Unraveling the Properties of Phage Display Fab Libraries and Their Use in the Selection of Gliadin-Specific Probes by Applying High-Throughput Nanopore Sequencing.

Directed evolution is a pivotal strategy for new antibody discovery, which allowed the generation of high-affinity Fabs against gliadin from two antibody libraries in our previous studies. One of the libraries was exclusively derived from celiac patients' mRNA (immune library) while the other was obtained through a protein engineering approach (semi-immune library). Recent advances in high-throughput DNA sequencing techniques are revolutionizing research across genomics, epigenomics, and transcriptomics. In the present work, an Oxford Nanopore in-lab sequencing device was used to comprehensively characterize the composition of the constructed libraries, both at the beginning and throughout the phage-mediated selection processes against gliadin. A customized analysis pipeline was used to select high-quality reads, annotate chain distribution, perform sequence analysis, and conduct statistical comparisons between the different selection rounds. Some immunological attributes of the most representative phage variants after the selection process were also determined. Sequencing results revealed the successful transfer of the celiac immune response features to the immune library and the antibodies derived from it, suggesting the crucial role of these features in guiding the selection of high-affinity recombinant Fabs against gliadin. In summary, high-throughput DNA sequencing has improved our understanding of the selection processes aimed at generating molecular binders against gliadin.

Multiplex Functional Characterization of Protein Variant Libraries in Mammalian Cells with Single-Copy Genomic Integration and High-Throughput DNA Sequencing.

Sequencing-based, massively parallel genetic assays have enabled simultaneous characterization of the genotype-phenotype relationships for libraries encoding thousands of unique protein variants. Since plasmid transfection and lentiviral transduction have characteristics that limit multiplexing with pooled libraries, we developed a mammalian synthetic biology platform that harnesses the Bxb1 bacteriophage DNA recombinase to insert single promoterless plasmids encoding a transgene of interest into a pre-engineered "landing pad" site within the cell genome. The transgene is expressed behind a genomically integrated promoter, ensuring only one transgene is expressed per cell, preserving a strict genotype-phenotype link. Upon selecting cells based on a desired phenotype, the transgene can be sequenced to ascribe each variant a phenotypic score. We describe how to create and utilize landing pad cells for large-scale, library-based genetic experiments. Using the provided examples, the experimental template can be adapted to explore protein variants in diverse biological problems within mammalian cells.

Building the taxonomic profile of the Riniaie Marwah hot spring of Kishtwar in Jammu and Kashmir: the first high-throughput sequencing-based metagenome study.

Background and Objectives: Rinaie Marwah hot spring Kishtwar (RMHSK) is one of the geothermal springs located at 33°51'51″N 75°32'07″E with an elevation of 2134 meters above sea level in Jammu and Kashmir, India. We aimed to study the microbial diversity of this geothermal spring using metagenomics. Materials and Methods: In the present study, physiochemical parameters including temperature (65-75°C), pH (6. 9-8. 8), hardness (250 ppm), and mineral content was measured along with the microbial diversity using Illumina MiSeq metagenome-based 16s amplicon sequencing (V3-V4). The sequence reads were classified taxonomically into 31 phyla, 71 classes, 152 orders, 256 families, 410 genus, and 665 species. QIIME 2 (Quantitative Insights into Microbial Ecology), an extensible, powerful, and decentralized analytical tool, was used for taxonomic analysis. Results: Bacteroidota (32. 57%) was the dominant phylum, Bacteroidia (32. 51%) the dominant class, Bacteroidales (16. 6%) the dominant order, and Lentimicrobiaceae (14. 23%) was the dominant family per the abundance analysis. Shannon (2. 28) and Chao 1 (87. 0) diversity indices support the existence of higher microbial diversity in RMHSK (50717 OTUs). Conclusion: The microbial diversity of RMHSK is reported for the first time through a metagenomic study. Identification of microorganisms with characteristics that are relevant to industries.

Corynebacterium bovis infection after autologous fat grafting in breast augmentation: a case report.

