A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana.

Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes.

Wastewater-based epidemiology applied at the building-level reveals distinct virome profiles based on the age of the contributing individuals.

BACKGROUND: Human viruses released into the environment can be detected and characterized in wastewater. The study of wastewater virome offers a consolidated perspective on the circulation of viruses within a population. Because the occurrence and severity of viral infections can vary across a person's lifetime, studying the virome in wastewater samples contributed by various demographic segments can provide valuable insights into the prevalence of viral infections within these segments. In our study, targeted enrichment sequencing was employed to characterize the human virome in wastewater at a building-level scale. This was accomplished through passive sampling of wastewater in schools, university settings, and nursing homes in two cities in Catalonia. Additionally, sewage from a large urban wastewater treatment plant was analysed to serve as a reference for examining the collective excreted human virome. RESULTS: The virome obtained from influent wastewater treatment plant samples showcased the combined viral presence from individuals of varying ages, with astroviruses and human bocaviruses being the most prevalent, followed by human adenoviruses, polyomaviruses, and papillomaviruses. Significant variations in the viral profiles were observed among the different types of buildings studied. Mamastrovirus 1 was predominant in school samples, salivirus and human polyomaviruses JC and BK in the university settings while nursing homes showed a more balanced distribution of viral families presenting papillomavirus and picornaviruses and, interestingly, some viruses linked to immunosuppression. CONCLUSIONS: This study shows the utility of building-level wastewater-based epidemiology as an effective tool for monitoring the presence of viruses circulating within specific age groups. It provides valuable insights for public health monitoring and epidemiological studies.

Phylogenomics of Neogastropoda: The Backbone Hidden in the Bush.

The molluskan order Neogastropoda encompasses over 15,000 almost exclusively marine species playing important roles in benthic communities and in the economies of coastal countries. Neogastropoda underwent intensive cladogenesis in the early stages of diversification, generating a "bush" at the base of their evolutionary tree, which has been hard to resolve even with high throughput molecular data. In the present study to resolve the bush, we use a variety of phylogenetic inference methods and a comprehensive exon capture dataset of 1817 loci (79.6% data occupancy) comprising 112 taxa of 48 out of 60 Neogastropoda families. Our results show consistent topologies and high support in all analyses at (super)family level, supporting monophyly of Muricoidea, Mitroidea, Conoidea, and, with some reservations, Olivoidea and Buccinoidea. Volutoidea and Turbinelloidea as currently circumscribed are clearly paraphyletic. Despite our analyses consistently resolving most backbone nodes, 3 prove problematic: First, the uncertain placement of Cancellariidae, as the sister group to either a Ficoidea-Tonnoidea clade or to the rest of Neogastropoda, leaves monophyly of Neogastropoda unresolved. Second, relationships are contradictory at the base of the major "core Neogastropoda" grouping. Third, coalescence-based analyses reject monophyly of the Buccinoidea in relation to Vasidae. We analyzed phylogenetic signal of targeted loci in relation to potential biases, and we propose the most probable resolutions in the latter 2 recalcitrant nodes. The uncertain placement of Cancellariidae may be explained by orthology violations due to differential paralog loss shortly after the whole genome duplication, which should be resolved with a curated set of longer loci.

Phylogenomic analyses using a new 1013-gene Vitaceae bait-set support major groups of North American Vitis.

A set of newly designed Vitaceae baits targeting 1013 genes was employed to explore phylogenetic relationships among North American Vitis. Eurasian Vitis taxa including Vitis vinifera were found to be nested within North American Vitis subgenus Vitis. North American Vitis subgenus Vitis can be placed into nine main groups: the Monticola group, the Occidentales group, the Californica group, the Vinifera group (introduced from Eurasia), the Mustangensis group, the Palmata group, the Aestivalis group, the Labrusca group, and the Cinerea group. Strong cytonuclear discordances were detected in North American Vitis, with many species non-monophyletic in the plastid phylogeny, while monophyletic in the nuclear phylogeny. The phylogenomic analyses support recognizing four distinct species in the Vitis cinerea complex in North America: V. cinerea, V. baileyana, V. berlandieri, and V. simpsonii. Such treatment will better serve the conservation of wild Vitis diversity in North America. Yet the evolutionary history of Vitis is highly complex, with the concordance analyses indicating conflicting signals across the phylogeny. Cytonuclear discordances and Analyses using the Species Networks applying Quartets (SNaQ) method support extensive hybridizations in North American Vitis. The results further indicate that plastid genomes alone are insufficient for resolving the evolutionary history of plant groups that have undergone rampant hybridization, like the case in North American Vitis. Nuclear gene data are essential for species delimitation, identification and reconstructing evolutionary relationships; therefore, they are imperative for plant phylogenomic studies.

