The detection and identification of species of fungi in the environment using molecular methods heavily depends on reliable reference sequence databases. However, these databases are largely incomplete in terms of taxon coverage, and a significant effort is required from herbaria and living fungal collections for the mass-barcoding of well-identified and well-curated fungal specimens or strains. Here, a PacBio amplicon sequencing approach is applied to recent lichen herbarium specimens for the sequencing of the fungal ITS barcode, allowing a higher throughput sample processing than Sanger sequencing, which often required the use of cloning. Out of 96 multiplexed samples, a full-length ITS sequence of the target lichenised fungal species was recovered for 85 specimens. In addition, sequences obtained for co-amplified fungi gave an interesting insight into the diversity of endolichenic fungi. Challenges encountered at both the laboratory and bioinformatic stages are discussed, and cost and quality are compared with Sanger sequencing. With increasing data output and reducing sequencing cost, PacBio amplicon sequencing is seen as a promising approach for the generation of reference sequences for lichenised fungi as well as the characterisation of lichen-associated fungal communities.
Construction of full-length Japanese reference panel of class I HLA genes with single-molecule, real-time sequencing.
Human leukocyte antigen (HLA) is a gene complex known for its exceptional diversity across populations, importance in organ and blood stem cell transplantation, and associations of specific alleles with various diseases. We constructed a Japanese reference panel of class I HLA genes (ToMMo HLA panel), comprising a distinct set of HLA-A, HLA-B, HLA-C, and HLA-H alleles, by single-molecule, real-time (SMRT) sequencing of 208 individuals included in the 1070 whole-genome Japanese reference panel (1KJPN). For high-quality allele reconstruction, we developed a novel pipeline, Primer-Separation Assembly and Refinement Pipeline (PSARP), in which the SMRT sequencing and additional short-read data were used. The panel consisted of 139 alleles, which were all extended from known IPD-IMGT/HLA sequences, contained 40 with novel variants, and captured more than 96.5% of allelic diversity in 1KJPN. These newly available sequences would be important resources for research and clinical applications including high-resolution HLA typing, genetic association studies, and analyzes of cis-regulatory elements.
Longitudinal HIV sequencing reveals reservoir expression leading to decay which is obscured by clonal expansion.
After initiating antiretroviral therapy (ART), a rapid decline in HIV viral load is followed by a long period of undetectable viremia. Viral outgrowth assay suggests the reservoir continues to decline slowly. Here, we use full-length sequencing to longitudinally study the proviral landscape of four subjects on ART to investigate the selective pressures influencing the dynamics of the treatment-resistant HIV reservoir. We find intact and defective proviruses that contain genetic elements favoring efficient protein expression decrease over time. Moreover, proviruses that lack these genetic elements, yet contain strong donor splice sequences, increase relatively to other defective proviruses, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by the expansion of proviral clones. Paradoxically, clonal expansion may also be enhanced by HIV expression that leads to splicing between HIV donor splice sites and downstream human exons.
Conventional and single-molecule targeted sequencing method for specific variant detection in IKBKG while bypassing the IKBKGP1 pseudogene.
In addition to Sanger sequencing, next-generation sequencing of gene panels and exomes has emerged as a standard diagnostic tool in many laboratories. However, these captures can miss regions, have poor efficiency, or capture pseudogenes, which hamper proper diagnoses. One such example is the primary immunodeficiency-associated gene IKBKG. Its pseudogene IKBKGP1 makes traditional capture methods aspecific. We therefore developed a long-range PCR method to efficiently target IKBKG, as well as two associated genes (IRAK4 and MYD88), while bypassing the IKBKGP1 pseudogene. Sequencing accuracy was evaluated using both conventional short-read technology and a newer long-read, single-molecule sequencer. Different mapping and variant calling options were evaluated in their capability to bypass the pseudogene using both sequencing platforms. Based on these evaluations, we determined a robust diagnostic application for unambiguous sequencing and variant calling in IKBKG, IRAK4, and MYD88. This method allows rapid identification of selected primary immunodeficiency diseases in patients suffering from life-threatening invasive pyogenic bacterial infections. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
In comparison to humans and chimpanzees, gorillas show low diversity at MHC class I genes (Gogo), as reflected by an overall reduced level of allelic variation as well as the absence of a functionally important sequence motif that interacts with killer cell immunoglobulin-like receptors (KIR). Here, we use recently generated large-scale genomic sequence data for a reassessment of allelic diversity at Gogo-C, the gorilla orthologue of HLA-C. Through the combination of long-range amplifications and long-read sequencing technology, we obtained, among the 35 gorillas reanalyzed, three novel full-length genomic sequences including a coding region sequence that has not been previously described. The newly identified Gogo-C*03:01 allele has a divergent recombinant structure that sets it apart from other Gogo-C alleles. Domain-by-domain phylogenetic analysis shows that Gogo-C*03:01 has segments in common with Gogo-B*07, the additional B-like gene that is present on some gorilla MHC haplotypes. Identified in ~ 50% of the gorillas analyzed, the Gogo-C*03:01 allele exclusively encodes the C1 epitope among Gogo-C allotypes, indicating its important function in controlling natural killer cell (NK cell) responses via KIR. We further explored the hypothesis whether gorillas experienced a selective sweep which may have resulted in a general reduction of the gorilla MHC class I repertoire. Our results provide little support for a selective sweep but rather suggest that the overall low Gogo class I diversity can be best explained by drastic demographic changes gorillas experienced in the ancient and recent past.