In this report, we present a case study of a rare human bacterium, Corynebacterium bovis, which caused an infection in a patient who had undergone autologous fat-based breast augmentation using cryopreserved fat. This infection occurred during a secondary fat grafting procedure. To identify the bacteria causing the infection, we used high-throughput DNA sequencing technology since this bacterium is seldomly reported in human infections. The patient was successfully treated with intravenous imipenem. We also discuss potential factors that may have contributed to this unusual bacterial infection and propose that DNA sequencing can be a useful tool in cases where standard culture techniques fail to identify the causative agent. Additionally, we highlight the importance of further research on the cryopreservation of fat. In summary, this case highlights the possibility of rare bacterial infections occurring after fat grafting procedures and emphasizes the importance of identifying the causative agent through advanced techniques such as DNA sequencing. Further research is needed to improve our understanding of the risks associated with cryopreservation of fat and to identify ways to prevent these types of infections in the future.

DNA metabarcoding assessment of Neotropical ichthyoplankton communities is marker-dependent.

The study of ichthyoplankton is paramount to understanding fish assemblages' reproductive dynamics. DNA metabarcoding has been applied as a rapid, cost-effective, and accurate taxonomy tool, allowing the identification of multiple individuals simultaneously. However, there remain significant challenges when using DNA metabarcoding, such as molecular marker choice according to the taxonomic resolution and length of the fragment to be sequenced, primer bias, incomplete reference databases, and qualitative inference incongruences. Here, 30 ichthyoplankton pools collected from a Neotropical river were identified at a molecular level using DNA metabarcoding to compare the resolution, sensibility, specificity, and relative read abundance (RRA) recovery of three molecular markers: the standard COI fragment (650 pb, with each end analyzed individually) and two short 12S rRNA genes markers (≅200 bp - NeoFish and MiFish markers). The combined use of the three markers increased the genera detection rates by 25%-87.5%, allowing an increased taxonomic coverage and robust taxonomic identification of complex Neotropical ichthyoplankton communities. RRA is marker-dependent, indicating caution is still needed while inferring species abundance based on DNA metabarcoding data when using PCR-dependent protocols.

Genetic Variants Identified by Whole Exome Sequencing in a Large Italian Family with High Plasma Levels of Factor VIII and Von Willebrand Factor.

High plasma levels of factor VIII (FVIII) and von Willebrand factor (VWF) have been indicated as independent risk factors for venous thromboembolism. However, the genetic factors responsible for their increase remain poorly known. In a large Italian family with high FVIII/VWF levels and thrombotic episodes, whole exome sequencing (WES) was performed on 12 family members to identify variants/genes involved in FVIII/VWF increase. Twenty variants spread over a 8300 Kb region on chromosome 5 were identified in 12 genes, including the low frequency rs13158382, located upstream of the MIR143/145 genes, which might affect miR-143/145 transcription or processing. The expression of miR-143/145 and VWF mRNA were evaluated in the peripheral blood mononuclear cells of six family members. Members with the variant (n = 3) showed lower levels of both miRNAs and higher levels of VWF mRNA compared to members without the variant (n = 3). An analysis of genetic and expression data from a larger cohort of individuals from the 1000 Genomes and GEUVADIS project confirmed a statistically significant reduction (p-value = 0.023) in miR-143 in heterozygous (n = 35) compared to homozygous wild-type individuals (n = 386). This family-based study identified a new genetic variant potentially involved in VWF increase by affecting miR-143/145 expression.

Analysis of the genetic contribution to thoracic aortic aneurysm or dissection in a prospective cohort of patients with familial and sporadic cases in East China.