Evaluation of Dried Blood and Cerebrospinal Fluid Filter Paper Spots for Storing and Transporting Clinical Material for the Molecular Diagnosis of Invasive Meningococcal Disease.

To improve the storage and transport of clinical specimens for the diagnosis of Neisseria meningitidis (Nm) infections in resource-limited settings, we have evaluated the performance of dried blood spot (DBS) and dried cerebrospinal fluid spot (DCS) assays. DBS and DCS were prepared on filter paper from liquid specimens previously tested for Nm in the United Kingdom. Nm was detected and genogrouped by real-time PCR performed on crude genomic DNA extracted from the DBS (n = 226) and DCS (n = 226) specimens. Targeted whole-genome sequencing was performed on a subset of specimens, DBS (n = 4) and DCS (n = 6). The overall agreement between the analysis of liquid and dried specimens was (94.2%; 95% CI 90.8−96.7) for blood and (96.4%; 95% CI 93.5−98.0) for cerebrospinal fluid. Relative to liquid specimens as the reference, the DBS and DCS assays had sensitivities of (89.1%; 95% CI 82.7−93.8) and (94.2%; 95% CI 88.9−97.5), respectively, and both assays had specificities above 98%. A genogroup was identified by dried specimen analysis for 81.9% of the confirmed meningococcal infections. Near full-length Nm genome sequences (>86%) were obtained for all ten specimens tested which allowed determination of the sequence type, clonal complex, presence of antimicrobial resistance and other meningococcal genotyping. Dried blood and CSF filter spot assays offer a practical alternative to liquid specimens for the molecular and genomic characterisation of invasive meningococcal diseases in low-resource settings.

Targeted enrichment of novel chloroplast-based probes reveals a large-scale phylogeny of 412 bamboos.

BACKGROUND: The subfamily Bambusoideae belongs to the grass family Poaceae and has significant roles in culture, economy, and ecology. However, the phylogenetic relationships based on large-scale chloroplast genomes (CpGenomes) were elusive. Moreover, most of the chloroplast DNA sequencing methods cannot meet the requirements of large-scale CpGenome sequencing, which greatly limits and impedes the in-depth research of plant genetics and evolution. RESULTS: To develop a set of bamboo probes, we used 99 high-quality CpGenomes with 6 bamboo CpGenomes as representative species for the probe design, and assembled 15 M unique sequences as the final pan-chloroplast genome. A total of 180,519 probes for chloroplast DNA fragments were designed and synthesized by a novel hybridization-based targeted enrichment approach. Another 468 CpGenomes were selected as test data to verify the quality of the newly synthesized probes and the efficiency of the probes for chloroplast capture. We then successfully applied the probes to synthesize, enrich, and assemble 358 non-redundant CpGenomes of woody bamboo in China. Evaluation analysis showed the probes may be applicable to chloroplasts in Magnoliales, Pinales, Poales et al. Moreover, we reconstructed a phylogenetic tree of 412 bamboos (358 in-house and 54 published), supporting a non-monophyletic lineage of the genus Phyllostachys. Additionally, we shared our data by uploading a dataset of bamboo CpGenome into CNGB ( https://db.cngb.org/search/project/CNP0000502/ ) to enrich resources and promote the development of bamboo phylogenetics. CONCLUSIONS: The development of the CpGenome enrichment pipeline and its performance on bamboos recommended an inexpensive, high-throughput, time-saving and efficient CpGenome sequencing strategy, which can be applied to facilitate the phylogenetics analysis of most green plants.

An updated infra-familial classification of Sapindaceae based on targeted enrichment data.

