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Abstract
BackgroundMeasuring DNA replication dynamics with high throughput and single-molecule resolution is critical for understanding both the basic biology behind how cells replicate their DNA and how DNA replication can be used as a therapeutic target for diseases like cancer. In recent years, the detection of base analogues in Oxford Nanopore Technologies (ONT) sequencing reads has become a promising new method to supersede existing single-molecule methods such as DNA fibre analysis: ONT sequencing yields long reads with high throughput, and sequenced molecules can be mapped to the genome using standard sequence alignment software.ResultsThis paper introduces DNAscent v2, software that uses a residual neural network to achieve fast, accurate detection of the thymidine analogue BrdU with single-nucleotide resolution. DNAscent v2 also comes equipped with an autoencoder that interprets the pattern of BrdU incorporation on each ONT-sequenced molecule into replication fork direction to call the location of replication origins termination sites. DNAscent v2 surpasses previous versions of DNAscent in BrdU calling accuracy, origin calling accuracy, speed, and versatility across different experimental protocols. Unlike NanoMod, DNAscent v2 positively identifies BrdU without the need for sequencing unmodified DNA. Unlike RepNano, DNAscent v2 calls BrdU with single-nucleotide resolution and detects more origins than RepNano from the same sequencing data. DNAscent v2 is open-source and available at https://github.com/MBoemo/DNAscent.ConclusionsThis paper shows that DNAscent v2 is the new state-of-the-art in the high-throughput, single-molecule detection of replication fork dynamics. These improvements in DNAscent v2 mark an important step towards measuring DNA replication dynamics in large genomes with single-molecule resolution. Looking forward, the increase in accuracy in single-nucleotide resolution BrdU calls will also allow DNAscent v2 to branch out into other areas of genome stability research, particularly the detection of DNA repair.
Highlights
Measuring DNA replication dynamics with high throughput and single-molecule resolution is critical for understanding both the basic biology behind how cells replicate their DNA and how DNA replication can be used as a therapeutic target for diseases like cancer
DNAscent (v1 and earlier) uses a hidden Markov model to assign a likelihood of BrdU to each thymidine [9], RepNano uses a convolutional neural network to estimate the fraction of thymidines substituted for BrdU in rolling 96-bp windows [10], and NanoMod compares modified and unmodified DNA to detect base analogues [7, 8]
DNAscent v2 outperformed the previous versions of DNAscent by a wide margin in all four samples
Summary
Measuring DNA replication dynamics with high throughput and single-molecule resolution is critical for understanding both the basic biology behind how cells replicate their DNA and how DNA replication can be used as a therapeutic target for diseases like cancer. The detection of base analogues in Oxford Nanopore Technologies (ONT) sequencing reads has become a promising new method to supersede existing single-molecule methods such as DNA fibre analysis: ONT sequencing yields long reads with high throughput, and sequenced molecules can be mapped to the genome using standard sequence alignment software. The high-throughput detection of replication fork movement with single-molecule resolution is critical for understanding how a cell replicates its DNA, which is important for diseases like cancer where DNA replication is a therapeutic target [2]. Sequencing with ONT and detecting the position of these bases reveals a footprint of replication fork movement on each sequenced molecule, allowing this method to answer questions that would have been traditionally addressed with DNA fibre analysis but with higher-throughput and the ability to map each sequenced read to the genome. DNAscent (v1 and earlier) uses a hidden Markov model to assign a likelihood of BrdU to each thymidine [9], RepNano uses a convolutional neural network to estimate the fraction of thymidines substituted for BrdU in rolling 96-bp windows [10], and NanoMod compares modified and unmodified DNA to detect base analogues [7, 8]
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