BACKGROUND: Thoracic aortic aneurysm or dissections (TAADs) represent a group of life-threatening diseases. Genetic aetiology can affect the age of onset, clinical phenotype, and timing of intervention. We conducted a prospective trial to determine the prevalence of pathogenic variants in TAAD patients and to elucidate the traits related to harbouring the pathogenic variants. One hundred and one unrelated TAAD patients underwent genetic sequencing and analysis for 23 TAAD-associated genes using a targeted PCR and next-generation sequencing-based panel. RESULTS: A total of 47 variants were identified in 52 TAAD patients (51.5%), including 5 pathogenic, 1 likely pathogenic and 41 variants of uncertain significance. The pathogenic or likely pathogenic (P/LP) variants in 4 disease-causing genes were carried by 1 patient with familial and 5 patients with sporadic TAAD (5.9%). In addition to harbouring one variant causing familial TAAD, the FBN1 gene harboured half of the P/LP variants causing sporadic TAAD. Individuals with an age of onset less than 50 years or normotension had a significantly increased genetic risk. CONCLUSIONS: TAAD patients with a younger age at diagnosis or normotension were more likely to carry a P/LP variant; thus, routine genetic testing will be beneficial to a better prognosis through genetically personalized care prior to acute rupture or dissection.

Pharmacovariome scanning using whole pharmacogene resequencing coupled with deep computational analysis and machine learning for clinical pharmacogenomics.

BACKGROUND: This pilot study aims to identify and functionally assess pharmacovariants in whole exome sequencing data. While detection of known variants has benefited from pharmacogenomic-dedicated bioinformatics tools before, in this paper we have tested novel deep computational analysis in addition to artificial intelligence as possible approaches for functional analysis of unknown markers within less studied drug-related genes. METHODS: Pharmacovariants from 1800 drug-related genes from 100 WES data files underwent (a) deep computational analysis by eight bioinformatic algorithms (overall containing 23 tools) and (b) random forest (RF) classifier as the machine learning (ML) approach separately. ML model efficiency was calculated by internal and external cross-validation during recursive feature elimination. Protein modelling was also performed for predicted highly damaging variants with lower frequencies. Genotype-phenotype correlations were implemented for top selected variants in terms of highest possibility of being damaging. RESULTS: Five deleterious pharmacovariants in the RYR1, POLG, ANXA11, CCNH, and CDH23 genes identified in step (a) and subsequent analysis displayed high impact on drug-related phenotypes. Also, the utilization of recursive feature elimination achieved a subset of 175 malfunction pharmacovariants in 135 drug-related genes that were used by the RF model with fivefold internal cross-validation, resulting in an area under the curve of 0.9736842 with an average accuracy of 0.9818 (95% CI: 0.89, 0.99) on predicting whether a carrying individuals will develop adverse drug reactions or not. However, the external cross-validation of the same model indicated a possible false positive result when dealing with a low number of observations, as only 60 important variants in 49 genes were displayed, giving an AUC of 0.5384848 with an average accuracy of 0.9512 (95% CI: 0.83, 0.99). CONCLUSION: While there are some technologies for functionally assess not-interpreted pharmacovariants, there is still an essential need for the development of tools, methods, and algorithms which are able to provide a functional prediction for every single pharmacovariant in both large-scale datasets and small cohorts. Our approaches may bring new insights for choosing the right computational assessment algorithms out of high throughput DNA sequencing data from small cohorts to be used for personalized drug therapy implementation.

Co-Transplantation of Barcoded Lymphoid-Primed Multipotent (LMPP) and Common Lymphocyte (CLP) Progenitors Reveals a Major Contribution of LMPP to the Lymphoid Lineage.

T cells have the potential to maintain immunological memory and self-tolerance by recognizing antigens from pathogens or tumors. In pathological situations, failure to generate de novo T cells causes immunodeficiency resulting in acute infections and complications. Hematopoietic stem cells (HSC) transplantation constitutes a valuable option to restore proper immune function. However, delayed T cell reconstitution is observed compared to other lineages. To overcome this difficulty, we developed a new approach to identify populations with efficient lymphoid reconstitution properties. To this end, we use a DNA barcoding strategy based on the insertion into a cell chromosome of a lentivirus (LV) carrying a non-coding DNA fragment named barcode (BC). These will segregate through cell divisions and be present in cells' progeny. The remarkable characteristic of the method is that different cell types can be tracked simultaneously in the same mouse. Thus, we in vivo barcoded LMPP and CLP progenitors to test their ability to reconstitute the lymphoid lineage. Barcoded progenitors were co-grafted in immuno-compromised mice and their fate analyzed by evaluating the BC composition in transplanted mice. The results highlight the predominant role of LMPP progenitors for lymphoid generation and reveal valuable novel insights to be reconsidered in clinical transplantation assays.

