined from RAD sequences. The Isterfoss population is shown to be most closely related to its geographical neighbors, yet clearly morphologically and genetically distinct and homogeneous. We conclude that this population is in the process of sympatric speciation, with temporal isolation being the most important direct barrier to gene flow. The shift in reproductive season results from the particular temperature and water level regime in the Isterfoss rapids. The distinct adult body shape and loss of flight are hypothesized to be an adaptation to the unusual habitat. Ecological diversification on small spatial and temporal scales is one of the likely causes of the high diversity of aquatic insects.

Sperm performance in conspecific and heterospecific female fluid.

Divergent sexual selection within allopatric populations may result in divergent sexual phenotypes, which can act as reproductive barriers between populations upon secondary contact. This hypothesis has been most tested on traits involved in precopulatory sexual selection, with less work focusing on traits that act after copulation and before fertilization (i.e., postcopulatory prezygotic traits), particularly in internally fertilizing vertebrates. However, postcopulatory sexual selection within species can also drive trait divergence, resulting in reduced performance of heterospecific sperm within the female reproductive tract. Such incompatibilities, arising as a by-product of divergent postcopulatory sexual selection in allopatry, can represent reproductive barriers, analogous to species-assortative mating preferences. Here, we tested for postcopulatory prezygotic reproductive barriers between three pairs of taxa with diverged sperm phenotypes and moderate-to-high opportunity for postcopulatory sexual selection (barn swallows Hirundo rustica versus sand martins Riparia riparia, two subspecies of bluethroats, Luscinia svecica svecica versus L. s. namnetum, and great tits Parus major versus blue tits Cyanistes caeruleus). We tested sperm swimming performance in fluid from the outer reproductive tract of females, because the greatest reduction in sperm number in birds occurs as sperm swim across the vagina. Contrary to our expectations, sperm swam equally well in fluid from conspecific and heterospecific females, suggesting that postcopulatory prezygotic barriers do not act between these taxon pairs, at this stage between copulation and fertilization. We therefore suggest that divergence in sperm phenotypes in allopatry is insufficient to cause widespread postcopulatory prezygotic barriers in the form of impaired sperm swimming performance in passerine birds.

Linked selection and recombination rate variation drive the evolution of the genomic landscape of differentiation across the speciation continuum of Ficedula flycatchers.

Speciation is a continuous process during which genetic changes gradually accumulate in the genomes of diverging species. Recent studies have documented highly heterogeneous differentiation landscapes, with distinct regions of elevated differentiation ("differentiation islands") widespread across genomes. However, it remains unclear which processes drive the evolution of differentiation islands; how the differentiation landscape evolves as speciation advances; and ultimately, how differentiation islands are related to speciation. Here, we addressed these questions based on population genetic analyses of 200 resequenced genomes from 10 populations of four Ficedula flycatcher sister species. We show that a heterogeneous differentiation landscape starts emerging among populations within species, and differentiation islands evolve recurrently in the very same genomic regions among independent lineages. Contrary to expectations from models that interpret differentiation islands as genomic regions involved in reproductive isolation that are shielded from gene flow, patterns of sequence divergence (d(xy) and relative node depth) do not support a major role of gene flow in the evolution of the differentiation landscape in these species. Instead, as predicted by models of linked selection, genome-wide variation in diversity and differentiation can be explained by variation in recombination rate and the density of targets for selection. We thus conclude that the heterogeneous landscape of differentiation in Ficedula flycatchers evolves mainly as the result of background selection and selective sweeps in genomic regions of low recombination. Our results emphasize the necessity of incorporating linked selection as a null model to identify genome regions involved in adaptation and speciation.

Evolutionary analysis of the female-specific avian W chromosome.

The typically repetitive nature of the sex-limited chromosome means that it is often excluded from or poorly covered in genome assemblies, hindering studies of evolutionary and population genomic processes in non-recombining chromosomes. Here, we present a draft assembly of the non-recombining region of the collared flycatcher W chromosome, containing 46 genes without evidence of female-specific functional differentiation. Survival of genes during W chromosome degeneration has been highly non-random and expression data suggest that this can be attributed to selection for maintaining gene dose and ancestral expression levels of essential genes. Re-sequencing of large population samples revealed dramatically reduced levels of within-species diversity and elevated rates of between-species differentiation (lineage sorting), consistent with low effective population size. Concordance between W chromosome and mitochondrial DNA phylogenetic trees demonstrates evolutionary stable matrilineal inheritance of this nuclear-cytonuclear pair of chromosomes. Our results show both commonalities and differences between W chromosome and Y chromosome evolution.

Increased divergence but reduced variation on the Z chromosome relative to autosomes in Ficedula flycatchers: differential introgression or the faster-Z effect?

Recent multilocus studies of congeneric birds have shown a pattern of elevated interspecific divergence on the Z chromosome compared to the autosomes. In contrast, intraspecifically, birds exhibit less polymorphism on the Z chromosome relative to the autosomes. We show that the four black-and-white Ficedula flycatcher species show greater genetic divergence on the Z chromosome than on the autosomes, and that the ratios of intraspecific polymorphism at Z-linked versus autosomal markers are below the neutral expectation of 75%. In all species pairs, we found more fixed substitutions and fewer shared polymorphisms on the Z chromosome than on the autosomes. Finally, using isolation with migration (IMa) models we estimated gene flow among the four closely related flycatcher species. The results suggest that different pattern of evolution of Z chromosomes and autosomes is best explained by the faster-Z hypothesis, since the estimated long-term gene flow parameters were close to zero in all comparisons.

Deep sympatric mitochondrial divergence without reproductive isolation in the common redstart Phoenicurus phoenicurus.

Mitochondrial DNA usually shows low sequence variation within and high sequence divergence among species, which makes it a useful marker for phylogenetic inference and DNA barcoding. A previous study on the common redstart (Phoenicurus phoenicurus) revealed two very different mtDNA haplogroups (5% K2P distance). This divergence is comparable to that among many sister species; however, both haplogroups coexist and interbreed in Europe today. Herein, we describe the phylogeographic pattern of these lineages and test hypotheses for how such high diversity in mtDNA has evolved. We found no evidence for mitochondrial pseudogenes confirming that both haplotypes are of mitochondrial origin. When testing for possible reproductive barriers, we found no evidence for lineage-specific assortative mating and no difference in sperm morphology, indicating that they are not examples of cryptic species, nor likely to reflect the early stages of speciation. A gene tree based on a short fragment of cytochrome c oxidase subunit 1 from the common redstart and 10 other Phoenicurus species, showed no introgression from any of the extant congenerics. However, introgression from an extinct congeneric cannot be excluded. Sequences from two nuclear introns did not show a similar differentiation into two distinct groups. Mismatch distributions indicated that the lineages have undergone similar demographic changes. Taken together, these results confirm that deeply divergent mitochondrial lineages can coexist in biological species. Sympatric mtDNA divergences are relatively rare in birds, but the fact that they occur argues against the use of threshold mtDNA divergences in species delineation.

Deep sympatric mtDNA divergence in the autumnal moth (Epirrita autumnata).

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species-level taxonomy. We found five COI sub-clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub-clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.