NASCarD (Nanopore Adaptive Sampling with Carrier DNA): A Rapid, PCR-Free Method for SARS-CoV-2 Whole-Genome Sequencing in Clinical Samples.

Whole-genome sequencing (WGS) represents the main technology for SARS-CoV-2 lineage characterization in diagnostic laboratories worldwide. The rapid, near-full-length sequencing of the viral genome is commonly enabled by high-throughput sequencing of PCR amplicons derived from cDNA molecules. Here, we present a new approach called NASCarD (Nanopore Adaptive Sampling with Carrier DNA), which allows a low amount of nucleic acids to be sequenced while selectively enriching for sequences of interest, hence limiting the production of non-target sequences. Using COVID-19 positive samples available during the omicron wave, we demonstrate how the method may lead to >99% genome completeness of the SARS-CoV-2 genome sequences within 7 h of sequencing at a competitive cost. The new approach may have applications beyond SARS-CoV-2 sequencing for other DNA or RNA pathogens in clinical samples.

External Quality Assessment of SARS-CoV-2 Sequencing: an ESGMD-SSM Pilot Trial across 15 European Laboratories.

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.

Characterization of a haplotype-reference panel for genotyping by low-pass sequencing in Swiss Large White pigs.

BACKGROUND: The key-ancestor approach has been frequently applied to prioritize individuals for whole-genome sequencing based on their marginal genetic contribution to current populations. Using this approach, we selected 70 key ancestors from two lines of the Swiss Large White breed that have been selected divergently for fertility and fattening traits and sequenced their genomes with short paired-end reads. RESULTS: Using pedigree records, we estimated the effective population size of the dam and sire line to 72 and 44, respectively. In order to assess sequence variation in both lines, we sequenced the genomes of 70 boars at an average coverage of 16.69-fold. The boars explained 87.95 and 95.35% of the genetic diversity of the breeding populations of the dam and sire line, respectively. Reference-guided variant discovery using the GATK revealed 26,862,369 polymorphic sites. Principal component, admixture and fixation index (FST) analyses indicated considerable genetic differentiation between the lines. Genomic inbreeding quantified using runs of homozygosity was higher in the sire than dam line (0.28 vs 0.26). Using two complementary approaches, we detected 51 signatures of selection. However, only six signatures of selection overlapped between both lines. We used the sequenced haplotypes of the 70 key ancestors as a reference panel to call 22,618,811 genotypes in 175 pigs that had been sequenced at very low coverage (1.11-fold) using the GLIMPSE software. The genotype concordance, non-reference sensitivity and non-reference discrepancy between thus inferred and Illumina PorcineSNP60 BeadChip-called genotypes was 97.60, 98.73 and 3.24%, respectively. The low-pass sequencing-derived genomic relationship coefficients were highly correlated (r > 0.99) with those obtained from microarray genotyping. CONCLUSIONS: We assessed genetic diversity within and between two lines of the Swiss Large White pig breed. Our analyses revealed considerable differentiation, even though the split into two populations occurred only few generations ago. The sequenced haplotypes of the key ancestor animals enabled us to implement genotyping by low-pass sequencing which offers an intriguing cost-effective approach to increase the variant density over current array-based genotyping by more than 350-fold.

Infertility due to defective sperm flagella caused by an intronic deletion in DNAH17 that perturbs splicing.

Artificial insemination in pig (Sus scrofa domesticus) breeding involves the evaluation of the semen quality of breeding boars. Ejaculates that fulfill predefined quality requirements are processed, diluted and used for inseminations. Within short time, eight Swiss Large White boars producing immotile sperm that had multiple morphological abnormalities of the sperm flagella were noticed at a semen collection center. The eight boars were inbred on a common ancestor suggesting that the novel sperm flagella defect is a recessive trait. Transmission electron microscopy cross-sections revealed that the immotile sperm had disorganized flagellar axonemes. Haplotype-based association testing involving microarray-derived genotypes at 41,094 SNPs of six affected and 100 fertile boars yielded strong association (P = 4.22 × 10-15) at chromosome 12. Autozygosity mapping enabled us to pinpoint the causal mutation on a 1.11 Mb haplotype located between 3,473,632 and 4,587,759 bp. The haplotype carries an intronic 13-bp deletion (Chr12:3,556,401-3,556,414 bp) that is compatible with recessive inheritance. The 13-bp deletion excises the polypyrimidine tract upstream exon 56 of DNAH17 (XM_021066525.1: c.8510-17_8510-5del) encoding dynein axonemal heavy chain 17. Transcriptome analysis of the testis of two affected boars revealed that the loss of the polypyrimidine tract causes exon skipping which results in the in-frame loss of 89 amino acids from DNAH17. Disruption of DNAH17 impairs the assembly of the flagellar axoneme and manifests in multiple morphological abnormalities of the sperm flagella. Direct gene testing may now be implemented to monitor the defective allele in the Swiss Large White population and prevent the frequent manifestation of a sterilizing sperm tail disorder in breeding boars.

Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing.

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.

A Sample-to-Report Solution for Taxonomic Identification of Cultured Bacteria in the Clinical Setting Based on Nanopore Sequencing.

Amplicon sequencing of the 16S rRNA gene is commonly used for the identification of bacterial isolates in diagnostic laboratories and mostly relies on the Sanger sequencing method. The latter, however, suffers from a number of limitations, with the most significant being the inability to resolve mixed amplicons when closely related species are coamplified from a mixed culture. This often leads to either increased turnaround time or absence of usable sequence data. Short-read next-generation sequencing (NGS) technologies could solve the mixed amplicon issue but would lack both cost efficiency at low throughput and fast turnaround times. Nanopore sequencing developed by Oxford Nanopore Technologies (ONT) could solve those issues by enabling a flexible number of samples per run and an adjustable sequencing time. Here, we report on the development of a standardized laboratory workflow combined with a fully automated analysis pipeline LORCAN (long read consensus analysis), which together provide a sample-to-report solution for amplicon sequencing and taxonomic identification of the resulting consensus sequences. Validation of the approach was conducted on a panel of reference strains and on clinical samples consisting of single or mixed rRNA amplicons associated with various bacterial genera by direct comparison to the corresponding Sanger sequences. Additionally, simulated read and amplicon mixtures were used to assess LORCAN's behavior when dealing with samples with known cross-contamination levels. We demonstrate that by combining ONT amplicon sequencing results with LORCAN, the accuracy of Sanger sequencing can be closely matched (>99.6% sequence identity) and that mixed samples can be resolved at the single-base resolution level. The presented approach has the potential to significantly improve the flexibility, reliability, and availability of amplicon sequencing in diagnostic settings.

NGS-Based S. aureus Typing and Outbreak Analysis in Clinical Microbiology Laboratories: Lessons Learned From a Swiss-Wide Proficiency Test.

Whole genome sequencing (WGS) enables high resolution typing of bacteria up to the single nucleotide polymorphism (SNP) level. WGS is used in clinical microbiology laboratories for infection control, molecular surveillance and outbreak analyses. Given the large palette of WGS reagents and bioinformatics tools, the Swiss clinical bacteriology community decided to conduct a ring trial (RT) to foster harmonization of NGS-based bacterial typing. The RT aimed at assessing methicillin-susceptible Staphylococcus aureus strain relatedness from WGS and epidemiological data. The RT was designed to disentangle the variability arising from differences in sample preparation, SNP calling and phylogenetic methods. Nine laboratories participated. The resulting phylogenetic tree and cluster identification were highly reproducible across the laboratories. Cluster interpretation was, however, more laboratory dependent, suggesting that an increased sharing of expertise across laboratories would contribute to further harmonization of practices. More detailed bioinformatic analyses unveiled that while similar clusters were found across laboratories, these were actually based on different sets of SNPs, differentially retained after sample preparation and SNP calling procedures. Despite this, the observed number of SNP differences between pairs of strains, an important criterion to determine strain relatedness given epidemiological information, was similar across pipelines for closely related strains when restricting SNP calls to a common core genome defined by S. aureus cgMLST schema. The lessons learned from this pilot study will serve the implementation of larger-scale RT, as a mean to have regular external quality assessments for laboratories performing WGS analyses in a clinical setting.

Viral Metagenomics in the Clinical Realm: Lessons Learned from a Swiss-Wide Ring Trial.