PREMISE: The economically important, cosmopolitan soapberry family (Sapindaceae) comprises ca. 1900 species in 144 genera. Since the seminal work of Radlkofer, several authors have attempted to overcome challenges presented by the family's complex infra-familial classification. With the advent of molecular systematics, revisions of the various proposed groupings have provided significant momentum, but we still lack a formal classification system rooted in an evolutionary framework. METHODS: Nuclear DNA sequence data were generated for 123 genera (86%) of Sapindaceae using target sequence capture with the Angiosperms353 universal probe set. HybPiper was used to produce aligned DNA matrices. Phylogenetic inferences were obtained using coalescence-based and concatenated methods. The clades recovered are discussed in light of both benchmark studies to identify synapomorphies and distributional evidence to underpin an updated infra-familial classification. KEY RESULTS: Coalescence-based and concatenated phylogenetic trees had identical topologies and node support, except for the placement of Melicoccus bijugatus Jacq. Twenty-one clades were recovered, which serve as the basis for a revised infra-familial classification. CONCLUSIONS: Twenty tribes are recognized in four subfamilies: two tribes in Hippocastanoideae, two in Dodonaeoideae, and 16 in Sapindoideae (no tribes are recognized in the monotypic subfamily Xanthoceratoideae). Within Sapindoideae, six new tribes are described: Blomieae Buerki & Callm.; Guindilieae Buerki, Callm. & Acev.-Rodr.; Haplocoeleae Buerki & Callm.; Stadmanieae Buerki & Callm.; Tristiropsideae Buerki & Callm.; and Ungnadieae Buerki & Callm. This updated classification provides a backbone for further research and conservation efforts on this family.

Recent advances in selective photothermal therapy of tumor.

Photothermal therapy (PTT), which converts light energy to heat energy, has become a new research hotspot in cancer treatment. Although researchers have investigated various ways to improve the efficiency of tumor heat ablation to treat cancer, PTT may cause severe damage to normal tissue due to the systemic distribution of photothermal agents (PTAs) in the body and inaccurate laser exposure during treatment. To further improve the survival rate of cancer patients and reduce possible side effects on other parts of the body, it is still necessary to explore PTAs with high selectivity and precise treatment. In this review, we summarized strategies to improve the treatment selectivity of PTT, such as increasing the accumulation of PTAs at tumor sites and endowing PTAs with a self-regulating photothermal conversion function. The views and challenges of selective PTT were discussed, especially the prospects and challenges of their clinical applications.

Xdrop: Targeted sequencing of long DNA molecules from low input samples using droplet sorting.

Long-read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore are ideal for genome-gap closure, solving structural rearrangements and sequencing through repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic-based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of DNA. Xdrop is based on the isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus 18 integrated into the genome of human HeLa cells. Analysis of the sequencing reads resolved three HPV18-chr8 integrations at base-pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. Further, we enriched the complete TP53 locus in a leukemia cell line and could successfully phase coexisting mutations using PacBio sequencing. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex genomic regions.

Full Molecular Typing of Neisseria meningitidis Directly from Clinical Specimens for Outbreak Investigation.

Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis worldwide and an occasional cause of meningococcal urethritis. When isolates are unavailable for surveillance or outbreak investigations, molecular characterization of pathogens needs to be performed directly from clinical specimens, such as cerebrospinal fluid (CSF), blood, or urine. However, genome sequencing of specimens is challenging because of low bacterial and high human DNA abundances. We developed selective whole-genome amplification (SWGA), an isothermal multiple-displacement amplification-based method, to efficiently enrich, sequence, and de novo assemble N. meningitidis DNA from clinical specimens with low bacterial loads. SWGA was validated with 12 CSF specimens from invasive meningococcal disease cases and 12 urine specimens from meningococcal urethritis cases. SWGA increased the mean proportion of N. meningitidis reads by 2 to 3 orders of magnitude, enabling identification of at least 90% of the 1,605 N. meningitidis core genome loci for 50% of the specimens. The validated method was used to investigate two meningitis outbreaks recently reported in Togo and Burkina Faso. Twenty-seven specimens with low bacterial loads were processed by SWGA before sequencing, and 12 of 27 were successfully assembled to obtain the full molecular typing and vaccine antigen profile of the N. meningitidis pathogen, thus enabling thorough characterization of outbreaks. This method is particularly important for enhancing molecular surveillance in regions with low culture rates. SWGA produces enough reads for phylogenetic and allelic analysis at a low cost. More importantly, the procedure can be extended to enrich other important human bacterial pathogens.

