A method to enrich and purify centromeric DNA from human cells

Gamba, Riccardo; Mazzucco, Giulia; Wilhelm, Thérèse; Chardon, Florian; Velikovsky, Leonid; Picotto, Julien; Doksani, Ylli; Fachinetti, Daniele

doi:10.1101/2021.09.24.461328

Cited by 4 publications

(4 citation statements)

References 35 publications

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“…If the sequencing cost and time for sufficient coverage becomes prohibitive, a few enrichment strategies can be used. A restriction enzyme-based approach like AlphaHOR-RES relies on preferential digestion of DNA outside of target regions followed by size selection to maintain larger on-target fragments 9,20 . The ONT Cas9 Sequencing Kit (SQC-CS9109) is another option to selectively ligate adapters to targeted regions during library preparation.…”

Section: Methodsmentioning

confidence: 99%

“…Without enrichment for regions of interest, DiMeLo-seq will sequence the whole genome uniformly, requiring deep sequencing to achieve sufficient coverage of specific target regions. However, there are options to enrich for regions of interest with DiMeLo-seq like AlphaHOR-RES 9 , other restriction-enzyme-based enrichment approaches 20 , and the Oxford Nanopore Technologies Cas9-based targeted library preparation kit (SQK-CS9109). Adaptive sampling methods could also be used, such as Readfish 21 , UNCALLED 22 , and the adaptive sampling that is built into ONT’s MinKNOW software.…”

Section: Introductionmentioning

confidence: 99%

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Mapping protein-DNA interactions with DiMeLo-seq

Maslan

Altemose

Mishra

et al. 2022

Preprint

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We recently developed Directed Methylation with Long-read sequencing (DiMeLo-seq) to map protein-DNA interactions genome wide. DiMeLo-seq maps multiple interaction sites on single DNA molecules, profiles protein binding in the context of endogenous DNA methylation, and maps protein-DNA interactions in repetitive regions of the genome that are difficult to study with short-read methods. Adenines in the vicinity of a protein of interest are methylated in situ by tethering the Hia5 methyltransferase to an antibody using protein A. Protein-DNA interactions are then detected by direct readout of adenine methylation with long-read, single-molecule, DNA sequencing platforms such as Nanopore sequencing. Here, we present a detailed protocol and guidance for performing DiMeLo-seq. This protocol can be run on nuclei from fresh, lightly fixed, or frozen cells. The protocol requires 1 day for performing in situ targeted methylation, 1-5 days for library preparation depending on desired fragment length, and 1-3 days for Nanopore sequencing depending on desired sequencing depth. The protocol requires basic molecular biology skills and equipment, as well as access to a Nanopore sequencer. We also provide a Python package, dimelo, for analysis of DiMeLo-seq data.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mapping protein-DNA interactions with DiMeLo-seq

Maslan

Altemose

Mishra

et al. 2022

Preprint

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“…Long-read sequencing has been successfully applied for the detection of copy number variants and DNA methylation of repetitive sequences, including STRs, transposable elements 18,20,28,32,34,48 , centromeres 50 , peri-centromeres, and telomeres 51,52 across a wide range of samples. Nanopore long-reads can simultaneously detect mosaic DNA methylation and somatic sequence variation 53,54 .…”

Section: Multi-modal Analysis Of Intractable Repetitive Sequences For...mentioning

confidence: 99%

MASTR-seq: Multiplexed Analysis of Short Tandem Repeats with sequencing

Su,

Chandradoss,

Malachowski

et al. 2024

Preprint

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More than 60 human disorders have been linked to unstable expansion of short tandem repeat (STR) tracts. STR length and the extent of DNA methylation is linked to disease pathology and can be mosaic in a cell type-specific manner in several repeat expansion disorders. Mosaic phenomenon have been difficult to study to date due to technical bias intrinsic to repeat sequences and the need for multi-modal measurements at single-allele resolution. Nanopore long-read sequencing accurately measures STR length and DNA methylation in the same single molecule but is cost prohibitive for studies assessing a target locus across multiple experimental conditions or patient samples. Here, we describe MASTR-seq, Multiplexed Analysis of Short Tandem Repeats, for cost-effective, high-throughput, accurate, multi-modal measurements of DNA methylation and STR genotype at single-allele resolution. MASTR-seq couples long-read sequencing, Cas9-mediated target enrichment, and PCR-free multiplexed barcoding to achieve a >ten-fold increase in on-target read mapping for 8-12 pooled samples in a single MinION flow cell. We provide a detailed experimental protocol and computational tools and present evidence that MASTR-seq quantifies tract length and DNA methylation status for CGG and CAG STR loci in normal-length and mutation-length human cell lines. The MASTR-seq protocol takes approximately eight days for experiments and one additional day for data processing and analyses.

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“…Using DiMeLo-seq one can now map CENP-A nucleosomes confidently onto homogeneously repetitive DNA sequences. Challenges of low centromeric coverage due to the low representation of centromere sequences in the genome can be overcome by restriction enzyme based digestion of non-centromeric DNA sequences and selective sequencing of centromeric DNA ( Gamba et al, 2021 ; Altemose et al, 2022b ). The next frontier in understanding centromere inheritance and maintenance at the single molecule level is to be able to track CENP-A position using DiMeLo-seq or other approaches before and after CENP-A deposition (in G1-phase) and before and after CENP-A distribution (in S-phase) on the same chromatin fiber.…”

Section: Centromeres Are Epigenetically Definedmentioning

confidence: 99%

Centromere Identity and the Regulation of Chromosome Segregation

Sundararajan

Straight

2022

Front. Cell Dev. Biol.

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Eukaryotes segregate their chromosomes during mitosis and meiosis by attaching chromosomes to the microtubules of the spindle so that they can be distributed into daughter cells. The complexity of centromeres ranges from the point centromeres of yeast that attach to a single microtubule to the more complex regional centromeres found in many metazoans or holocentric centromeres of some nematodes, arthropods and plants, that bind to dozens of microtubules per kinetochore. In vertebrates, the centromere is defined by a centromere specific histone variant termed Centromere Protein A (CENP-A) that replaces histone H3 in a subset of centromeric nucleosomes. These CENP-A nucleosomes are distributed on long stretches of highly repetitive DNA and interspersed with histone H3 containing nucleosomes. The mechanisms by which cells control the number and position of CENP-A nucleosomes is unknown but likely important for the organization of centromeric chromatin in mitosis so that the kinetochore is properly oriented for microtubule capture. CENP-A chromatin is epigenetically determined thus cells must correct errors in CENP-A organization to prevent centromere dysfunction and chromosome loss. Recent improvements in sequencing complex centromeres have paved the way for defining the organization of CENP-A nucleosomes in centromeres. Here we discuss the importance and challenges in understanding CENP-A organization and highlight new discoveries and advances enabled by recent improvements in the human genome assembly.

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A method to enrich and purify centromeric DNA from human cells

Cited by 4 publications

References 35 publications

Mapping protein-DNA interactions with DiMeLo-seq

Mapping protein-DNA interactions with DiMeLo-seq

MASTR-seq: Multiplexed Analysis of Short Tandem Repeats with sequencing

Centromere Identity and the Regulation of Chromosome Segregation

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