Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

Wei, Shan; Weiss, Zachary R; Williams, Zev

doi:10.1534/g3.118.200087

Cited by 28 publications

(19 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, cost and time efficiency of a sequencing protocol can be improved through multiplexing of more samples in a single sequencing run. 3,47 In this study, samples (Run 1 = 10, Run 2 = 5) were simultaneously multiplexed (i.e., pooled and then sequenced in one run) while maintaining IBV genotyping from data collected. Since the MinION flow cells were not exhausted, and can be washed and re-used, the AmpSeq method also has the potential for sequential multiplexing.…”

Section: Discussionmentioning

confidence: 99%

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

Butt

Erwood

Zhang

et al. 2019

Preprint

View full text Add to dashboard Cite

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of infectious bronchitis is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. While serotyping by definition requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV and it is inefficient at detecting mixed isolates. This paper describes a MinION-based AmpSeq method that genetically typed IBV from clinical samples, including samples with multiple isolates. Total RNA was extracted from fifteen tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples.AmpSeq accurately detected and genotyped both IBV lineages in three of five samples containing two IBV lineages. Additionally, one sample contained three IBV lineages, and AmpSeq accurately detected two of the three. Strain identification, including detection of different strains from the same lineage, was also possible with this AmpSeq method. The results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.Infectious bronchitis virus is an enveloped, pleomorphic gammacoronavirus with an unsegmented, single-stranded, positive-sense, 26-27.8 Kb, RNA genome that encodes the nonstructural polyprotein,1a and 1b, and several structural proteins: spike (S), envelop (E), membrane (M) and nucleocapsid (N). 38,41 In addition, two accessory genes, expressing 3a, 3b and 5a and 5b, respectively, have also been described. 6,14,38 The S protein is highly glycosylated and post-translational cleavage leads to two subunits: S1 and S2. 10,48 Besides acting as the viral attachment protein, the S1 protein is a major target of neutralizing antibodies. 7 As with many attachment proteins that are targets of virus-neutralizing antibodies, the S1 subunit is highly diverse with almost 50% of the amino acids differing among IBV serotypes. 2,21,38 Such variation leads to important biological differences between IBV serotypes and the emergence of novel variants. More than 60 serotypes of IBV have been reported, but the most common serotypes of All rights reserved. No reuse allowed without permission.Massachusetts. 16 This genetic diversity leads to emergence of new serotypes and a lack of complete cross-serotype protection by vaccines. 29 The correlation of IBV genotypes and serotypes of IBV has been reported 45 ; therefore, accurate genotypic identification o...

show abstract

Section: Discussionmentioning

confidence: 99%

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

Butt

Erwood

Zhang

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…The demultiplexed sequences from each sample were subjected to parallel blat (pBlat) alignment to human reference genome GRCh37 and uniquely mapped reads were screened by pslReps from the UCSC suite (19) using parameter -minCover=0.40, -minAli=0.80, -nearTop=0.001and -singleHit. Numbers of sequences assigned to each chromosome were subjected to a modified Zscore method for aneuploidy detection as described in our former study (16,17). Chromosomes with ≥ 25% copy number changes comparing to a normal male reference were considered not normal chromosomes, and they were eliminated in calculation of standard deviation for normal chromosomes (SD_normal) to increase the detection sensitivity for WGA PGS samples.…”

Section: Discussionmentioning

confidence: 99%

“…For each sample, ~260ng WGA amplified products were also subjected to MinION PGS assay using rapid multiplex library preparation according to our recently developed protocol ( Figure 1) (17). The full protocol is included in supplementary methods.…”

Section: Library Preparationmentioning

confidence: 99%

“…We developed a method for rapid library preparation and sequencing of short DNA fragments (cite: Genomics paper) and demonstrated that this method can provide rapid and aCGH-concordant aneuploidy detection from chorionic villus cells retrieved from products of miscarriage (16). We then developed a method to barcode and multiplex multiple samples within a single sequencing run (17). NGS technologies typically require days in order for results to become available, precluding a fresh embryo transfer when a biopsy is obtained at the blastocyst stage of development.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Rapid preimplantation genetic screening (PGS) using a handheld, nanopore-based, DNA sequencer

