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DNA copy number measurement of genome replication dynamics by high-throughput sequencing – the sort-seq, sync-seq and MFA-seq family

ABSTRACT: DNA copy number measurement of genome replication dynamics by high-throughput sequencing – the sort-seq, sync-seq and MFA-seq family

PROVIDER: PRJNA557675 | ENA |

REPOSITORIES: ENA

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DNA copy number measurement of genome replication dynamics by high-throughput sequencing – the sort-seq, sync-seq and MFA-seq family

Project description:Genome replication follows a defined temporal programme that can change during cellular differentiation and disease onset. DNA replication results in an increase in DNA copy number that can be measured by high-throughput sequencing (HTS). Here we present a protocol to determine genome replication dynamics using DNA copy number measurements. Cell populations can be obtained in three variants of the method. First, sort-seq uses fluorescence activated cell sorting (FACS) to isolate replicating and non-replicating subpopulations from asynchronous cells. Second, sync-seq uses cell synchronisation to obtain samples before and during DNA replication. Third, marker frequency analysis (MFA-seq) assesses copy number differences in rapidly replicating asynchronous cells. These approaches have been used to reveal genome replication dynamics in prokaryotes, archaea, and a wide range of eukaryotes, including mammalian cells. The whole protocol can be performed in 7-10 days.

2019-08-01 | GSE135178 | GEO

SORT-seq of Synovial Tissue Dentritic Cells from Rheumatoid arthritis

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Single-cell measurement of plasmid copy number and promoter activity.

Project description:Accurate measurements of promoter activities are crucial for predictably building genetic systems. Here we report a method to simultaneously count plasmid DNA, RNA transcripts, and protein expression in single living bacteria. From these data, the activity of a promoter in units of RNAP/s can be inferred. This work facilitates the reporting of promoters in absolute units, the variability in their activity across a population, and their quantitative toll on cellular resources, all of which provide critical insights for cellular engineering.

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Project description:We present SMURF-seq, a protocol to efficiently sequence short DNA molecules on a long-read sequencer by randomly ligating them to form long molecules. Applying SMURF-seq using the Oxford Nanopore MinION yields up to 30 fragments per read, providing an average of 6.2 and up to 7.5 million mappable fragments per run, increasing information throughput for read-counting applications. We apply SMURF-seq on the MinION to generate copy number profiles. A comparison with profiles from Illumina sequencing reveals that SMURF-seq attains similar accuracy. More broadly, SMURF-seq expands the utility of long-read sequencers for read-counting applications.

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Project description:The copy number variation in beta-defensin genes on human chromosome 8 has been proposed to underlie susceptibility to inflammatory disorders, but presents considerable challenges for accurate typing on the scale required for adequately powered case-control studies. In this work, we have used accurate methods of copy number typing based on the paralogue ratio test (PRT) to assess beta-defensin copy number in more than 1500 UK DNA samples including more than 1000 cases of Crohn's disease. A subset of 625 samples was typed using both PRT-based methods and standard real-time PCR methods, from which direct comparisons highlight potentially serious shortcomings of a real-time PCR assay for typing this variant. Comparing our PRT-based results with two previous studies based only on real-time PCR, we find no evidence to support the reported association of Crohn's disease with either low or high beta-defensin copy number; furthermore, it is noteworthy that there are disagreements between different studies on the observed frequency distribution of copy number states among European controls. We suggest safeguards to be adopted in assessing and reporting the accuracy of copy number measurement, with particular emphasis on integer clustering of results, to avoid reporting of spurious associations in future case-control studies.

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