Shotgun metagenomics using next generation sequencing (NGS) is a promising technique to analyze both DNA and RNA microbial material from patient samples. Mostly used in a research setting, it is now increasingly being used in the clinical realm as well, notably to support diagnosis of viral infections, thereby calling for quality control and the implementation of ring trials (RT) to benchmark pipelines and ensure comparable results. The Swiss NGS clinical virology community therefore decided to conduct a RT in 2018, in order to benchmark current metagenomic workflows used at Swiss clinical virology laboratories, and thereby contribute to the definition of common best practices. The RT consisted of two parts (increments), in order to disentangle the variability arising from the experimental compared to the bioinformatics parts of the laboratory pipeline. In addition, the RT was also designed to assess the impact of databases compared to bioinformatics algorithms on the final results, by asking participants to perform the bioinformatics analysis with a common database, in addition to using their own in-house database. Five laboratories participated in the RT (seven pipelines were tested). We observed that the algorithms had a stronger impact on the overall performance than the choice of the reference database. Our results also suggest that differences in sample preparation can lead to significant differences in the performance, and that laboratories should aim for at least 5-10 Mio reads per sample and use depth of coverage in addition to other interpretation metrics such as the percent of coverage. Performance was generally lower when increasing the number of viruses per sample. The lessons learned from this pilot study will be useful for the development of larger-scale RTs to serve as regular quality control tests for laboratories performing NGS analyses of viruses in a clinical setting.

Evolution in action: habitat transition from sediment to the pelagial leads to genome streamlining in Methylophilaceae.

The most abundant aquatic microbes are small in cell and genome size. Genome-streamlining theory predicts gene loss caused by evolutionary selection driven by environmental factors, favouring superior competitors for limiting resources. However, evolutionary histories of such abundant, genome-streamlined microbes remain largely unknown. Here we reconstruct the series of steps in the evolution of some of the most abundant genome-streamlined microbes in freshwaters ("Ca. Methylopumilus") and oceans (marine lineage OM43). A broad genomic spectrum is visible in the family Methylophilaceae (Betaproteobacteria), from sediment microbes with medium-sized genomes (2-3 Mbp genome size), an occasionally blooming pelagic intermediate (1.7 Mbp), and the most reduced pelagic forms (1.3 Mbp). We show that a habitat transition from freshwater sediment to the relatively oligotrophic pelagial was accompanied by progressive gene loss and adaptive gains. Gene loss has mainly affected functions not necessarily required or advantageous in the pelagial or is encoded by redundant pathways. Likewise, we identified genes providing adaptations to oligotrophic conditions that have been transmitted horizontally from pelagic freshwater microbes. Remarkably, the secondary transition from the pelagial of lakes to the oceans required only slight modifications, i.e., adaptations to higher salinity, gained via horizontal gene transfer from indigenous microbes. Our study provides first genomic evidence of genome reduction taking place during habitat transitions. In this regard, the family Methylophilaceae is an exceptional model for tracing the evolutionary history of genome streamlining as such a collection of evolutionarily related microbes from different habitats is rare in the microbial world.

The transcriptome response of the ruminal methanogen Methanobrevibacter ruminantium strain M1 to the inhibitor lauric acid.

OBJECTIVE: Lauric acid (C12) is a medium-chain fatty acid that inhibits growth and production of the greenhouse gas methane by rumen methanogens such as Methanobrevibacter ruminantium. To understand the inhibitory mechanism of C12, a transcriptome analysis was performed in M. ruminantium strain M1 (DSM 1093) using RNA-Seq. RESULTS: Pure cell cultures in the exponential growth phase were treated with 0.4 mg/ml C12, dissolved in dimethyl sulfoxide (DMSO), for 1 h and transcriptomic changes were compared to DMSO-only treated cells (final DMSO concentration 0.2%). Exposure to C12 resulted in differential expression of 163 of the 2280 genes in the M1 genome (maximum log2-fold change 6.6). Remarkably, C12 hardly affected the expression of genes involved in methanogenesis. Instead, most affected genes encode cell-surface associated proteins (adhesion-like proteins, membrane-associated transporters and hydrogenases), and proteins involved in detoxification or DNA-repair processes. Enrichment analysis on the genes regulated in the C12-treated group showed a significant enrichment for categories 'cell surface' and 'mobile elements' (activated by C12), and for the categories 'regulation' and 'protein fate' (represssed). These results are useful to generate and test specific hypotheses on the mechanism how C12 affects rumen methanogens.

