June 1, 2021  |  

Improving the reference with a diversity panel of sequence-resolved structural variation

Although the accuracy of the human reference genome is critical for basic and clinical research, structural variants (SVs) have been difficult to assess because data capable of resolving them have been limited. To address potential bias, we sequenced a diversity panel of nine human genomes to high depth using long-read, single-molecule, real-time sequencing data. Systematically identifying and merging SVs =50 bp in length for these nine and one public genome yielded 83,909 sequence-resolved insertions, deletions, and inversions. Among these, 2,839 (2.0 Mbp) are shared among all discovery genomes with an additional 13,349 (6.9 Mbp) present in the majority of humans, indicating minor alleles or errors in the reference, which is partially explained by an enrichment for GC-content and repetitive DNA. Genotyping 83% of these in 290 additional genomes confirms that at least one allele of the most common SVs in unique euchromatin are now sequence-resolved. We observe a 9-fold increase within 5 Mbp of chromosome telomeric ends and correlation with de novo single-nucleotide variant mutations showing that such variation is nonrandomly distributed defining potential hotspots of mutation. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. To illustrate the utility of sequence-resolved SVs in resequencing experiments, we mapped 30 diverse high-coverage Illumina-sequenced samples to GRCh38 with and without contigs containing SV insertions as alternate sequences, and we found these additional sequences recover 6.4% of unmapped reads. For reads mapped within the SV insertion, 25.7% have a better mapping quality, and 18.7% improved by 1,000-fold or more. We reveal 72,964 occurrences of 15,814 unique variants that were not discoverable with the reference sequence alone, and we note that 7% of the insertions contain an SV in at least one sample indicating that there are additional alleles in the population that remain to be discovered. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity. We present a summary of our findings and discuss ideas for revealing variation that was once difficult to ascertain.


June 1, 2021  |  

Microbiome profiling at the strain level using rRNA amplicons

Strain level microbiome profiling is needed for a full understanding of how microbial communities influence human health. Microbiome profiling of rRNA gene amplicons is a well-understood method that is rapid and inexpensive, but standard 16S rRNA gene methods generally cannot differentiate closely related strains. Whole genome/shotgun microbiome profiling is considered a higher-resolution alternative, but with decreased throughput and significantly increased sequencing costs and analysis burden. With both methods there are also challenges with microbial lysis, DNA preparation, and taxonomic analysis. Specialized microbiome-focused protocols were developed to achieve strain-level taxonomic differentiation using a rapid, high throughput rRNA gene assay. The protocol integrates lysis and DNA preparation improvements with a unique high information content amplicon and associated novel database to enable taxonomic differentiation of closely related microbial strains.


April 21, 2020  |  

Streptococcus oralis subsp. dentisani Produces Monolateral Serine-Rich Repeat Protein Fibrils, One of Which Contributes to Saliva Binding via Sialic Acid.

Our studies reveal that the oral colonizer and cause of infective endocarditis Streptococcus oralis subsp. dentisani displays a striking monolateral distribution of surface fibrils. Furthermore, our data suggest that these fibrils impact the structure of adherent bacterial chains. Mutagenesis studies indicate that these fibrils are dependent on three serine-rich repeat proteins (SRRPs), here named fibril-associated protein A (FapA), FapB, and FapC, and that each SRRP forms a different fibril with a distinct distribution. SRRPs are a family of bacterial adhesins that have diverse roles in adhesion and that can bind to different receptors through modular nonrepeat region domains. Amino acid sequence and predicted structural similarity searches using the nonrepeat regions suggested that FapA may contribute to interspecies interactions, that FapA and FapB may contribute to intraspecies interactions, and that FapC may contribute to sialic acid binding. We demonstrate that a fapC mutant was significantly reduced in binding to saliva. We confirmed a role for FapC in sialic acid binding by demonstrating that the parental strain was significantly reduced in adhesion upon addition of a recombinantly expressed, sialic acid-specific, carbohydrate binding module, while the fapC mutant was not reduced. However, mutation of a residue previously shown to be essential for sialic acid binding did not decrease bacterial adhesion, leaving the precise mechanism of FapC-mediated adhesion to sialic acid to be defined. We also demonstrate that the presence of any one of the SRRPs is sufficient for efficient biofilm formation. Similar structures were observed on all infective endocarditis isolates examined, suggesting that this distribution is a conserved feature of this S. oralis subspecies.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Long-read sequence and assembly of segmental duplications.

We have developed a computational method based on polyploid phasing of long sequence reads to resolve collapsed regions of segmental duplications within genome assemblies. Segmental Duplication Assembler (SDA; https://github.com/mvollger/SDA ) constructs graphs in which paralogous sequence variants define the nodes and long-read sequences provide attraction and repulsion edges, enabling the partition and assembly of long reads corresponding to distinct paralogs. We apply it to single-molecule, real-time sequence data from three human genomes and recover 33-79 megabase pairs (Mb) of duplications in which approximately half of the loci are diverged (<99.8%) compared to the reference genome. We show that the corresponding sequence is highly accurate (>99.9%) and that the diverged sequence corresponds to copy-number-variable paralogs that are absent from the human reference genome. Our method can be applied to other complex genomes to resolve the last gene-rich gaps, improve duplicate gene annotation, and better understand copy-number-variant genetic diversity at the base-pair level.


April 21, 2020  |  

Single-Molecule Sequencing: Towards Clinical Applications.

In the past several years, single-molecule sequencing platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being tested for clinical applications. They offer exceptionally long reads that permit direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. This includes disease-causing long repetitive elements, extreme GC content regions, and complex gene loci. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA. Here, we review how these technologies are opening up new clinical avenues that are being applied to pathogenic microorganisms and viruses, constitutional disorders, pharmacogenomics, cancer, and more.Copyright © 2018 Elsevier Ltd. All rights reserved.


April 21, 2020  |  

Characterizing the major structural variant alleles of the human genome.

In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity. Copyright © 2018 Elsevier Inc. All rights reserved.


April 21, 2020  |  

Immunogenetic factors driving formation of ultralong VH CDR3 in Bos taurus antibodies.

The antibody repertoire of Bos taurus is characterized by a subset of variable heavy (VH) chain regions with ultralong third complementarity determining regions (CDR3) which, compared to other species, can provide a potent response to challenging antigens like HIV env. These unusual CDR3 can range to over seventy highly diverse amino acids in length and form unique ß-ribbon ‘stalk’ and disulfide bonded ‘knob’ structures, far from the typical antigen binding site. The genetic components and processes for forming these unusual cattle antibody VH CDR3 are not well understood. Here we analyze sequences of Bos taurus antibody VH domains and find that the subset with ultralong CDR3 exclusively uses a single variable gene, IGHV1-7 (VHBUL) rearranged to the longest diversity gene, IGHD8-2. An eight nucleotide duplication at the 3′ end of IGHV1-7 encodes a longer V-region producing an extended F ß-strand that contributes to the stalk in a rearranged CDR3. A low amino acid variability was observed in CDR1 and CDR2, suggesting that antigen binding for this subset most likely only depends on the CDR3. Importantly a novel, potentially AID mediated, deletional diversification mechanism of the B. taurus VH ultralong CDR3 knob was discovered, in which interior codons of the IGHD8-2 region are removed while maintaining integral structural components of the knob and descending strand of the stalk in place. These deletions serve to further diversify cysteine positions, and thus disulfide bonded loops. Hence, both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual cattle VH ultralong CDR3 repertoire.


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