Next-generation sequencing of 35 RHD variants in 16 253 serologically D− pregnant women in the Finnish population

Abstract Fetal RHD screening for targeted routine antenatal anti-D prophylaxis has been implemented in many countries, including Finland, since the 2010s. Comprehensive knowledge of the RHD polymorphism in the population is essential for the performance and safety of the anti-D prophylaxis program. During the first 3 years of the national screening program in Finland, over 16 000 samples from RhD− women were screened for fetal RHD; among them, 79 samples (0.5%) containing a maternal variant allele were detected. Of the detected maternal variants, 35 cases remained inconclusive using the traditional genotyping methods and required further analysis by next-generation sequencing (NGS) of the whole RHD gene to uncover the variant allele. In addition to the 13 RHD variants that have been previously reported in different populations, 8 novel variants were also detected, indicating that there is more variation of RHD in the RhD− Finnish population than has been previously known. Three of the novel alleles were identified in multiple samples; thus, they are likely specific to the original Finnish population. National screening has thus provided new information about the diversity of RHD variants in the Finnish population. The results show that NGS is a powerful method for genotyping the highly polymorphic RHD gene compared with traditional methods that rely on the detection of specific nucleotides by polymerase chain reaction amplification.</jats:p

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

The broad-band properties of the intermediate synchrotron peaked BL Lac S2 0109+22 from radio to VHE gamma-rays

The Major Atmospheric Gamma-ray Imaging Cherenkov (MAGIC) telescopes observed S2 0109+22 in 2015 July during its flaring activity in high-energy gamma-rays observed by Fermi-Large Area Telescope. We analyse the MAGIC data to characterize the very high energy (VHE) gamma-ray emission of S2 0109+22, which belongs to the subclass of intermediate synchrotron peak (ISP) BL Lacertae (BL Lac) objects. We study the multifrequency emission in order to investigate the source classification. Finally, we compare the source long-term behaviour to other VHE gamma-ray emitting (TeV) blazars. We performed a temporal and spectral analysis of the data centred around the MAGIC interval of observation (MJD 57225-57231). Long-term radio and optical data have also been investigated using the discrete correlation function. The redshift of the source is estimated through optical host-galaxy imaging and also using the amount of VHE gamma-ray absorption. The quasi-simultaneous multifrequency spectral energy distribution (SED) is modelled with the conventional one-zone synchrotron self-Compton (SSC) model. MAGIC observations resulted in the detection of the source at a significance level of 5.3 sigma. The VHE gamma-ray emission of S2 0109+22 is variable on a daily time scale. VHE gamma-ray luminosity of the source is lower than the average of TeV BL Lacs. The optical polarization and long-term optical/radio behaviour of the source are different from the general population of TeV blazars. All these findings agree with the classification of the source as an ISP BL Lac object. We estimate the source redshift as z = 0.36 +/- 0.07. The SSC parameters describing the SED are rather typical for blazars

Detection of persistent VHE gamma-ray emission from PKS 1510-089 by the MAGIC telescopes during low states between 2012 and 2017

Context. PKS 1510-089 is a flat spectrum radio quasar strongly variable in the optical and GeV range. To date, very high-energy (VHE, >100 GeV) emission has been observed from this source either during long high states of optical and GeV activity or during short flares.Aims. We search for low-state VHE gamma-ray emission from PKS 1510-089. We characterize and model the source in a broadband context, which would provide a baseline over which high states and flares could be better understood.Methods. PKS 1510-089 has been monitored by the MAGIC telescopes since 2012. We use daily binned Fermi-LAT flux measurements of PKS 1510-089 to characterize the GeV emission and select the observation periods of MAGIC during low state of activity. For the selected times we compute the average radio, IR, optical, UV, X-ray, and gammaray emission to construct a low-state spectral energy distribution of the source. The broadband emission is modeled within an external Compton scenario with a stationary emission region through which plasma and magnetic fields are flowing. We also perform the emissio-model-independent calculations of the maximum absorption in the broad line region (BLR) using two different models.Results. The MAGIC telescopes collected 75 hr of data during times when the Fermi-LAT flux measured above 1 GeV was below 3 x10(-8)cm(-2) s(-1) , which is the threshold adopted for the definition of a low gamma-ray activity state. The data show a strongly significant (9.50-sigma ) VHE gamma-ray emission at the level of (4.27 +/- 0.61(stat)) x 10(-12) cm(-2) s(-1) above 150 GeV, a factor of 80 lower than the highest flare observed so far from this object. Despite the lower flux, the spectral shape is consistent with earlier detections in the VHE band. The broadband emission is compatible with the external Compton scenario assuming a large emission region located beyond the BLR. For the first time the gamma-ray data allow us to place a limit on the location of the emission region during a low gamma ray state of a FSRQ. For the used model of the BLR, the 95% confidence level on the location of the emission region allows us to place it at a distance >74% of the outer radius of the BLR

Unraveling the Complex Behavior of Mrk 421 with Simultaneous X-Ray and VHE Observations during an Extreme Flaring Activity in 2013 April*