Microdiversification in genome-streamlined ubiquitous freshwater Actinobacteria.

Actinobacteria of the acI lineage are the most abundant microbes in freshwater systems, but there are so far no pure living cultures of these organisms, possibly because of metabolic dependencies on other microbes. This, in turn, has hampered an in-depth assessment of the genomic basis for their success in the environment. Here we present genomes from 16 axenic cultures of acI Actinobacteria. The isolates were not only of minute cell size, but also among the most streamlined free-living microbes, with extremely small genome sizes (1.2-1.4 Mbp) and low genomic GC content. Genome reduction in these bacteria might have led to auxotrophy for various vitamins, amino acids and reduced sulphur sources, thus creating dependencies to co-occurring organisms (the 'Black Queen' hypothesis). Genome analyses, moreover, revealed a surprising degree of inter- and intraspecific diversity in metabolic pathways, especially of carbohydrate transport and metabolism, and mainly encoded in genomic islands. The striking genotype microdiversification of acI Actinobacteria might explain their global success in highly dynamic freshwater environments with complex seasonal patterns of allochthonous and autochthonous carbon sources. We propose a new order within Actinobacteria ('Candidatus Nanopelagicales') with two new genera ('Candidatus Nanopelagicus' and 'Candidatus Planktophila') and nine new species.

Abrupt stop of deep water turnover with lake warming: Drastic consequences for algal primary producers.

After strong fertilization in the 20th century, many deep lakes in Central Europe are again nutrient poor due to long-lasting restoration (re-oligotrophication). In line with reduced phosphorus and nitrogen loadings, total organismic productivity decreased and lakes have now historically low nutrient and biomass concentrations. This caused speculations that restoration was overdone and intended fertilizations are needed to ensure ecological functionality. Here we show that recent re-oligotrophication processes indeed accelerated, however caused by lake warming. Rising air temperatures strengthen thermal stabilization of water columns which prevents thorough turnover (holomixis). Reduced mixis impedes down-welling of oxygen rich epilimnetic (surface) and up-welling of phosphorus and nitrogen rich hypolimnetic (deep) water. However, nutrient inputs are essential for algal spring blooms acting as boost for annual food web successions. We show that repeated lack (since 1977) and complete stop (since 2013) of holomixis caused drastic epilimnetic phosphorus depletions and an absence of phytoplankton spring blooms in Lake Zurich (Switzerland). By simulating holomixis in experiments, we could induce significant vernal algal blooms, confirming that there would be sufficient hypolimnetic phosphorus which presently accumulates due to reduced export. Thus, intended fertilizations are highly questionable, as hypolimnetic nutrients will become available during future natural or artificial turnovers.

Genetic and genomic analysis of hyperthelia in Brown Swiss cattle.

Supernumerary teats (SNT) are any abnormal teats found on a calf in addition to the usual and functional 4 teats. The presence of SNT has also been termed "hyperthelia" since the end of the 19th century. Supernumerary teats can act as an incubator for bacteria, infecting the whole udder, and can interfere with the positioning of the milking machine, and consequently, have economic relevance. Different types of SNT are observed at different positions on the udder. Caudal teats are in the rear, ramal teats are attached to another teat, and intercalary teats are found between 2 regular teats. Not all teats are equally developed; some are completely functional but most are rudimentary and not attached to any mammary gland tissue. Recently, different studies showed the poly/oligogenic character of these malformations in cattle as well as in other mammalian species. The objective of this study was to analyze the genetic architecture and incidence of hyperthelia in Swiss Brown Swiss cattle using both traditional genetic evaluation as well as imputed whole genome sequence variant information. First, phenotypes collected over the last 20 yr were used together with pedigree information for estimation of genetic variance. Second, breeding values of Brown Swiss bulls were estimated applying the BLUP algorithm. The BLUP-EBV were deregressed and used as phenotypes in genome-wide association studies. The gene LGR5 on chromosome 5 was identified as a candidate for the presence of SNT. Using alternative trait coding, genomic regions on chromosome 17 and 20 were also identified as being involved in the development of SNT with their own supernumerary mammary gland tissue. Implementing knowledge gained in this study as a routine application allows a more accurate evaluation of the trait and reduction of SNT prevalence in the Swiss Brown Swiss cattle population.

