Biology subjects

Neumann, R.

Publications and source records attributed to Neumann, R..

5 recordsLinked to original sources

bioRxiv2024

Targeted Long-Read Bisulfite Sequencing for Promoter Methylation Analysis in Severe Preterm Birth

DNA methylation plays a critical role in the dynamics of gene expression regulation and the development of various disorders. Whole-genome bisulfite sequencing can provide single base resolution of CpG methylation levels and is the "gold standard" for DNA methylation quantification, but it also has a high cost. In contrast, targeted sequencing is optimal when focusing on specific candidate regions, while providing sufficient sequencing depth. Here, we present a targeted bisulfite sequencing approach to study the methylation status of regions of interest. We amplify selected regions from bisulfite-treated DNA and sequence them using Nanopore sequencing. In this work, we applied this workflow to candidate gene promoters for severe premature labor in a Latin American population. We successfully amplified fragments over 1 Kb in length using long PCR conditions for 12 genes that were barcoded per sample and pooled to be sequenced on MinION flow cells. This approach achieved high sequencing depths, ensuring reliable DNAm estimation. We found significant hypomethylation of the MIR155HG gene promoter in severe preterm birth samples, which is concordant with reported gene expression changes. We demonstrate that combining bisulfite DNA treatment with pooled long-read sequencing is a cost- and time-effective method to evaluate DNAm in several targeted regions and several samples in parallel. This study provides proof-of-concept for larger studies, demonstrating the applicability and high scalability of our assay to any locus of interest. Our experience suggests that this approach can be easily transferred to other diagnostic questions.

genetics↗

bioRxiv2023

Defining cat mitogenome variation and accounting for numts via multiplex amplification and Nanopore sequencing

Hair shed by domestic cats is a potentially useful source of forensic evidence. Analysable hair DNA is predominantly mitochondrial, but the recent domestication history of cats means that mtDNA diversity is low. A 402-bp control region segment is usually sequenced, defining only a small number of distinct mitotypes in populations. Previously, we used a long-amplicon approach to sequence whole mitogenomes in a sample of blood DNAs from 119 UK cats, greatly increasing observed diversity and reducing random match probabilities. To exploit this variation for forensic analysis, we here describe a multiplex system that amplifies the cat mitogenome in 60 overlapping amplicons of mean length 360 bp, followed by Nanopore sequencing. Variants detected in multiplex sequence data from hair completely mirror those from long-amplicon data from blood from the same individuals. However, applying the multiplex to matched blood DNA reveals additional sequence variants which derive from the major feline nuclear mitochondrial insertion sequence (numt), which covers 7.9 kb of the 17-kb mitogenome and exists in multiple tandem copies. We use long-amplicon Nanopore sequencing to investigate numt variation in a set of cats, together with an analysis of published genome sequences, and show that numt arrays are variable in both structure and sequence, thus providing a potential source of uncertainty when nuclear DNA predominates in a sample. Forensic application of the test was demonstrated by matching hairs from a cat with skeletal remains from its putative mother, both of which shared a globally common mitotype at the control region. The random match probability (RMP) in this case with the CR 402-bp segment was 0.21 and this decreased to 0.03 when considering the whole mitogenome. The developed multiplex and sequencing approach, when applied to cat hair where nuclear DNA is scarce, can provide a reliable and highly discriminating source of forensic genetic evidence. The confounding effect of numt co-amplification in degraded samples where mixed sequences are observed can be mitigated by variant phasing, and by comparison with numt sequence diversity data, such as those presented here.

genetics↗

bioRxiv2023

Mitogenome sequences of domestic cats demonstrate lineage expansions and dynamic mutation processes in a mitochondrial minisatellite

As a population genetic tool, mitochondrial DNA is commonly divided into the [~]1-kb control region (CR), in which single nucleotide variant (SNV) diversity is relatively high, and the coding region, in which selective constraint is greater and diversity lower, but which provides an informative phylogeny. In some species, the CR contains variable tandemly repeated sequences that are understudied due to heteroplasmy. Domestic cats (Felis catus) have a recent origin and therefore traditional CR-based analysis of populations yields only a small number of haplotypes. To increase resolution we used Nanopore sequencing to analyse 119 cat mitogenomes via a long-amplicon approach. This greatly improves discrimination (from 15 to 87 distinct haplotypes) and defines a phylogeny showing similar starlike topologies within all cat haplogroups, likely reflecting post-domestication expansion. We sequenced RS2, a CR tandem array of 80-bp repeat units, placing RS2 array structures within the phylogeny and increasing overall haplotype diversity. Repeat number varies between 3 and 12 (median: 4) with over 30 different repeat unit types differing largely by SNVs. Five SNVs show evidence of independent recurrence within the phylogeny, and seven are involved in at least 11 instances of rapid spread along repeat arrays within haplogroups. In defining mitogenome variation our study provides key information for the forensic genetic analysis of cat hair evidence, and for the first time a phylogenetically informed picture of tandem repeat variation that reveals remarkably dynamic mutation processes at work in the mitochondrion.