An enhanced target-enrichment bait set for Hexacorallia provides phylogenomic resolution of the staghorn corals (Acroporidae) and close relatives.

Targeted enrichment of genomic DNA can profoundly increase the phylogenetic resolution of clades and inform taxonomy. Here, we redesign a custom bait set previously developed for the cnidarian class Anthozoa to more efficiently target and capture ultraconserved elements (UCEs) and exonic loci within the subclass Hexacorallia. We test this enhanced bait set (targeting 2476 loci) on 99 specimens of scleractinian corals spanning both the "complex" (Acroporidae, Agariciidae) and "robust" (Fungiidae) clades. Focused sampling in the staghorn corals (genus Acropora) highlights the ability of sequence capture to inform the taxonomy of a clade previously deficient in molecular resolution. A mean of 1850 (±298) loci were captured per taxon (955 UCEs, 894 exons), and a 75% complete concatenated alignment of 96 samples included 1792 loci (991 UCE, 801 exons) and ~1.87 million base pairs. Maximum likelihood and Bayesian analyses recovered robust molecular relationships and revealed that species-level relationships within the Acropora are incongruent with traditional morphological groupings. Both UCE and exon datasets delineated six well-supported clades within Acropora. The enhanced bait set will facilitate investigations of the evolutionary history of many important groups of reef corals, particularly where previous molecular marker development has been unsuccessful.

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing.

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with single molecule, real-time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods.

Evolutionary dynamism in bryophytes: Phylogenomic inferences confirm rapid radiation in the moss family Funariaceae.

Rapid diversifications of plants are primarily documented and studied in angiosperms, which are perceived as evolutionarily dynamic. Recent studies have, however, revealed that bryophytes have also undergone periods of rapid radiation. The speciose family Funariaceae, including the model taxon Physcomitrella patens, is one such lineage. Here, we infer relationships among major lineages within the Entosthodon-Physcomitrium complex from virtually complete organellar exomes (i.e., 123 genes) obtained through high throughput sequencing of genomic libraries enriched in these loci via targeted locus capture. Based on these extensive exonic data we (1) reconstructed a robust backbone topology of the Funariaceae, (2) confirmed the monophyly of Funaria and the polyphyly of Entosthodon, Physcomitrella, and Physcomitrium, and (3) argue for the occurrence of a rapid radiation within the Entosthodon-Physcomitrium complex that began 28 mya and gave rise more than half of the species diversity of the family. This diversification may have been triggered by a whole genome duplication and coincides with global Eocene cooling that continued through the Oligocene and Miocene. The Funariaceae join a growing list of bryophyte lineages whose history is marked by at least one burst of diversification, and our study thereby strengthens the view that bryophytes are evolutionarily dynamic lineages and that patterns and processes characterizing the evolution of angiosperms may be universal among land plants.

Targeted Enrichment for Pathogen Detection and Characterization in Three Felid Species.

Traditional diagnostic assays often lack sensitivity and can be difficult to multiplex across many pathogens. Next-generation sequencing (NGS) can overcome some of these problems but has limited application in the detection of low-copy-number pathogens in complex samples. Targeted genome capture (TGC) utilizes oligonucleotide probes to enrich specific nucleic acids in heterogeneous extracts and can therefore increase the proportion of NGS reads for low-abundance targets. While earlier studies have demonstrated the utility of this technology for detection of novel pathogens in human clinical samples, the capacity and practicality of TGC-NGS in a veterinary diagnostic setting have not yet been evaluated. Here we report the use of TGC-NGS assays for the detection and characterization of diverse feline pathogen taxa. We detected 31 pathogens comprising nine pathogen taxa in 28 felid samples analyzed. This included 20 pathogens detected via traditional PCR and 11 additional pathogens that had not been previously detected in the same samples. Most of the pathogens detected were sequenced at sufficient breadth and depth to confidently classify them at the species or subspecies level. Target nucleic acids were enriched from a low of 58-fold to 56 million-fold relative to host nucleic acids. Despite the promising performance of these assays, a number of pathogens detected by conventional PCR or serology were not isolated by TGC-NGS, suggesting that further validation is required before this technology can be used in lieu of quality-controlled standard assays. We conclude that TGC-NGS offers great potential as a broad multiplex pathogen characterization assay in veterinary diagnostic and research settings.