The ecology of pelagic freshwater methylotrophs assessed by a high-resolution monitoring and isolation campaign.

Methylotrophic planktonic bacteria fulfill a particular role in the carbon cycle of lakes via the turnover of single-carbon compounds. We studied two planktonic freshwater lineages (LD28 and PRD01a001B) affiliated with Methylophilaceae (Betaproteobacteria) in Lake Zurich, Switzerland, by a combination of molecular and cultivation-based approaches. Their spatio-temporal distribution was monitored at high resolution (n=992 samples) for 4 consecutive years. LD28 methylotrophs constituted up to 11 × 10(7) cells l(-1) with pronounced peaks in spring and autumn-winter, concomitant with blooms of primary producers. They were rare in the warm water layers during summer but abundant in the cold hypolimnion, hinting at psychrophilic growth. Members of the PRD01a001B lineage were generally less abundant but also had maxima in spring. More than 120 axenic strains from these so far uncultivated lineages were isolated from the pelagic zone by dilution to extinction. Phylogenetic analysis separated isolates into two distinct genotypes. Isolates grew slowly (µmax=0.4 d(-1)), were of conspicuously small size, and were indeed psychrophilic, with higher growth yield at low temperatures. Growth was enhanced upon addition of methanol and methylamine to sterile lake water. Genomic analyses of two strains confirmed a methylotrophic lifestyle with a reduced set of genes involved in C1 metabolism. The very small and streamlined genomes (1.36 and 1.75 Mb) shared several pathways with the marine OM43 lineage. As the closest described taxa (Methylotenera sp.) are only distantly related to either set of isolates, we propose a new genus with two species, that is, 'Candidatus Methylopumilus planktonicus' (LD28) and 'Candidatus Methylopumilus turicensis' (PRD01a001B).

Fluorescence in situ hybridization and sequential catalyzed reporter deposition (2C-FISH) for the flow cytometric sorting of freshwater ultramicrobacteria.

Flow cytometric sorting is a powerful tool to physically separate cells within mixed microbial communities. If combined with phylogenetic staining (fluorescence in situ hybridization, FISH) it allows to specifically sort defined genotypic microbial populations from complex natural samples. However, the targeted enrichment of freshwater ultramicrobacteria, such as members of the LD12 clade of Alphaproteobacteria (SAR11-IIIb), is still challenging. Current FISH protocols, even in combination with signal amplification by catalyzed reporter deposition (CARD), are not sufficiently sensitive for the distinction of these bacteria from background noise by flow cytometry, presumably due to their low ribosome content and small cell sizes. We, therefore, modified a CARD based flow sorting protocol with the aim of increasing its sensitivity to a level sufficient for ultramicrobacteria. This was achieved by a second signal amplification step mediated by horseradish peroxidase labeled antibodies targeted to the fluorophores that were previously deposited by CARD-FISH staining. The protocol was tested on samples from an oligo-mesotrophic lake. Ultramicrobacteria affiliated with LD12 Alphaproteobacteria could be successfully sorted to high purity by flow cytometry. The ratios of median fluorescence signal to background ranged around 20, and hybridization rates determined by flow cytometry were comparable to those obtained by fluorescence microscopy. Potential downstream applications of our modified cell staining approach range from the analysis of microdiversity within 16S rRNA-defined populations to that of functional properties, such as the taxon-specific incorporation rates of organic substrates.

Seasonal growth potential of rare lake water bacteria suggest their disproportional contribution to carbon fluxes.

We studied the seasonal growth potential of opportunistic bacterial populations in Lake Zurich (Switzerland) by a series of grazer-free dilution culture assays. Pronounced shifts in the composition of the bacterial assemblages were observed within one doubling of total cell numbers, from initially abundant Actinobacteria to other fast-growing microbial lineages. Small populations with growth potentials far above community average were detected throughout the year with striking seasonal differences in their respective taxonomic affiliations. Members of Cytophaga-Flavobacteria (CF) were disproportionally proliferating only during phytoplankton blooms in spring and summer, while Beta- and Gammaproteobacteria showed superior growth at all other occasions. Growth rates of Alphaproteobacteria and esp. Sphingomonadaceae were significantly correlated to water temperatures and were far above community average in summer. Within the genus Flavobacterium, two species-like populations showed a tendency for fast growth in most experiments, while four others were exclusively proliferating either during a spring or during a summer phytoplankton bloom. Their high growth potentials but low in situ abundances hint at a tight control by bacterivorous grazers and at a consequently accelerated carbon flux to higher trophic levels.

