Modbamtools: Analysis of single-molecule epigenetic data for long-range profiling, heterogeneity, and clustering

Razaghi, Roham; Hook, Paul W.; Ou, Shujun; Schatz, Michael C.; Hansen, Kasper D.; Timp, Winston

doi:10.1101/2022.07.07.499188

Cited by 14 publications

(8 citation statements)

References 21 publications

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“…We produced phased 5-methylcytosine calls aligned to GRCh38 and T2T-CHM13 references. Initial methylation calls were produced de novo using Remora (https://github.com/nanoporetech/remora) incorporated in Guppy 6.1.2; afterward reads were aligned and phased using PEPPER-Margin-DeepVariant (Shafin et al, 2021) and annotated with modbamtools v0.4.8 (Razaghi et al, 2022) and modbam2bed v0.6.3 (https://github.com/epi2me-labs/modbam2bed). Regional haplotype methylation in gene promoters and SVs were calculated using “modbamtools calcMeth”.…”

Section: Methodsmentioning

confidence: 99%

Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation

Kolmogorov

Billingsley

Mastoras

et al. 2023

Preprint

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Long-read sequencing technologies substantially overcome the limitations of short-reads but to date have not been considered as feasible replacement at scale due to a combination of being too expensive, not scalable enough, or too error-prone. Here, we develop an efficient and scalable wet lab and computational protocol for Oxford Nanopore Technologies (ONT) long-read sequencing that seeks to provide a genuine alternative to short-reads for large-scale genomics projects. We applied our protocol to cell lines and brain tissue samples as part of a pilot project for the NIH Center for Alzheimer's and Related Dementias (CARD). Using a single PromethION flow cell, we can detect SNPs with F1-score better than Illumina short-read sequencing. Small indel calling remains to be difficult inside homopolymers and tandem repeats, but is comparable to Illumina calls elsewhere. Further, we can discover structural variants with F1-score comparable to state-of the-art methods involving Pacific Biosciences HiFi sequencing and trio information (but at a lower cost and greater throughput). Using ONT based phasing, we can then combine and phase small and structural variants at megabase scales. Our protocol also produces highly accurate, haplotype-specific methylation calls. Overall, this makes large-scale long-read sequencing projects feasible; the protocol is currently being used to sequence thousands of brain-based genomes as a part of the NIH CARD initiative. We provide the protocol and software as open-source integrated pipelines for generating phased variant calls and assemblies.

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Section: Methodsmentioning

confidence: 99%

Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation

Kolmogorov

Billingsley

Mastoras

et al. 2023

Preprint

Self Cite

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show abstract

“…Then, we used PEPPER-Margin-DeepVariant (v.0.8) 131 to call small variants (<50bp) and phase our variant calls and alignments. We then used our phased alignment, to produce haplotype-specific methylation calls using Modbamtools (v0.4.8) 132 and Nanopore’s modbam2bed (GitHub - epi2me-labs/modbam2bed ). Lastly, structural variants (SVs) were called using Sniffles2 (v2.2) 133 with default settings.…”

Section: Methodsmentioning

confidence: 99%

iSCORE-PD: an isogenic stem cell collection to research Parkinson’s Disease

Busquets,

Li,

Syed

et al. 2024

Preprint

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Parkinson's disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer the uniique potential to advance our understanding of PD etiology by providing disease-relevant cell-types carrying patient mutations along with isogenic control cells. To facilitate this experimental approach, we generated a collection of 55 cell lines genetically engineered to harbor mutations in genes associated with monogenic PD (SNCA A53T, SNCA A30P, PRKN Ex3del, PINK1 Q129X, DJ1/PARK7 Ex1-5del, LRRK2 G2019S, ATP13A2 FS, FBXO7 R498X/FS, DNAJC6 c.801 A>G+FS, SYNJ1 R258Q/FS, VPS13C A444P, VPS13C W395C, GBA1 IVS2+1). All mutations were generated in a fully characterized and sequenced female human embryonic stem cell (hESC) line (WIBR3; NIH approval number NIHhESC-10-0079) using CRISPR/Cas9 or prime editing-based approaches. We implemented rigorous quality controls, including high density genotyping to detect structural variants and confirm the genomic integrity of each cell line. This systematic approach ensures the high quality of our stem cell collection, highlights differences between conventional CRISPR/Cas9 and prime editing and provides a roadmap for how to generate gene-edited hPSCs collections at scale in an academic setting. We expect that our isogenic stem cell collection will become an accessible platform for the study of PD, which can be used by investigators to understand the molecular pathophysiology of PD in a human cellular setting.

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“…Comparisons were restricted to CpG islands with 50 or more CpG positions containing non-N bases in all 76 sample datasets, comprising 20,836 regions. Methylation for the SLC29A3 DMR was visualized with modbamtools v0.4.8 (Razaghi et al 2022).…”

Section: Methylation Analysismentioning

confidence: 99%

Nanopore sequencing of 1000 Genomes Project samples to build a comprehensive catalog of human genetic variation

Gustafson,

Gibson,

Damaraju

et al. 2024

Preprint

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Less than half of individuals with a suspected Mendelian condition receive a precise molecular diagnosis after comprehensive clinical genetic testing. Improvements in data quality and costs have heightened interest in using long-read sequencing (LRS) to streamline clinical genomic testing, but the absence of control datasets for variant filtering and prioritization has made tertiary analysis of LRS data challenging. To address this, the 1000 Genomes Project ONT Sequencing Consortium aims to generate LRS data from at least 800 of the 1000 Genomes Project samples. Our goal is to use LRS to identify a broader spectrum of variation so we may improve our understanding of normal patterns of human variation. Here, we present data from analysis of the first 100 samples, representing all 5 superpopulations and 19 subpopulations. These samples, sequenced to an average depth of coverage of 37x and sequence read N50 of 54 kbp, have high concordance with previous studies for identifying single nucleotide and indel variants outside of homopolymer regions. Using multiple structural variant (SV) callers, we identify an average of 24,543 high-confidence SVs per genome, including shared and private SVs likely to disrupt gene function as well as pathogenic expansions within disease-associated repeats that were not detected using short reads. Evaluation of methylation signatures revealed expected patterns at known imprinted loci, samples with skewed X-inactivation patterns, and novel differentially methylated regions. All raw sequencing data, processed data, and summary statistics are publicly available, providing a valuable resource for the clinical genetics community to discover pathogenic SVs.

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Modbamtools: Analysis of single-molecule epigenetic data for long-range profiling, heterogeneity, and clustering

Cited by 14 publications

References 21 publications

Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation

Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation

iSCORE-PD: an isogenic stem cell collection to research Parkinson’s Disease

Nanopore sequencing of 1000 Genomes Project samples to build a comprehensive catalog of human genetic variation

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