Examining Different Analysis Protocols Targeting Hospital Sanitary Facility Microbiomes.

Indoor spaces exhibit microbial compositions that are distinctly dissimilar from one another and from outdoor spaces. Unique in this regard, and a topic that has only recently come into focus, is the microbiome of hospitals. While the benefits of knowing exactly which microorganisms propagate how and where in hospitals are undoubtedly beneficial for preventing hospital-acquired infections, there are, to date, no standardized procedures on how to best study the hospital microbiome. Our study aimed to investigate the microbiome of hospital sanitary facilities, outlining the extent to which hospital microbiome analyses differ according to sample-preparation protocol. For this purpose, fifty samples were collected from two separate hospitals-from three wards and one hospital laboratory-using two different storage media from which DNA was extracted using two different extraction kits and sequenced with two different primer pairs (V1-V2 and V3-V4). There were no observable differences between the sample-preservation media, small differences in detected taxa between the DNA extraction kits (mainly concerning Propionibacteriaceae), and large differences in detected taxa between the two primer pairs V1-V2 and V3-V4. This analysis also showed that microbial occurrences and compositions can vary greatly from toilets to sinks to showers and across wards and hospitals. In surgical wards, patient toilets appeared to be characterized by lower species richness and diversity than staff toilets. Which sampling sites are the best for which assessments should be analyzed in more depth. The fact that the sample processing methods we investigated (apart from the choice of primers) seem to have changed the results only slightly suggests that comparing hospital microbiome studies is a realistic option. The observed differences in species richness and diversity between patient and staff toilets should be further investigated, as these, if confirmed, could be a result of excreted antimicrobials.

Bioinformatic approaches for studying the microbiome of fermented food.

High-throughput DNA sequencing-based approaches continue to revolutionise our understanding of microbial ecosystems, including those associated with fermented foods. Metagenomic and metatranscriptomic approaches are state-of-the-art biological profiling methods and are employed to investigate a wide variety of characteristics of microbial communities, such as taxonomic membership, gene content and the range and level at which these genes are expressed. Individual groups and consortia of researchers are utilising these approaches to produce increasingly large and complex datasets, representing vast populations of microorganisms. There is a corresponding requirement for the development and application of appropriate bioinformatic tools and pipelines to interpret this data. This review critically analyses the tools and pipelines that have been used or that could be applied to the analysis of metagenomic and metatranscriptomic data from fermented foods. In addition, we critically analyse a number of studies of fermented foods in which these tools have previously been applied, to highlight the insights that these approaches can provide.

Gut Microbiota Associated with Gestational Health Conditions in a Sample of Mexican Women.