The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity.

BACKGROUND: The ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. While emerging sequencing platforms are capable of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kbp. The DNA enrichment methodology described herein, Region-Specific Extraction (RSE), produces DNA segments in excess of 20 kbp in length. Coupling this enrichment method to appropriate sequencing platforms will significantly enhance the ability to generate complete and accurate sequence characterization of any genomic region without the need for reference-based assembly. RESULTS: RSE is a long-range DNA target capture methodology that relies on the specific hybridization of short (20-25 base) oligonucleotide primers to selected sequence motifs within the DNA target region. These capture primers are then enzymatically extended on the 3'-end, incorporating biotinylated nucleotides into the DNA. Streptavidin-coated beads are subsequently used to pull-down the original, long DNA template molecules via the newly synthesized, biotinylated DNA that is bound to them. We demonstrate the accuracy, simplicity and utility of the RSE method by capturing and sequencing a 4 Mbp stretch of the major histocompatibility complex (MHC). Our results show an average depth of coverage of 164X for the entire MHC. This depth of coverage contributes significantly to a 99.94 % total coverage of the targeted region and to an accuracy that is over 99.99 %. CONCLUSIONS: RSE represents a cost-effective target enrichment method capable of producing sequencing templates in excess of 20 kbp in length. The utility of our method has been proven to generate superior coverage across the MHC as compared to other commercially available methodologies, with the added advantage of producing longer sequencing templates amenable to DNA sequencing on recently developed platforms. Although our demonstration of the method does not utilize these DNA sequencing platforms directly, our results indicate that the capture of long DNA fragments produce superior coverage of the targeted region.

Single-locus enrichment without amplification for sequencing and direct detection of epigenetic modifications.

A gene-level targeted enrichment method for direct detection of epigenetic modifications is described. The approach is demonstrated on the CGG-repeat region of the FMR1 gene, for which large repeat expansions, hitherto refractory to sequencing, are known to cause fragile X syndrome. In addition to achieving a single-locus enrichment of nearly 700,000-fold, the elimination of all amplification steps removes PCR-induced bias in the repeat count and preserves the native epigenetic modifications of the DNA. In conjunction with the single-molecule real-time sequencing approach, this enrichment method enables direct readout of the methylation status and the CGG repeat number of the FMR1 allele(s) for a clonally derived cell line. The current method avoids potential biases introduced through chemical modification and/or amplification methods for indirect detection of CpG methylation events.

Ancient whole genome enrichment using baits built from modern DNA.

We report metrics from complete genome capture of nuclear DNA from extinct mammoths using biotinylated RNAs transcribed from an Asian elephant DNA extract. Enrichment of the nuclear genome ranged from 1.06- to 18.65-fold, to an apparent maximum threshold of ∼80% on-target. This projects an order of magnitude less costly complete genome sequencing from long-dead organisms, even when a reference genome is unavailable for bait design.

Development of targeted whole genome sequencing approaches for Crimean-Congo haemorrhagic fever virus (CCHFV).

