scholarly journals Rapid DNA Re-Identification for Cell Line Authentication and Forensics

2017 ◽  
Author(s):  
Sophie Zaaijer ◽  
Assaf Gordon ◽  
Daniel Speyer ◽  
Robert Piccone ◽  
Yaniv Erlich
Keyword(s):  
Cell Line ◽  
Reference Data ◽  
Personal Identification ◽  
Cell Line Authentication ◽  
Identification Schemes ◽  
Human Dna ◽  
Oxford Nanopore ◽  
Hands On ◽  
Using Data ◽  
Oxford Nanopore Technologies

AbstractDNA re-identification is used for a broad range of applications, ranging from cell line authentication to crime scene sample identification. However, current re-identification schemes suffer from high latency. Here, we describe a rapid, inexpensive, and portable strategy to re-identify human DNA called MinION sketching. Using data from Oxford Nanopore Technologies’ sequencer, MinION sketching requires only 3min of sequencing and ∼91 random SNPs to identify a sample, enabling near real-time applications of DNA re-identification. This method capitalizes on the vastly growing availability of genomic reference data for individuals and cancer cell lines. Hands-on preparation of the samples can be reduced to <1 hour. This empowers the application of MinION sketching in research settings for routine cell line authentication or in forensics.Software is available at https://github.com/TeamErlich/personal-identification-pipeline

scholarly journals Rapid re-identification of human samples using portable DNA sequencing

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sophie Zaaijer ◽  
Assaf Gordon ◽  
Daniel Speyer ◽  
Robert Piccone ◽  
Simon Cornelis Groen ◽  
...  
Keyword(s):  
Cell Line ◽  
Human Cell ◽  
Reference Data ◽  
Clinical Settings ◽  
Tissue Cultures ◽  
Cell Line Authentication ◽  
Periodic Cell ◽  
Identification Schemes ◽  
Human Dna ◽  
Real Time Applications

DNA re-identification is used for a broad suite of applications, ranging from cell line authentication to forensics. However, current re-identification schemes suffer from high latency and limited access. Here, we describe a rapid, inexpensive, and portable strategy to robustly re-identify human DNA called 'MinION sketching'. MinION sketching requires as few as 3 min of sequencing and 60-300 random SNPs to re-identify a sample enabling near real-time applications of DNA re-identification. Our method capitalizes on the rapidly growing availability of genomic reference data for cell lines, tissues in biobanks, and individuals. This empowers the application of MinION sketching in research and clinical settings for periodic cell line and tissue authentication. Importantly, our method enables considerably faster and more robust cell line authentication relative to current practices and could help to minimize the amount of irreproducible research caused by mix-ups and contamination in human cell and tissue cultures.

scholarly journals Detecting DNA Methylation using the Oxford Nanopore Technologies MinION sequencer

2016 ◽  
Author(s):  
Jared T Simpson ◽  
Rachael Workman ◽  
P. C. Zuzarte ◽  
Matei David ◽  
L. J. Dursi ◽  
...  
Keyword(s):  
Methylation Status ◽  
Cpg Islands ◽  
Nanopore Sequencing ◽  
Methylated Dna ◽  
Human Dna ◽  
Oxford Nanopore ◽  
Electrolytic Current ◽  
Low Coverage ◽  
Oxford Nanopore Technologies ◽  
Unmethylated Cytosine

AbstractNanopore sequencing instruments measure the change in electric current caused by DNA transiting through the pore. In experimental and prototype nanopore sequencing devices it has been shown that the electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine. Here we quantify the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. Using synthetically methylated DNA we are able to train a hidden Markov model to distinguish 5-methylcytosine from unmethylated cytosine in DNA. We demonstrate by sequencing natural human DNA, without any special library preparation, that global patterns of methylation can be detected from low-coverage sequencing and that the methylation status of CpG islands can be reliably predicted from single MinION reads. Our trained model and prediction software is open source and freely available to the community under the MIT license.

scholarly journals DNA Thermo-Protection Facilitates Whole Genome Sequencing of Mycobacteria Direct from Clinical Samples by the Nanopore Platform

2020 ◽  
Author(s):  
Sophie George ◽  
Yifei Xu ◽  
Gillian Rodger ◽  
Marcus Morgan ◽  
Nicholas D. Sanderson ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Low Cost ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Whole Genome ◽  
Genome Coverage ◽  
Human Dna ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

ABSTRACTMycobacterium tuberculosis (MTB) is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for Oxford Nanopore Technologies (ONT) sequencing direct from MTB positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat-inactivation (99°C/30min) and enrichment for Mycobacteria DNA was achieved using an equal volume of thermo-protection buffer (4M KCl, 0.05M HEPES buffer pH7.5, 0.1% DTT). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermo-degradation, which renders it a poor template for sequencing. Initial validation employed Mycobacteria DNA (extracted or intracellular). Next, mock clinical samples (infection-negative human sputum spiked 0-105 BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat-inactivation. DNA was extracted and sequenced. Human DNA degraded faster than Mycobacteria DNA, resulting in target enrichment. Four replicate experiments each demonstrated detection at 101 BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) was achieved at 104 BCG cells/ml; >91% coverage (1x depth) at 103 BCG cells/ml. Final validation employed MTB positive clinical samples (n=20), revealed initial sample volumes ≥1ml typically yielded higher mean depth of MTB genome coverage, the overall range 0.55-81.02. A mean depth of 3 gave >96% one-fold TB genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% five-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of MTB genomes was facilitated by a low cost thermo-protection buffer.

