Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort

Cheung, Warren A.; Johnson, Adam F.; Rowell, William J.; Farrow, Emily; Hall, Richard; Cohen, Ana S A; Means, John C.; Zion, Tricia; Portik, Daniel M.; Saunders, Christopher T.; Koseva, Boryana; Bi, Chengpeng; Truong, Tina; Schwendinger-Schreck, Carl; Yoo, Byunggil; Johnston, Jeff; Gibson, Margaret; Evrony, Gilad D.; Rizzo, William B.; Thiffault, Isabelle; Younger, Scott T.; Wenger, Aaron M.; Grundberg, Elin; Pastinen, Tomi

doi:10.1038/s41467-023-38782-1

Cited by 16 publications

(6 citation statements)

References 43 publications

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“…We generated or analyzed data from testis tissue of chimpanzee, gorilla, bonobo, Sumatran and Bornean orangutan (Makova et al 2023) and from pooled human fetal brain tissue (Supplementary Table S3). Additionally, we analyzed a very deep pool of ∼500 million human FLNC recently generated from induced pluripotent stem cells (iPSCs) (Cheung et al 2023). We mapped FLNC reads to both haplotypes of the respective species of origin genome assemblies, allowing only high-quality mappings, and tracking all best map assignments versus multiple mappings among the paralogous copies for each species (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Sequencing and assembly data are available in NCBI under the following BioProject names: owl monkey: PRJNA941350; macaque: PRJNA877605; marmoset: PRJNA941358; gorilla: PRJNA916732 and PRJNA916733; bonobo: PRJNA916735 and PRJNA916734; chimpanzee: PRJNA916736 and PRJNA916737. Human iPSC Iso-Seq libraries were generated in Cheung et al 2023 and made available in the dbGAP database under accession code phs002206.v4.p1. Fetal brain tissue sequenced is from BioSample SAMN09459150, and sequencing data made available under SRA SUB14282898.…”

Section: Data Accessmentioning

confidence: 99%

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Independent expansion, selection and hypervariability of theTBC1D3gene family in humans

Guitart,

Porubsky,

Yoo

et al. 2024

Preprint

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TBC1D3 is a primate-specific gene family that has expanded in the human lineage and has been implicated in neuronal progenitor proliferation and expansion of the frontal cortex. The gene family and its expression have been challenging to investigate because it is embedded in high identity and highly variable segmental duplications. We sequenced and assembled the gene family using long-read sequencing data from 34 humans and 11 nonhuman primate species. Our analysis shows that this particular gene family has independently duplicated in at least five primate lineages, and the duplicated loci are enriched at sites of large-scale chromosomal rearrangements on chromosome 17. We find that most humans vary along two TBC1D3 clusters where human haplotypes are highly variable in copy number, differing by as many as 20 copies, and structure (structural heterozygosity 90%). We also show evidence of positive selection, as well as a significant change in the predicted human TBC1D3 protein sequence. Lastly, we find that, despite multiple duplications, human TBC1D3 expression is limited to a subset of copies and, most notably, from a single paralog group: TBC1D3-CDKL. These observations may help explain why a gene potentially important in cortical development can be so variable in the human population.

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Section: Resultsmentioning

confidence: 99%

Section: Data Accessmentioning

confidence: 99%

Independent expansion, selection and hypervariability of theTBC1D3gene family in humans

Guitart,

Porubsky,

Yoo

et al. 2024

Preprint

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“…Recent work has shown that long-read sequencing can haplotype-resolve epigenetic variation ( e.g. CpG methylation, protein-DNA interactions) in complex genomic regions [60,61]. To assess the potential for long-read epigenetic assays to characterize IGH in a haplotype-resolved manner through use of an IGH-personalized reference, we determined the mappability of k-mers ranging from 1,000 - 25,000 bp for NA12878 and NA19240 ( Figure 5 ).…”

Section: Resultsmentioning

confidence: 99%

Addressing technical pitfalls in pursuit of molecular factors that mediate immunoglobulin gene regulation

