Forensic tri-allelic SNP genotyping using nanopore sequencing

Cornelis, Senne; Gansemans, Yannick; Plaetsen, Ann-Sophie Vander; Weymaere, Jana; Willems, Sander; Deforce, Dieter; Nieuwerburgh, Filip Van

doi:10.1016/j.fsigen.2018.11.012

Cited by 38 publications

(24 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The performance of the software used for base calling and further downstream analyses is also crucial, and is the subject of continuous improvements. The platform already evolved in such a way that the MinION sequencer is now capable of correctly genotyping forensic SNP loci [14,15]. The first attempts to assess nanopore sequencing for STR-profiling have been reported recently [16,17].…”

Section: Introductionmentioning

confidence: 99%

Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling

Tytgat

Gansemans

Weymaere

et al. 2020

Genes

Self Cite

View full text Add to dashboard Cite

Nanopore sequencing for forensic short tandem repeats (STR) genotyping comes with the advantages associated with massively parallel sequencing (MPS) without the need for a high up-front device cost, but genotyping is inaccurate, partially due to the occurrence of homopolymers in STR loci. The goal of this study was to apply the latest progress in nanopore sequencing by Oxford Nanopore Technologies in the field of STR genotyping. The experiments were performed using the state of the art R9.4 flow cell and the most recent R10 flow cell, which was specifically designed to improve consensus accuracy of homopolymers. Two single-contributor samples and one mixture sample were genotyped using Illumina sequencing, Nanopore R9.4 sequencing, and Nanopore R10 sequencing. The accuracy of genotyping was comparable for both types of flow cells, although the R10 flow cell provided improved data quality for loci characterized by the presence of homopolymers. We identify locus-dependent characteristics hindering accurate STR genotyping, providing insights for the design of a panel of STR loci suited for nanopore sequencing. Repeat number, the number of different reference alleles for the locus, repeat pattern complexity, flanking region complexity, and the presence of homopolymers are identified as unfavorable locus characteristics. For single-contributor samples and for a limited set of the commonly used STR loci, nanopore sequencing could be applied. However, the technology is not mature enough yet for implementation in routine forensic workflows.

show abstract

Section: Introductionmentioning

confidence: 99%

Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling

Tytgat

Gansemans

Weymaere

et al. 2020

Genes

Self Cite

View full text Add to dashboard Cite

show abstract

“…A large proportion of long MinION reads represented self-chimeras that were not recognized by the chimera filtering software. This issue was common on PacBio RSII instrument (Tedersoo et al,77 2018), but it was not observed in the current Sequel runs. Since MinION reads are typically mapped to reference, 78…”

Section: Discussion 54mentioning

confidence: 84%

Relative performance of Oxford Nanopore MinION vs. Pacific Biosciences Sequel third-generation sequencing platforms in identification of agricultural and forest pathogens

Loit¹,

Adamson

Bahram

et al. 2019

Preprint

View full text Add to dashboard Cite

Culture-based molecular characterization methods have revolutionized detection of pathogens, yet 22 these methods are either slow or imprecise. The second-generation sequencing tools have much improved 23 precision and sensitivity of detection, but the analysis processes are costly and take several days. Of third-24 generation techniques, the portable Oxford Nanopore MinION device has received much attention because of its 25 small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performance of two 26 third-generation sequencing instruments, MinION and Pacific Biosciences Sequel in identification and 27 diagnostics of pathogens from conifer needles and potato leaves and tubers. We demonstrate that Sequel is 28 efficient in metabarcoding of complex samples, whereas MinION is not suited for this purpose due to the high 29 error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification 30 of dominant pathogenic organisms from plant tissues following both amplicon-based and metagenomics-based 31 approaches. Using the PCR-free approach with shortened extraction and incubation times, we performed the 32 entire MinION workflow from sample preparation through DNA extraction, sequencing, bioinformatics and 33 interpretation in two and half hours. We advocate the use of MinION for rapid diagnostics of pathogens, but care 34 needs to be taken to control or account for all potential technical biases. 35 36 IMPORTANCE We develop new and rapid protocols for MinION-based third-generation diagnostics of plant 37 pathogens that greatly improves the speed and precision of diagnostics. Due to high error rate and technical biases 38 in MinION, PacBio Sequel platform is more useful for amplicon-based metabarcoding from complex biological 39 samples. 40 41

show abstract

“… As the MinION flow cells possessed suboptimal number of active pores, the sequencing time might be overestimated. Nanopore sequencing accuracy might be improved with ‘1D 2 ′ chemistry, at the expense of lower sequencing depth [ 30 ]. Nanopolish could not improve the detection of minor variants, which might be caused by high error rate of Nanopore raw data.…”

Section: Discussionmentioning

confidence: 99%

Rapid and economical drug resistance profiling with Nanopore MinION for clinical specimens with low bacillary burden of Mycobacterium tuberculosis

Chan

Chung

et al. 2020

BMC Res Notes

View full text Add to dashboard Cite

Objective We designed and tested a Nanopore sequencing panel for direct tuberculosis drug resistance profiling. The panel targeted 10 resistance-associated loci. We assessed the feasibility of amplifying and sequencing these loci from 23 clinical specimens with low bacillary burden. Results At least 8 loci were successfully amplified from the majority for predicting first- and second-line drug resistance (14/23, 60.87%), and the 12 specimens yielding all 10 targets were sequenced with Nanopore MinION and Illumina MiSeq. MinION sequencing data was corrected by Nanopolish and recurrent variants were filtered. A total of 67,082 bases across all consensus sequences were analyzed, with 67,019 bases called by both MinION and MiSeq as wildtype. For the 41 single nucleotide variants (SNVs) called by MiSeq with 100% variant allelic frequency (VAF), 39 (95.1%) were called by MinION. For the 22 mixed bases called by MiSeq, a SNV with the highest VAF (70%) was called by MinION. With short assay time, reasonable reagent cost as well as continuously improving sequencing chemistry and signal correction pipelines, this Nanopore method can be a viable option for direct tuberculosis drug resistance profiling in the near future.

show abstract

Forensic tri-allelic SNP genotyping using nanopore sequencing

Cited by 38 publications

References 32 publications

Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling

Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling

Relative performance of Oxford Nanopore MinION vs. Pacific Biosciences Sequel third-generation sequencing platforms in identification of agricultural and forest pathogens

Rapid and economical drug resistance profiling with Nanopore MinION for clinical specimens with low bacillary burden of Mycobacterium tuberculosis

Contact Info

Product

Resources

About