Gestational diabetes (GD), pre-gestational diabetes (PD), and pre-eclampsia (PE) are morbidities affecting gestational health which have been associated with dysbiosis of the mother's gut microbiota. This study aimed to assess the extent of change in the gut microbiota diversity, short-chain fatty acids (SCFA) production, and fecal metabolites profile in a sample of Mexican women affected by these disorders. Fecal samples were collected from women with GD, PD, or PE in the third trimester of pregnancy, along with clinical and biochemical data. Gut microbiota was characterized by high-throughput DNA sequencing of V3-16S rRNA gene libraries; SCFA and metabolites were measured by High-Pressure Liquid Chromatography (HPLC) and (Fourier Transform Ion Cyclotron Mass Spectrometry (FT-ICR MS), respectively, in extracts prepared from feces. Although the results for fecal microbiota did not show statistically significant differences in alfa diversity for GD, PD, and PE concerning controls, there was a difference in beta diversity for GD versus CO, and a high abundance of Proteobacteria, followed by Firmicutes and Bacteroidota among gestational health conditions. DESeq2 analysis revealed bacterial genera associated with each health condition; the Spearman's correlation analyses showed selected anthropometric, biochemical, dietary, and SCFA metadata associated with specific bacterial abundances, and although the HPLC did not show relevant differences in SCFA content among the studied groups, FT-ICR MS disclosed the presence of interesting metabolites of complex phenolic, valeric, arachidic, and caprylic acid nature. The major conclusion of our work is that GD, PD, and PE are associated with fecal bacterial microbiota profiles, with distinct predictive metagenomes.

The Entero-Mammary Pathway and Perinatal Transmission of Gut Microbiota and SARS-CoV-2.

COVID-19 is a severe respiratory disease threatening pregnant women, which increases the possibility of adverse pregnancy outcomes. Several recent studies have demonstrated the ability of SARS-CoV-2 to infect the mother enterocytes, disturbing the gut microbiota diversity. The aim of this study was to characterize the entero-mammary microbiota of women in the presence of the virus during delivery. Fifty mother−neonate pairs were included in a transversal descriptive work. The presence of SARS-CoV-2 RNA was detected in nasopharyngeal, mother rectal swabs (MRS) and neonate rectal swabs (NRS) collected from the pairs, and human colostrum (HC) samples collected from mothers. The microbiota diversity was characterized by high-throughput DNA sequencing of V3-16S rRNA gene libraries prepared from HC, MRS, and NRS. Data were analyzed with QIIME2 and R. Our results indicate that several bacterial taxa are highly abundant in MRS positive for SARS-CoV-2 RNA. These bacteria mostly belong to the Firmicutes phylum; for instance, the families Bifidobacteriaceae, Oscillospiraceae, and Microbacteriaceae have been previously associated with anti-inflammatory effects, which could explain the capability of women to overcome the infection. All samples, both positive and negative for SARS-CoV-2, featured a high abundance of the Firmicutes phylum. Further data analysis showed that nearly 20% of the bacterial diversity found in HC was also identified in MRS. Spearman correlation analysis highlighted that some genera of the Proteobacteria and Actinobacteria phyla were negatively correlated with MRS and NRS (p < 0.005). This study provides new insights into the gut microbiota of pregnant women and their potential association with a better outcome during SARS-CoV-2 infection.

Chronic-Antibiotics Induced Gut Microbiota Dysbiosis Rescues Memory Impairment and Reduces ß-Amyloid Aggregation in a Preclinical Alzheimer's Disease Model.

Alzheimer's disease (AD) is a multifactorial pathology characterized by ß-amyloid (Aß) deposits, Tau hyperphosphorylation, neuroinflammatory response, and cognitive deficit. Changes in the bacterial gut microbiota (BGM) have been reported as a possible etiological factor of AD. We assessed in offspring (F1) 3xTg, the effect of BGM dysbiosisdysbiosis in mothers (F0) at gestation and F1 from lactation up to the age of 5 months on Aß and Tau levels in the hippocampus, as well as on spatial memory at the early symptomatic stage of AD. We found that BGM dysbiosisdysbiosis with antibiotics (Abx) treatment in F0 was vertically transferred to their F1 3xTg mice, as observed on postnatal day (PD) 30 and 150. On PD150, we observed a delay in spatial memory impairment and Aß deposits, but not in Tau and pTau protein in the hippocampus at the early symptomatic stage of AD. These effects are correlated with relative abundance of bacteria and alpha diversity, and are specific to bacterial consortia. Our results suggest that this specific BGM could reduce neuroinflammatory responses related to cerebral amyloidosis and cognitive deficit and activate metabolic pathways associated with the biosynthesis of triggering or protective molecules for AD.