Direct-gap gain and optical absorption in germanium correlated to the density of photoexcited carriers, doping, and strain.

Direct-gap gain up to 850 cm(-1) at 0.74 eV is measured and modeled in optically pumped Ge-on-Si layers for photoexcited carrier densities of 2.0 × 10(20) cm(-3). The gain spectra are correlated to carrier density via plasma-frequency determinations from reflection spectra. Despite significant gain, optical amplification cannot take place, because the carriers also generate pump-induced absorption of ≈7000 cm(-1). Parallel studies of III-V direct-gap InGaAs layers validate our spectroscopy and modeling. Our self-consistent results contradict current explanations of lasing in Ge-on-Si cavities.

Porcine arthrogryposis multiplex congenita (AMC): new diagnostic test and narrowed candidate region.

In the Swiss Large White pig population a genetically caused arthrogryposis multiplex congenita (AMC) variant was identified. The disease is autosomal recessively inherited and is a fatal defect. Affected piglets are of normal size, but show malformed and permanently contracted joints in their legs. Often the spinal cord is curved and the lower jaw is shortened. Originally, AMC was mapped to a 5 Mb region on pig chromosome 5 (SSC5) between microsatellite markers SW152 and SW904. In order to detect unaffected carriers a diagnostic test using markers within the candidate region was developed. However, two independent recombination events occurred in a diseased and in a healthy piglet. Therefore, we selected 24 consecutive markers (3 microsatellites, 19 SNPs and 2 indels) in the candidate region, and determined the haplotypes in the two pedigrees with the recombinations. The parents and five offspring were investigated. In consequence, we were able to narrow down the candidate region and map AMC between SNPs ALGA0032767 and DRGA0006010 on SSC5 which span around 2.32 Mb. The candidate region shares homology to human chromosome 12. However, we are still lacking good candidate genes. A PCR-RFLP was developed and is used as an improved genetic test for AMC.

Characterisation of five candidate genes within the ETEC F4ab/ac candidate region in pigs.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) that express the F4ab and F4ac fimbriae is a major contributor to diarrhoea outbreaks in the pig breeding industry, infecting both newborn and weaned piglets. Some pigs are resistant to this infection, and susceptibility is inherited as a simple dominant Mendelian trait. Indentifying the genetics behind this trait will greatly benefit pig welfare as well as the pig breeding industry by providing an opportunity to select against genetically susceptible animals, thereby reducing the number of diarrhoea outbreaks. The trait has recently been mapped by haplotype sharing to a 2.5 Mb region on pig chromosome 13, a region containing 18 annotated genes. FINDINGS: The coding regions of five candidate genes for susceptibility to ETEC F4ab/ac infection (TFRC, ACK1, MUC20, MUC4 and KIAA0226), all located in the 2.5 Mb region, were investigated for the presence of possible causative mutations. A total of 34 polymorphisms were identified in either coding regions or their flanking introns. The genotyping data for two of those were found to perfectly match the genotypes at the ETEC F4ab/ac locus, a G to C polymorphism in intron 11 of TFRC and a C to T silent polymorphism in exon 22 of KIAA0226. Transcriptional profiles of the five genes were investigated in a porcine tissue panel including various intestinal tissues. All five genes were expressed in intestinal tissues at different levels but none of the genes were found differentially expressed between ETEC F4ab/ac resistant and ETEC F4ab/ac susceptible animals in any of the tested tissues. CONCLUSIONS: None of the identified polymorphisms are obvious causative mutations for ETEC F4ab/ac susceptibility, as they have no impact on the level of the overall mRNA expression nor predicted to influence the composition of the amino acids composition. However, we cannot exclude that the five tested genes are bona fide candidate genes for susceptibility to ETEC F4ab/ac infection since the identified polymorphism might affect the translational apparatus, alternative splice forms may exist and post translational mechanisms might contribute to disease susceptibility.