Homopolish: a method for the removal of systematic errors in nanopore sequencing by homologous polishing

Huang, Yao‐Ting; Liu, Po‐Yu; Shih, Pei-Wen

doi:10.1186/s13059-021-02282-6

Cited by 106 publications

(97 citation statements)

References 22 publications

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“…The developers of Homopolish tested the software’s polishing ability against bacterial genomes ( Enterococcus faecalis , Pseudomonas aeruginosa , Salmonella enterica , et al), a viral genome ( Lambda phage ), and a fungal genome ( Saccharomyces cerevisiae ). When combined with Medaka/HELEN, genome quality can exceed Q50 on R9.4 flow cells, achieving similar precision to that of hybrid assemblies with Unicycler [ 9 ]. Results showed that ONT-only sequencing could produce sufficiently high-quality microbial genomes for downstream analysis.…”

Section: Discussionmentioning

confidence: 99%

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Comparison of De Novo Assembly Strategies for Bacterial Genomes

Zhang

Jiang

Wang

et al. 2021

IJMS

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(1) Background: Short-read sequencing allows for the rapid and accurate analysis of the whole bacterial genome but does not usually enable complete genome assembly. Long-read sequencing greatly assists with the resolution of complex bacterial genomes, particularly when combined with short-read Illumina data. However, it is not clear how different assembly strategies affect genomic accuracy, completeness, and protein prediction. (2) Methods: we compare different assembly strategies for Haemophilus parasuis, which causes Glässer’s disease, characterized by fibrinous polyserositis and arthritis, in swine by using Illumina sequencing and long reads from the sequencing platforms of either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio). (3) Results: Assembly with either PacBio or ONT reads, followed by polishing with Illumina reads, facilitated high-quality genome reconstruction and was superior to the long-read-only assembly and hybrid-assembly strategies when evaluated in terms of accuracy and completeness. An equally excellent method was correction with Homopolish after the ONT-only assembly, which had the advantage of avoiding hybrid sequencing with Illumina. Furthermore, by aligning transcripts to assembled genomes and their predicted CDSs, the sequencing errors of the ONT assembly were mainly indels that were generated when homopolymer regions were sequenced, thus critically affecting protein prediction. Polishing can fill indels and correct mistakes. (4) Conclusions: The assembly of bacterial genomes can be directly achieved by using long-read sequencing techniques. To maximize assembly accuracy, it is essential to polish the assembly with homologous sequences of related genomes or sequencing data from short-read technology.

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Section: Discussionmentioning

confidence: 99%

“…The ONT assembly genome was polished using Illumina reads with Pilon and ONT reads with Medaka (v1.0.1 (accessed on 15 October 2020)). Then, Homopolish (v0.2.1 (accessed on 15 May 2020)) [ 9 ] was used to remove systematic indel errors from the genome polished by Medaka.…”

Section: Methodsmentioning

confidence: 99%

Comparison of De Novo Assembly Strategies for Bacterial Genomes

Zhang

Jiang

Wang

et al. 2021

IJMS

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“…Current read-based error correction and polishing methods for ONT data primarily target genome assembly 17,18 . Raw read polishing has proven to be extremely effective in generating high quality assemblies, however, information supporting low frequency (less than 0.5) intra-host variants is almost always lost during the process.…”

Section: Main Textmentioning

confidence: 99%

Rescuing Low Frequency Variants within Intra-Host Viral Populations directly from Oxford Nanopore sequencing data

Liu

Kearney

Mahmoud

et al. 2021

Preprint

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Infectious disease monitoring on Oxford Nanopore Technologies (ONT) platforms offers rapid turnaround times and low cost, exemplified by well over a half of million ONT SARS-COV-2 datasets. Tracking low frequency intra-host variants has provided important insights with respect to elucidating within host viral population dynamics and transmission. However, given the higher error rate of ONT, accurate identification of intra-host variants with low allele frequencies remains an open challenge with no viable solutions available. In response to this need, we present Variabel, a novel approach and first method designed for rescuing low frequency intra-host variants from ONT data alone. We evaluated Variabel on both within patient and across patient paired Illumina and ONT datasets; our results show that Variabel can accurately identify low frequency variants below 0.5 allele frequency, outperforming existing state-of-the-art ONT variant callers for this task. Variabel is open-source and available for download at: www.gitlab.com/treangenlab/variabel.

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“…Next, the filtered reads were assembled using Flye v2.8.3 ( 26 ), with five iterations of the polishing step (–iterations 5), generating a single circular draft assembly. The resulting assembly was aligned using bwa v0.7.17 ( https://github.com/lh3/bwa ) and polished sequentially using Racon v1.4.22 (-m 8 -x -6 -g -8 -w 500) ( 27 ), Medaka v1.4.3 ( https://github.com/nanoporetech/medaka ) (-m r941_min_hac_g507), and Homopolish v0.2.3 (-s bacteria.msh -m R9.4.pkl) ( 28 ). The quality of the genome assembly was evaluated using QUAST v5.1.0 ( 29 ).…”

Section: Announcementmentioning

confidence: 99%