Engelbrecht,

Rodriguez,

Watson

2024

Preprint

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The expressed antibody repertoire is a critical determinant of immune-related phenotypes. Antibody-encoding transcripts are distinct from other expressed genes because they are transcribed from somatically rearranged gene segments. Human antibodies are composed of two identical heavy and light chain polypeptides derived from genes in the immunoglobulin heavy chain (IGH) locus and one of two light chain loci. The combinatorial diversity that results from antibody gene rearrangement and the pairing of different heavy and light chains contributes to the immense diversity of the baseline antibody repertoire. During rearrangement, antibody gene selection is mediated by factors that influence chromatin architecture, promoter/enhancer activity, and V(D)J recombination. Interindividual variation in the composition of the antibody repertoire associates with germline variation in IGH, implicating polymorphism in antibody gene regulation. Determining how IGH variants directly mediate gene regulation will require integration of these variants with other functional genomic datasets. Here, we argue that standard approaches using short reads have limited utility for characterizing regulatory regions in IGH at haplotype-resolution. Using simulated and ChIP-seq reads, we define features of IGH that limit use of short reads and a single reference genome, namely 1) the highly duplicated nature of DNA sequence in IGH and 2) structural polymorphisms that are frequent in the population. We demonstrate that personalized diploid references enhance performance of short-read data for characterizing mappable portions of the locus, while also showing that long-read profiling tools will ultimately be needed to fully resolve functional impacts of IGH germline variation on expressed antibody repertoires.

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“…We also confirm the properties of SVs linking to QTLs impacting at greater distances and at a higher frequency. Hypermethylation in regulatory elements canonically leads to loss of activity, which we previously exploited in rare variant characterization of long-read sequences (Cheung et al 2023). The hypermethylating impact we observed for leading SV-mQTLs suggests an important role for studying SVs in gene silencing.…”

Section: Discussionmentioning

confidence: 99%

“…Expanding the non-reference results to a larger number of human genomes and epigenomes can expose population variation with potential new insights on trait variation and disease. The ability to survey epigenomes was recently augmented by long-read technologies that simultaneously characterize the sequence of personal genomes, resolving polymorphic SVs, together with their epigenomic status (Yue et al 2022; Cheung et al 2023; Sigurpalsdottir et al 2024).…”

Section: Introductionmentioning

confidence: 99%

Expanded methylome and quantitative trait loci detection by long-read profiling of personal DNA

Groza,

Ge,

Cheung

et al. 2024

Preprint

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Structural variants (SVs) are omnipresent in human DNA, yet their genotype and methylation status is rarely characterized due to previous limitations in genome assembly and detection of modified nucleotides. Because of this, the extent to which these regions act as quantitative-trait loci is also largely unknown.Here, we generated a pangenome graph summarizing the SVs in 782 de novo assembled genomes obtained from the Genomic Answers for Kids rare disease cohort, that captures 14.6 million CpGs in DNA segments that are absent from the CHM13v2 assembly (SV-CpGs), expanding their number by 43.6%. Next, using 435 methylomes from the same samples, we genotyped a total of 7.99 million SV-CpGs, of which 5.18 million (64.8%) were found to be methylated (SV-5mCpGs) in at least one sample.To understand the provenance and impact of these novel SV-CpGs, we noted that non-repeat sequences were the leading contributor of SV-CpGs (3.3 × 106), followed by centromeric satellites (1.58 × 106), simple repeats (1.19 × 106), Alus (0.67 × 106), satellites (0.39 × 106), L1s (0.27 × 106), and SVAs (0.19 × 106). Meanwhile, the methylation rate of SV-CpGs was the highest in repeat sequences. Moreover, in contrast to Alus and L1s, centromeric satellites, simple repeats and SVA sequences were overrepresented in SV-5mCpGs compared to reference CpGs. Similarly, we established that non-reference CpGs were more than twice (37% vs. 15%) as likely to be variable, showing intermediate methylation levels in the population.Lastly, to explore if SVs detected in this pangenome are potentially causal for functional variation in population we measured methylation quantitative trait loci (SV-mQTLs) using CHM13v2 as a backbone. This revealed over 230,464 methylation bins within 100 kbp of a common SV (>5% MAF) showing significant association (at 5% FDR) with methylation variation. Finally, we assessed how many of these SVs-mQTLs were the leading QTL variant compared to SNVs and identified 65,659 methylation bins (28.5%) where the leading variant was an SV.In conclusion, our results demonstrate that graph genome references providing full SV structures in combination with the associated methylation variation reveal tens-of-thousands of QTLs that are more accurately mapped in personal genomes, underscoring the importance of assembly-based analyses of human traits.

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Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort

Cited by 16 publications

References 43 publications

Independent expansion, selection and hypervariability of theTBC1D3gene family in humans

Independent expansion, selection and hypervariability of theTBC1D3gene family in humans

Addressing technical pitfalls in pursuit of molecular factors that mediate immunoglobulin gene regulation

Expanded methylome and quantitative trait loci detection by long-read profiling of personal DNA

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