Wei

Weiss²,

Gaur³

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

Objective: To determine if a handheld, nanopore-based DNA sequencer can be used for rapid preimplantation genetic screening (PGS).Design: Retrospective study.Setting: Academic medical center. Patient(s):Amplified genomic DNA from euploid and aneuploid trophectoderm biopsy samples (n=9) that was also tested using traditional next generation sequencing (NGS). Intervention(s):Short-read DNA library preparation and nanopore-based sequencing using a hand-held MinION sequencer. Main outcome measure(s):Comparison of cytogenetic testing result from NGS and nanoporebased sequencing and the time required for library preparation and sequencing. Result(s):Multiplexed short-read DNA library preparation was completed in 45 minutes.Sequencing times varied from 1 to 2 hours. These times compare favorably with NGS library preparation (>3.5 hours) and sequencing (>12 hours) times. Whole-chromosome aneuploidy screening results obtained from nanopore-based sequencing were identical to those obtained using NGS. Conclusion(s): Methods for PGS of embryos have evolved from FISH to microarrays and mostrecently to NGS. Here we report the first application of nanopore-based sequencing for PGS on trophecoderm biopsy samples using a rapid multiplex short-read nanopore sequencing library preparation. Aneuploidy screening could be performed on 5 samples in one nanopore flowcell with 1 to 2 hour sequencing times. Overall, nanopore sequencing is a promising tool to perform rapid PGS assay onsite with a rapid turnover time, enabling same day testing and embryo All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/274563 doi: bioRxiv preprint first posted online Mar. 1, 2018; 4 transfer thus obviating the need for complex, large and expensive DNA sequencers or frozen embryos.

show abstract

“…Despite these positive attributes, nanopore sequencing is ill-suited for important applications, such as the profiling of microRNA 3 or cell-free DNA 4 , which require sequencing of ultra-short DNA (< 100 bp). In fact, the shortest DNA successfully sequenced on a nanopore platform to date is 434 bp 5,6 .…”

mentioning

confidence: 99%

High-Fidelity Nanopore Sequencing of Ultra-Short DNA Sequences

Wilson

Eisenstein

Soh

2019

Preprint

View full text Add to dashboard Cite

One Sentence Summary:We introduce a simple method of accurately sequencing ultra-short (<100bp) target DNA on a nanopore sequencing platform. AbstractNanopore sequencing offers a portable and affordable alternative to sequencing-by-synthesis methods but suffers from lower accuracy and cannot sequence ultra-short DNA. This puts applications such as molecular diagnostics based on the analysis of cell-free DNA or singlenucleotide variants (SNV) out of reach. To overcome these limitations, we report a nanopore-based sequencing strategy in which short target sequences are first circularized and then amplified via rolling-circle amplification to produce long stretches of concatemeric repeats. These can be sequenced on the Oxford Nanopore Technology's (ONT) MinION platform, and the resulting repeat sequences aligned to produce a highly-accurate consensus that reduces the high error-rate present in the individual repeats. Using this approach, we demonstrate for the first time the ability to obtain unbiased and accurate nanopore data for target DNA sequences of < 100 bp. Critically, this approach is sensitive enough to achieve SNV discrimination in mixtures of sequences and even enables quantitative detection of specific variants present at ratios of < 10%. Our method is simple, cost-effective, and only requires well-established processes. It therefore expands the utility of nanopore sequencing for molecular diagnostics and other applications, especially in resourcelimited settings.

show abstract

Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

Cited by 28 publications

References 23 publications

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

Rapid preimplantation genetic screening (PGS) using a handheld, nanopore-based, DNA sequencer

High-Fidelity Nanopore Sequencing of Ultra-Short DNA Sequences

Contact Info

Product

Resources

About