Whole genome sequencing of marine organisms by Oxford Nanopore Technologies: Assessment and optimization of HMW‐DNA extraction protocols

Boughattas, Sonia; Albatesh, Dana; Al‐Khater, Albandari; Giraldes, Bruno Welter; Althani, Asma; Benslimane, Fatiha

doi:10.1002/ece3.8447

Cited by 11 publications

(13 citation statements)

References 29 publications

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“…Peripheral blood samples were collected, and high-molecular-weight genomic DNA was prepared using the SDS method followed by purification with the QIAGEN® Genomic kit (Cat#13,343, QIAGEN, Germany) according to the standard operating procedure provided by the manufacturer [ 31 ]. DNA degradation and contamination were monitored using 1% agarose gels.…”

Section: Methodsmentioning

confidence: 99%

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Nanopore sequencing for detecting reciprocal translocation carrier status in preimplantation genetic testing

Xia

Ding

et al. 2023

BMC Genomics

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Background Balanced reciprocal translocation (BRT) is one of the most common chromosomal abnormalities that causes infertility, recurrent miscarriage, and birth defects. Preimplantation genetic testing (PGT) is widely used to select euploid embryos for BRT carriers to increase the chance of a healthy live birth. Several strategies can be used to distinguish reciprocal translocation carrier embryos from those with a normal karyotype; however, these techniques are time-consuming and difficult to implement in clinical laboratories. In this study, nanopore sequencing was performed in two reciprocal translocation carriers, and the results were validated using the next-generation sequencing-based method named, “Mapping Allele with Resolved Carrier Status” (MaReCs). Results The translocation breakpoints in both reciprocal translocation carriers were accurately identified by nanopore sequencing and were in accordance with the results obtained using MaReCs. More than one euploid non-balanced translocation carrier embryo was identified in both patients. Amniocentesis results revealed normal karyotypes, consistent with the findings by MaReCs and nanopore sequencing. Conclusion Our results suggest that nanopore sequencing is a powerful strategy for accurately distinguishing non-translocation embryos from translocation carrier embryos and precisely localizing translocation breakpoints, which is essential for PGT and aids in reducing the propagation of reciprocal translocation in the population.

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Section: Methodsmentioning

confidence: 99%

“…to the standard operating procedure provided by the manufacturer [31]. DNA degradation and contamination were monitored using 1% agarose gels.…”

Section: Dna Extractionmentioning

confidence: 99%

Nanopore sequencing for detecting reciprocal translocation carrier status in preimplantation genetic testing

Xia

Ding

et al. 2023

BMC Genomics

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“…It's recommended to select fresh collected samples and avoid freeze-thaw. In addition, high molecular weight DNA extraction methods (Quick, 2019;Boughattas et al, 2021;Petersen et al, 2022;Trigodet et al, 2022) can be used to maintain longer read length, which may help to improve the enrichment effect of adaptive sequencing. This study has some limitations.…”

Section: Discussionmentioning

confidence: 99%

Application of nanopore adaptive sequencing in pathogen detection of a patient with Chlamydia psittaci infection

Lin

Dai

Zhang

et al. 2023

Front. Cell. Infect. Microbiol.

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IntroductionNanopore sequencing has been widely used in clinical metagenomic sequencing for pathogen detection with high portability and real-time sequencing. Oxford Nanopore Technologies has recently launched an adaptive sequencing function, which can enrich on-target reads through real-time alignment and eject uninteresting reads by reversing the voltage across the nanopore. Here we evaluated the utility of adaptive sequencing in clinical pathogen detection.MethodsNanopore adaptive sequencing and standard sequencing was performed on a same flow cell with a bronchoalveolar lavage fluid sample from a patient with Chlamydia psittacosis infection, and was compared with the previous mNGS results.ResultsNanopore adaptive sequencing identified 648 on-target stop receiving reads with the longest median read length(688bp), which account for 72.4% of all Chlamydia psittaci reads and 0.03% of total reads in enriched group. The read proportion matched to C. psittaci in the stop receiving group was 99.85%, which was much higher than that of the unblock (<0.01%) and fail to adapt (0.02%) groups. Nanopore adaptive sequencing generated similar data yield of C. psittaci compared with standard nanopore sequencing. The proportion of C. psittaci reads in adaptive sequencing is close to that of standard nanopore sequencing and mNGS, but generated lower genome coverage than mNGS.DiscussionNanopore adaptive sequencing can effectively identify target C. psittaci reads in real-time, but how to increase the targeted data of pathogens still needs to be further evaluated.

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“…This limited availability of genome assemblies highlights the challenges associated with obtaining high‐quality samples for omics studies of marine organisms, including the remoteness and inaccessibility of marine environments, as well as the difficulties associated with sample storage on board research vessels and transportation to the laboratory. Additionally, extracting pure and high molecular weight DNA from numerous marine organisms is a complex task, as it involves dealing with tissues of inconsistent consistency and the presence of contaminants, such as polysaccharides, polyphenols and other secondary metabolites that can hinder enzyme activity during DNA library preparation for whole‐genome sequencing (Boughattas et al., 2021; Ramakrishnan et al., 2017).…”

Section: Discussionmentioning

confidence: 99%

Comparative genomics reveals the dynamic evolutionary history of cement protein genes of barnacles from intertidal to deep‐sea hydrothermal vents

Lee,

Chan,

Kim

et al. 2023

Molecular Ecology Resources

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Thoracican barnacles are a diverse group of marine organisms for which the availability of genome assemblies is currently limited. In this study, we sequenced the genomes of two neolepadoid species (Ashinkailepas kermadecensis, Imbricaverruca yamaguchii) from hydrothermal vents, in addition to two intertidal species. Genome sizes ranged from 481 to 1054 Mb, with repetitive sequence contents of 21.2% to 50.7%. Concordance rates of orthologs and heterozygosity rates were between 82.4% and 91.7% and between 1.0% and 2.1%, respectively, indicating high genetic diversity and heterozygosity. Based on phylogenomic analyses, we revised the nomenclature of cement genes encoding cement proteins that are not homologous to any known proteins. The major cement gene, CP100A, was found in all thoracican species, including vent‐associated neolepadoids, and was hypothesised to be essential for thoracican settlement. Duplicated genes, CP100B and CP100C, were found only in balanids, suggesting potential functional redundancy or acquisition of new functions associated with the calcareous base. An ancestor of CP52 genes was duplicated dynamically among lepadids, pollicipedids with multiple copies on a single scaffold, and balanids with multiple sequential repeats of the conserved regions, but no CP52 genes were found in neolepadoids, providing insights into cement gene evolution among thoracican lineages. This study enhances our understanding of the adhesion mechanisms of thoracicans in underwater environments. The newly sequenced genomes provide opportunities for studying their evolution and ecology, shedding light on their adaptation to diverse marine environments, and contributing to our knowledge of barnacle biology with valuable genomic resources for further studies in this field.

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Whole genome sequencing of marine organisms by Oxford Nanopore Technologies: Assessment and optimization of HMW‐DNA extraction protocols

Cited by 11 publications

References 29 publications

Nanopore sequencing for detecting reciprocal translocation carrier status in preimplantation genetic testing

Nanopore sequencing for detecting reciprocal translocation carrier status in preimplantation genetic testing

Application of nanopore adaptive sequencing in pathogen detection of a patient with Chlamydia psittaci infection

Comparative genomics reveals the dynamic evolutionary history of cement protein genes of barnacles from intertidal to deep‐sea hydrothermal vents

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