Rapid, cost-effective identification of genetic variants in small candidate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. The application of Oxford Nanopore Technologies’ MinION sequencer has the potential to fulfil this requirement. We demonstrate a proof of concept for a multiplexing assay that pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals, which could be applied to gene-targeted diagnostics. A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons. The amplicons are family-specific, spanning a total of 30 loci in DNA isolated from 82 human neurodevelopmental cases and family members. The target regions were chosen for further interrogation because a potentially disease-causative variant had been identified in affected individuals following Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the minimap2 aligner software. Our multiplexing approach produced an interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interestingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (127×–19,626×; mean = 8321×, std err = 452.99) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 130 × depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory with a potential cost as much 200 × less than that from Sanger sequencing.
Migration is a fundamental aspect of leukocyte behavior and represents a significant therapeutic target clinically. However, most migration assays used in research are relatively low throughput and not easily compatible with rapid analysis or high-throughput screening (HTS) protocols required for drug screening assays. We therefore investigated the quantification of the migration of human leukocytes using the Molecular Devices high-content Discovery-1 platform or PerkinElmer ATPlite assay compared to manual counting. We have conducted extensive assay validation, investigating the detection limits, sensitivity, and precision of each method to count human leukocytes. Leukocyte migration assays were conducted using 96-well HTS-Transwell plates and the potent chemokine stromal cell-derived factor-1 (SDF-1). We reveal that the Discovery-1 and ATPlite methods developed here provide useful approaches to quantify leukocyte migration in an HTS manner with high levels of detection, sensitivity, and precision.
Objective: Rapid, cost-effective identification of genetic variants in small candiate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. Application of Oxford Nanopore Technologies' MinION sequencer has the potential to fulfil this requirement. We have developed a multiplexing assay which pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals which could be applied to gene-targeted diagnostics. Methods: A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons, spanning 30 loci in DNA isolated from 82 neurodevelopmental cases and family members. The target regions were chosen for further interegation because a potentially disease-causative variants had been identified in affected individuals by Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the guppy aligner software. Results: Our multiplexing approach produced interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interstingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (139x - 21,499x; mean = 9,050, std err = 538.21) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. Conclusion: We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 140x depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory.
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