Summary Hepatitis E virus genotype 3 (HEV-3) can lead to chronic infection in immunocompromised patients, and ribavirin is the treatment of choice. Recently, mutations in the polymerase gene have been associated with ribavirin failure but their frequency before treatment according to HEV-3 subtypes has not been studied on a large data set. We used single-molecule real-time sequencing technology to sequence 115 new complete genomes of HEV-3 infecting French patients. We analyzed phylogenetic relationships, the length of the polyproline region, and mutations in the HEV polymerase gene. Eighty-five (74%) were in the clade HEV-3efg, 28 (24%) in HEV-3chi clade, and 2 (2%) in HEV-3ra clade. Using automated partitioning of maximum likelihood phylogenetic trees, complete genomes were classified into subtypes. Polyproline region length differs within HEV-3 clades (from 189 to 315 nt). Investigating mutations in the polymerase gene, distinct polymorphisms between HEV-3 subtypes were found (G1634R in 95% of HEV-3e, G1634K in 56% of HEV-3ra, and V1479I in all HEV-3efg, clade HEV-3ra, and HEV-3k strains). Subtype-specific polymorphisms in the HEV-3 polymerase have been identified. Our study provides new complete genome sequences of HEV-3 that could be useful for comparing strains circulating in humans and the animal reservoir.
Detection of somatic mutations in human leukocyte antigen (HLA) genes using whole-exome sequencing (WES) is hampered by the high polymorphism of the HLA loci, which prevents alignment of sequencing reads to the human reference genome. We describe a computational pipeline that enables accurate inference of germline alleles of class I HLA-A, B and C genes and subsequent detection of mutations in these genes using the inferred alleles as a reference. Analysis of WES data from 7,930 pairs of tumor and healthy tissue from the same patient revealed 298 nonsilent HLA mutations in tumors from 266 patients. These 298 mutations are enriched for likely functional mutations, including putative loss-of-function events. Recurrence of mutations suggested that these ‘hotspot’ sites were positively selected. Cancers with recurrent somatic HLA mutations were associated with upregulation of signatures of cytolytic activity characteristic of tumor infiltration by effector lymphocytes, supporting immune evasion by altered HLA function as a contributory mechanism in cancer.
Comparisons of MHC gene content and diversity among closely related species can provide insights into the evolutionary mechanisms shaping immune system variation. After chimpanzees and bonobos, gorillas are humans’ closest living relatives; but in contrast, relatively little is known about the structure and variation of gorilla MHC class I genes (Gogo). Here, we combined long-range amplifications and long-read sequencing technology to analyze full-length MHC class I genes in 35 gorillas. We obtained 50 full-length genomic sequences corresponding to 15 Gogo-A alleles, 4 Gogo-Oko alleles, 21 Gogo-B alleles, and 10 Gogo-C alleles including 19 novel coding region sequences. We identified two previously undetected MHC class I genes related to Gogo-A and Gogo-B, respectively, thereby illustrating the potential of this approach for efficient and highly accurate MHC genotyping. Consistent with their phylogenetic position within the hominid family, individual gorilla MHC haplotypes share characteristics with humans and chimpanzees as well as orangutans suggesting a complex history of the MHC class I genes in humans and the great apes. However, the overall MHC class I diversity appears to be low further supporting the hypothesis that gorillas might have experienced a reduction of their MHC repertoire.
A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples.
VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally acquired immunity and, as such, is a leading vaccine candidate against placental malaria. This protein is very polymorphic and technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time consuming and costly, and that performs poorly when applied to clinical samples that are commonly polyclonal. A next-generation sequencing platform, Pacific Biosciences (PacBio), offers an alternative approach to overcome these issues.PCR primers were designed that target a 5 kb segment in the 5′ end of var2csa and the resulting amplicons were sequenced using PacBio sequencing. The primers were optimized using two laboratory strains and were validated on DNA from 43 clinical samples, extracted from dried blood spots on filter paper or from cryopreserved P. falciparum-infected erythrocytes. Sequence reads were assembled using the SMRT-analysis ConsensusTools module.Here, a PacBio sequencing-based approach for recovering a segment encoding the majority of VAR2CSA’s extracellular region is described; this segment includes the totality of the first four domains in the 5′ end of var2csa (~5 kb), from clinical malaria samples. The feasibility of the method is demonstrated, showing a high success rate from cryopreserved samples and more limited success from dried blood spots stored at room temperature, and characterized the genetic variation of the var2csa locus.This method will facilitate a detailed analysis of var2csa genetic variation and can be adapted to sequence other hypervariable P. falciparum genes.