A high-throughput DNA sequencing study of fecal bacteria of seven Mexican horse breeds.

Horses are non-ruminant, herbivorous mammals, been used through history for various purposes, with a gut microbiota from cecum to the colon, possessing remarkable fermentative capacity. We studied the fecal microbiota of Azteca, Criollo, Frisian, Iberian, Pinto, Quarter and Spanish horse breeds living in Mexico by next-generation DNA sequencing of 16S rRNA gene libraries. Dominant phyla Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, Fibrobacteres, Actinobacteria and Verrucomicrobia have different relative abundances among breeds, with contrasted alpha and beta diversities as well. Heatmap analysis revealed that Ruminococcaceae, Lachnospiraceae, Mogibacteriaceae families, and order Clostridiales are more abundant in Spanish, Azteca, Quarter and Criollo breeds. The LEfSe analysis displayed higher abundance of order Bacteroidales, family BS11, and genera Faecalibacterium, Comamonas, Collinsella, Acetobacter, and Treponema in Criollo, Azteca, Iberian, Spanish, Frisian, Pinto, and Quarter horse breeds. The conclusion is that dominant bacterial taxa, found in fecal samples of horse breeds living in Mexico, have different relative abundances.

FACETS: Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing.

Clinical sequencing studies routinely involve molecular profiling of patients for mutations and copy number alterations. However, detection of "actionable" aberrations to guide treatment decisions require accurate, tumor purity-, ploidy-, and clonal heterogeneity-adjusted integer copy number calls. In this chapter, we describe the FACETS algorithm, an Allele-Specific Copy Number (ASCN) analysis tool with a broad application to whole-genome, whole-exome, as well as targeted panel sequencing platforms to annotate the genome for the detection of copy number alterations including homozygous/heterozygous deletions, copy-neutral loss-of-heterozygosity (LOH) events, allele-specific gains/amplifications, and cellular fraction profiles.We will describe some methodological details on joint segmentation of total and allele-specific copy number, on the estimation of integer copy number calls adjusting for tumor purity, ploidy, and intratumor heterogeneity, along with comprehensive output and integrated visualization. We also provide a tutorial on the installation, application, and tips to run and interpret FACETS.

Clinical and genetic investigations in Chinese families with retinitis pigmentosa.

To describe clinical and genetic characteristics in a series of Chinese patients with non-syndromic retinitis pigmentosa, a total of 20 unrelated Chinese pedigrees with non-syndromic retinitis pigmentosa were evaluated. Complete ophthalmic examinations data including the Humphrey visual field, spectral domain-optical coherence tomography, full-field electroretinography, and fundus fluorescence were collected and analyzed. Targeted exome sequencing was utilized to investigate variations in 260 known genes of inherited retinal disease, including the 90 known causative retinitis pigmentosa genes. We initially identified the potential candidate variants in the pedigrees, then validated the variants using the Sanger sequencing and performed segregation analysis to verify that the variants constituted disease-causing mutations in these pedigrees. We detected three novel (likely) pathogenic and eight previously reported (likely) pathogenic variations in nine genes reported to be related to non-syndromic retinitis pigmentosa in nine of the pedigrees. We report clinical characteristics of Chinese patients with retinitis pigmentosa and novel mutations responsible for non-syndromic retinitis pigmentosa in Chinese pedigrees, expanding the number of gene mutations associated with this disorder and clarifying its genetic basis in the Chinese population. These data will help with rapid and efficient molecular diagnosis and the study of targeted treatment for retinitis pigmentosa in this population.

Ichthyoplankton DNA metabarcoding: Challenges and perspectives.