scholarly journals Democratizing DNA Fingerprinting

2016 ◽  
Author(s):  
Sophie Zaaijer ◽  
Assaf Gordon ◽  
Robert Piccone ◽  
Daniel Speyer ◽  
Yaniv Erlich
Keyword(s):  
Dna Fingerprinting ◽  
Low Input ◽  
The Public ◽  
Ethical Implications ◽  
Direct To Consumer ◽  
Human Dna ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

AbstractWe report a rapid, inexpensive, and portable strategy to re-identify human DNA using the MinION, a miniature sequencing sensor by Oxford Nanopore Technologies. Our strategy requires only 10-30 minutes of MinION sequencing, works with low input DNA, and enables familial searches. We also show that it can re-identify individuals from Direct-to-Consumer genomic datasets that are publicly available. We discuss potential forensic applications as well as the legal and ethical implications of a democratized DNA fingerprinting strategy available to the public.

scholarly journals Integration of mobile sequencers in an academic classroom

2015 ◽  
Author(s):  
Sophie Zaaijer ◽  
Yaniv Erlich
Keyword(s):  
Dna Sequencing ◽  
Rapid Development ◽  
Lessons Learned ◽  
Educational Opportunities ◽  
Educational Resources ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Hands On ◽  
Academic Class ◽  
Oxford Nanopore Technologies

The rapid development of DNA sequencing technologies creates new educational opportunities for hands-on training. We report our experience in integrating handheld DNA sequencers (Oxford Nanopore Technologies MinION) as part of an academic class. This manuscript describes lessons learned to facilitate successful integration and provides educational resources for the benefit of the community.

scholarly journals Phylogenomic Evidence of Reinfection and Persistence of SARS-CoV-2: First Report from Colombia

Vaccines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 282
Author(s):  
Juan David Ramírez ◽  
Marina Muñoz ◽  
Nathalia Ballesteros ◽  
Luz H. Patiño ◽  
Sergio Castañeda ◽  
...  
Keyword(s):  
Viral Persistence ◽  
Transmission Dynamics ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Paired Samples ◽  
Infection Dynamics ◽  
Oxford Nanopore ◽  
Novel Variants ◽  
First Time ◽  
Oxford Nanopore Technologies

The continuing evolution of SARS-CoV-2 and the emergence of novel variants have raised concerns about possible reinfection events and potential changes in the coronavirus disease 2019 (COVID-19) transmission dynamics. Utilizing Oxford Nanopore technologies, we sequenced paired samples of three patients with positive RT-PCR results in a 1–2-month window period, and subsequent phylogenetics and genetic polymorphism analysis of these genomes was performed. Herein, we report, for the first time, genomic evidence of one case of reinfection in Colombia, exhibiting different SARS-CoV-2 lineage classifications between samples (B.1 and B.1.1.269). Furthermore, we report two cases of possible viral persistence, highlighting the importance of deepening our understanding on the evolutionary intra-host traits of this virus throughout different timeframes of disease progression. These results emphasize the relevance of genomic surveillance as a tool for understanding SARS-CoV-2 infection dynamics, and how this may translate effectively to future control and mitigations efforts, such as the national vaccination program.

scholarly journals Haemophilus influenzae Meningitis Direct Diagnosis by Metagenomic Next-Generation Sequencing: A Case Report

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 461
Author(s):  
Madjid Morsli ◽  
Quentin Kerharo ◽  
Jeremy Delerce ◽  
Pierre-Hugues Roche ◽  
Lucas Troude ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Haemophilus Influenzae ◽  
Real Time ◽  
Real Time Pcr ◽  
Point Of Care ◽  
Next Generation ◽  
Oxford Nanopore ◽  
Sequence Encoding ◽  
Oxford Nanopore Technologies ◽  
Generation Sequencing

Current routine real-time PCR methods used for the point-of-care diagnosis of infectious meningitis do not allow for one-shot genotyping of the pathogen, as in the case of deadly Haemophilus influenzae meningitis. Real-time PCR diagnosed H. influenzae meningitis in a 22-year-old male patient, during his hospitalisation following a more than six-metre fall. Using an Oxford Nanopore Technologies real-time sequencing run in parallel to real-time PCR, we detected the H. influenzae genome directly from the cerebrospinal fluid sample in six hours. Furthermore, BLAST analysis of the sequence encoding for a partial DUF417 domain-containing protein diagnosed a non-b serotype, non-typeable H.influenzae belonging to lineage H. influenzae 22.1-21. The Oxford Nanopore metagenomic next-generation sequencing approach could be considered for the point-of-care diagnosis of infectious meningitis, by direct identification of pathogenic genomes and their genotypes/serotypes.

scholarly journals Genome and transcriptome of a pathogenic yeast, Candida nivariensis

2021 ◽  
Author(s):  
Yunfan Fan ◽  
Andrew N Gale ◽  
Anna Bailey ◽  
Kali Barnes ◽  
Kiersten Colotti ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Glabrata ◽  
Nanopore Sequencing ◽  
Cdna Sequencing ◽  
Pathogenic Yeast ◽  
Mitochondrial Sequence ◽  
Dna And Rna ◽  
Oxford Nanopore ◽  
Candida Nivariensis ◽  
Oxford Nanopore Technologies

Abstract We present a highly contiguous genome and transcriptome of the pathogenic yeast, Candida nivariensis. We sequenced both the DNA and RNA of this species using both the Oxford Nanopore Technologies (ONT) and Illumina platforms. We assembled the genome into an 11.8 Mb draft composed of 16 contigs with an N50 of 886 Kb, including a circular mitochondrial sequence of 28 Kb. Using direct RNA nanopore sequencing and Illumina cDNA sequencing, we constructed an annotation of our new assembly, supplemented by lifting over genes from Saccharomyces cerevisiae and Candida glabrata.

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