Crimean-Congo haemorrhagic fever (CCHF) is the most prevalent human tick-borne viral disease, with a reported case fatality rate of 30 % or higher. The virus contains a tri-segmented, negative-sense RNA genome consisting of the small (S), medium (M) and large (L) segments encoding respectively the nucleoprotein (NP), the glycoproteins precursor (GPC) and the viral RNA-dependent RNA polymerase (RDRP). CCHFV is one of the most genetically diverse arboviruses, with seven distinct lineages named after the region they were first reported in and based on S segment phylogenetic analysis. Due to the high genetic divergence of the virus, a single targeted tiling PCR strategy to enrich for viral nucleic acids prior to sequencing is difficult to develop, and previously we have developed and validated a tiling PCR enrichment method for the Europe 1 genetic lineage. We have developed a targeted, probe hybridisation capture method and validated its performance on clinical as well as cell-cultured material of CCHFV from different genetic lineages, including Europe 1, Europe 2, Africa 2 and Africa 3. The method produced over 95 % reference coverages with at least 10x sequencing depth. While we were only able to recover a single complete genome sequence from the tested Europe 1 clinical samples with the capture hybridisation protocol, the data provides evidence of its applicability to different CCHFV genetic lineages. CCHFV is an important tick-borne human pathogen with wide geographical distribution. Environmental as well as anthropogenic factors are causing increased CCHFV transmission. Development of strategies to recover CCHFV sequences from genetically diverse lineages of the virus is of paramount importance to monitor the presence of the virus in new areas, and in public health responses for CCHFV molecular surveillance to rapidly detect, diagnose and characterise currently circulating strains.

Recovery of complete genome sequences of Crimean-Congo haemorrhagic fever virus (CCHFV) directly from clinical samples: A comparative study between targeted enrichment and metagenomic approaches.

Crimean-Congo haemorrhagic fever (CCHF) is the most prevalent human tick-borne viral disease, endemic to the Balkans, Africa, Middle East and Asia. There are currently no licensed vaccines or effective antivirals against CCHF. CCHF virus (CCHFV) has a negative sense segmented tripartite RNA genome consisting of the small (S), medium (M) and large (L) segments. Depending on the segment utilised for genetic affiliation, there are up to 7 circulating lineages of CCHFV. The current lack of geographical representation of CCHFV sequences in various repositories highlights a requirement for increased CCHFV sequencing capabilities in endemic regions. We have optimised and established a multiplex PCR tiling methodology for the targeted enrichment of complete genomes of Europe 1 CCHFV lineage directly from clinical samples and compared its performance to a non-targeted enrichment approach on both short-read and long-read sequencing platforms. We have found a statistically significant increase in mapped viral sequencing reads produced with our targeted enrichment approach. This has allowed us to recover near complete S segment sequences and above 90% of the M and L segment sequences for samples with Ct values as high as 31.3. This study demonstrates the superiority of a targeted enrichment approach for recovery of CCHFV genomic sequences from samples with low virus titre. CCHFV is an important vector-borne human pathogen with wide geographical distribution. The validated methodology reported here adds value to front-line public health laboratories employing genomic sequencing for CCHFV Europe 1 lineage surveillance, particularly in the Balkan and Middle Eastern territories currently monitoring the spread of the pathogen. Tracking the genomic evolution of the virus across regions improves risk assessment and directly informs the development of diagnostics, therapeutics, and vaccines.

Evaluation of a custom designed hybridisation assay for whole genome sequencing of human adenoviruses direct from clinical samples.

BACKGROUND: Human Adenoviruses are a common cause of disease and can cause significant morbidity and mortality in immunocompromised patients. Nosocomial transmission events can occur with whole genome sequencing playing a crucial role. This study evaluates the performance of a custom designed SureSelectXT target enrichment assay based on 14 adenovirus genomes for sequencing direct from clinical samples. METHODS: Modifications were made to the SureSelectXT low input protocol to enhance performance for viral targets. Consensus sequences were generated using an in-house designed three stage bioinformatics pipeline. We assessed, percentage of on target reads, average depth of coverage and percentage genome coverage to determine assay performance across a range of sample matrices. RESULTS: Whole genome sequences were successfully generated for 91.6 % of samples assessed. Adenovirus DNA concentration was a good indicator of enrichment success. Highly specific enrichment was observed with only 6 % of samples showing < 50 % on target reads. Respiratory and faecal samples performed well where bloods showed higher levels of non-specific enrichment likely confounded by low adenovirus DNA concentrations. Protocol performance did not appear impacted by Adenovirus type or species. CONCLUSION: Overall performance of this modified SureSelectXT protocol appears in line with previously published works although there are some confounding factors requiring further investigation. The use of a small RNA bait set has the potential to reduce associated costs which can be prohibitive.