genomics↗

bioRxiv2021

Variation in Human Herpesvirus 6B telomeric integration, excision and transmission between tissues and individuals

Human herpesviruses 6A and 6B (HHV-6A/6B) are ubiquitous pathogens that persist lifelong in latent form and can cause severe conditions upon reactivation. They are spread by community-acquired infection of free virus (acqHHV6A/6B) and by germline transmission of inherited chromosomally- integrated HHV-6A/6B (iciHHV-6A/6B) in telomeres. We exploited a hypervariable region of the HHV- 6B genome to investigate the relationship between acquired and inherited virus and revealed predominantly maternal transmission of acqHHV-6B in families. Remarkably, we demonstrate that some copies of acqHHV-6B in saliva from healthy adults gained a telomere, indicative of integration and latency, and that the frequency of viral genome excision from telomeres in iciHHV-6B carriers is surprisingly high and varies between tissues. In addition, newly formed short telomeres generated by partial viral genome release are frequently lengthened, particularly in telomerase-expressing pluripotent cells. Consequently, iciHHV-6B carriers are mosaic for different iciHHV-6B structures, including circular extra-chromosomal forms that have the potential to reactivate. Finally, we show transmission of an HHV-6B strain from an iciHHV-6B mother to her non-iciHHV-6B son. Altogether we demonstrate that iciHHV-6B can readily transition between telomere-integrated and free virus forms.

genetics↗

bioRxiv2017

Inherited chromosomally integrated human herpesvirus 6 genomes are ancient, intact and potentially able to reactivate from telomeres

Human herpesviruses 6A and 6B (HHV6-A and HHV-6B; species Human herpesvirus 6A and Human herpesvirus 6B) have the capacity to integrate into telomeres, the essential capping structures of chromosomes that play roles in cancer and ageing. About 1% of people worldwide are carriers of chromosomally integrated HHV-6 (ciHHV-6), which is inherited as a genetic trait. Understanding the consequences of integration for the evolution of the viral genome, for the telomere and for the risk of disease associated with carrier status is hampered by a lack of knowledge about ciHHV-6 genomes. Here, we report an analysis of 28 ciHHV-6 genomes and show that they are significantly divergent from the few modern non-integrated HHV-6 strains for which complete sequences are currently available. In addition ciHHV-6B genomes in Europeans are more closely related to each other than to ciHHV-6B genomes from China and Pakistan, suggesting regional variation of the trait. Remarkably, at least one group of European ciHHV-6B carriers has inherited the same ciHHV-6B genome, integrated in the same telomere allele, from a common ancestor estimated to have existed 24,500 {+/-}10,600 years ago. Despite the antiquity of some, and possibly most, germline HHV-6 integrations, the majority of ciHHV-6B (95%) and ciHHV-6A (72%) genomes contain a full set of intact viral genes and therefore appear to have the capacity for viral gene expression and full reactivation.\n\nIMPORTANCEInheritance of HHV-6A or HHV-6B integrated into a telomere occurs at a low frequency in most populations studied to date but its characteristics are poorly understood. However, stratification of ciHHV-6 carriers in modern populations due to common ancestry is an important consideration for genome-wide association studies that aim to identify disease risks for these people. Here we present full sequence analysis of 28 ciHHV-6 genomes and show that ciHHV-6B in many carriers with European ancestry most likely originated from ancient integration events in a small number of ancestors. We propose that ancient ancestral origins for ciHHV-6A and ciHHV-6B are also likely in other populations. Moreover, despite their antiquity, all of the ciHHV-6 genomes appear to retain the capacity to express viral genes and most are predicted to be capable of full viral reactivation. These discoveries represent potentially important considerations in immune-compromised patients, in particular in organ transplantation and in stem cell therapy.

evolutionary biology↗