We report on a multiband variability and correlation study of the TeV blazar Mrk 421 during an exceptional flaring activity observed from 2013 April 11 to 19. The study uses, among others, data from GLAST-AGILE Support Program (GASP) of the Whole Earth Blazar Telescope (WEBT), Swift, Nuclear Spectroscopic Telescope Array (NuSTAR), Fermi Large Area Telescope, Very Energetic Radiation Imaging Telescope Array System (VERITAS), and Major Atmospheric Gamma Imaging Cherenkov (MAGIC). The large blazar activity and the 43 hr of simultaneous NuSTAR and MAGIC/VERITAS observations permitted variability studies on 15 minute time bins over three X-ray bands (3-7 keV, 7-30 keV, and 30-80 keV) and three very-high-energy (VHE; >0.1 TeV) gamma-ray bands (0.2-0.4 TeV, 0.4-0.8 TeV, and >0.8 TeV). We detected substantial flux variations on multi-hour and sub-hour timescales in all of the X-ray and VHE gamma-ray bands. The characteristics of the sub-hour flux variations are essentially energy independent, while the multi-hour flux variations can have a strong dependence on the energy of the X-rays and the VHE gamma-rays. The three VHE bands and the three X-ray bands are positively correlated with no time lag, but the strength and characteristics of the correlation change substantially over time and across energy bands. Our findings favor multi-zone scenarios for explaining the achromatic/chromatic variability of the fast/slow components of the light curves, as well as the changes in the flux-flux correlation on day-long timescales. We interpret these results within a magnetic reconnection scenario, where the multi-hour flux variations are dominated by the combined emission from various plasmoids of different sizes and velocities, while the sub-hour flux variations are dominated by the emission from a single small plasmoid moving across the magnetic reconnection layer

Rh blood group D antigen genotyping using a portable nanopore based sequencing device: proof of principle

BACKGROUND: Nanopore sequencing is direct sequencing of a single-stranded DNA molecule using biological pores. A portable nanopore-based sequencing device from Oxford Nanopore Technologies (MinION) depends on driving a DNA molecule through nanopores embedded in a membrane using a voltage. Changes in current are then measured by a sensor, thousands of times per second and translated to nucleobases.METHODS: Genomic DNA (gDNA) samples (n = 13) were tested for Rh blood group D antigen (RHD) gene zygosity using droplet digital PCR. The RHD gene was amplified in 6 overlapping amplicons using long-range PCR. Amplicons were purified, and the sequencing library was prepared following the 1D Native barcoding gDNA protocol. Sequencing was carried out with 1D flow cells R9 version. Data analysis included basecalling, aligning to the RHD reference sequence, and calling variants. Variants detected were compared to the results acquired previously by the Ion Personal Genome Machine (Ion PGM).RESULTS: Up to 500× sequence coverage across the RHD gene allowed accurate variant calling. Exonic changes in the RHD gene allowed RHD allele determination for all samples sequenced except 1 RHD homozygous sample, where 2 heterozygous RHD variant alleles are suspected. There were 3 known variant RHD alleles (RHD*01W.02, RHD*11, and RHD*15) and 6 novel RHD variant alleles, as previously seen in Ion PGM sequencing data for these samples.CONCLUSIONS: MinION was effective in blood group genotyping, provided enough sequencing data to achieve high coverage of the RHD gene, and enabled confident calling of variants and RHD allele determination.</p

Combinatorial analysis and algorithms for quasispecies reconstruction using next-generation sequencing

<p>Abstract</p> <p>Background</p> <p>Next-generation sequencing (NGS) offers a unique opportunity for high-throughput genomics and has potential to replace Sanger sequencing in many fields, including de-novo sequencing, re-sequencing, meta-genomics, and characterisation of infectious pathogens, such as viral quasispecies. Although methodologies and software for whole genome assembly and genome variation analysis have been developed and refined for NGS data, reconstructing a viral quasispecies using NGS data remains a challenge. This application would be useful for analysing intra-host evolutionary pathways in relation to immune responses and antiretroviral therapy exposures. Here we introduce a set of formulae for the combinatorial analysis of a quasispecies, given a NGS re-sequencing experiment and an algorithm for quasispecies reconstruction. We require that sequenced fragments are aligned against a reference genome, and that the reference genome is partitioned into a set of sliding windows (amplicons). The reconstruction algorithm is based on combinations of multinomial distributions and is designed to minimise the reconstruction of false variants, called <it>in-silico </it>recombinants.</p> <p>Results</p> <p>The reconstruction algorithm was applied to error-free simulated data and reconstructed a high percentage of true variants, even at a low genetic diversity, where the chance to obtain <it>in-silico </it>recombinants is high. Results on empirical NGS data from patients infected with hepatitis B virus, confirmed its ability to characterise different viral variants from distinct patients.</p> <p>Conclusions</p> <p>The combinatorial analysis provided a description of the difficulty to reconstruct a quasispecies, given a determined amplicon partition and a measure of population diversity. The reconstruction algorithm showed good performance both considering simulated data and real data, even in presence of sequencing errors.</p