DNA metabarcoding has been widely used to access and monitor species. However, several challenges remain open for its mainstream application in ecological studies, particularly when dealing with a quantitative approach. In a From the Cover article in this issue of Molecular Ecology, Mariac et al. (2021) report species-level ichthyoplankton dynamics for 97 fish species from two Amazon river basins using a clever quantitative metabarcoding approach employing a probe capture method. They clearly show that most species spawned during the floods, although ~20% also spawned mainly during the receding period and some other year-round, but interestingly, species from the same genus reproduced in distinct periods (i.e., inverse phenology). Opportunistically, Mariac et al. (2021) reported that during an intense hydrological anomaly, several species had a sharp reduction in spawning activity, demonstrating a quick response to environmental cues. This is an interesting result since the speed at which fish species can react to environmental changes, during the spawning period, is largely unknown. Thus, this study brings remarkable insights into basic life history information that is imperative for proposing strategies that could lead to a realistic framework for sustainable fisheries management practices and conservation, fundamental for an understudied and threatened realm, such as the Amazon River basin.

Genetic variants at the chromosomal region 2q21.3 underlying inhibitor development in patients with severe haemophilia A.

INTRODUCTION: Inhibitor development affects about 30% of patients with severe haemophilia A (HA) and results from different environmental and genetic risk factors. Previously, we identified the missense variant rs3754689 in the LCT gene linked with this predisposition. Since rs3754689 variant is benign and is located in a conserved haplotype region, we hypothesized that the association signal captured by this variant is located in coinherited, neighbouring genes. AIM: To identify novel genetic risk factors associated with inhibitor development in coding regions of R3HDM1, UBXN4, CXCR4, MCM6, DARS and miR128-1 genes. METHODS: Targeted sequencing was performed in 246 severe HA patients (72 with and 174 without inhibitor): 181 previously and 65 newly enrolled. RESULTS: Forty-one common and 152 rare variants passed the quality control. Logistic regression analysis of common variants identified rs3754689 and four additional variants (.011 < P < .047; FDR ranging .2-.38). Logistic regression analysis performed only in the 220 Italian patients showed similar results (.004 < P < .05; FDR ranging .12-.22). Three of these variants (rs3213892 and rs3816155 in the LCT intron 13 and rs961360 in the R3HDM1 intron10-exon11 junction) may affect the expression of UBXN4 and R3HDM1, respectively. Rare variants did not show association with inhibitor development. Identified variants were not replicated in the multi-ethnic SIPPET cohort of 230 severe HA patients. CONCLUSION: Due to the limited sample size that may be responsible of the high FDR values, we could not confirm with certainty the analysed association. Further evaluation of the expression levels of analysed genes will confirm or not their role in inhibitor development.

Molecular Characterization of Microbiota in Cerebrospinal Fluid From Patients With CSF Shunt Infections Using Whole Genome Amplification Followed by Shotgun Sequencing.

Understanding the etiology of cerebrospinal fluid (CSF) shunt infections and reinfections requires detailed characterization of associated microorganisms. Traditionally, identification of bacteria present in the CSF has relied on culture methods, but recent studies have used high throughput sequencing of 16S rRNA genes. Here we evaluated the method of shotgun DNA sequencing for its potential to provide additional genomic information. CSF samples were collected from 3 patients near the beginning and end of each of 2 infection episodes. Extracted total DNA was sequenced by: (1) whole genome amplification followed by shotgun sequencing (WGA) and (2) high-throughput sequencing of the 16S rRNA V4 region (16S). Taxonomic assignments of sequences from WGA and 16S were compared with one another and with conventional microbiological cultures. While classification of bacteria was consistent among the 3 approaches, WGA provided additional insights into sample microbiological composition, such as showing relative abundances of microbial versus human DNA, identifying samples of questionable quality, and detecting significant viral load in some samples. One sample yielded sufficient non-human reads to allow assembly of a high-quality Staphylococcus epidermidis genome, denoted CLIMB1, which we characterized in terms of its MLST profile, gene complement (including putative antimicrobial resistance genes), and similarity to other annotated S. epidermidis genomes. Our results demonstrate that WGA directly applied to CSF is a valuable tool for the identification and genomic characterization of dominant microorganisms in CSF shunt infections, which can facilitate molecular approaches for the development of better diagnostic and treatment methods.