Optimised multiplex amplicon sequencing for mutation identification using the MinION nanopore sequencer

Abstract

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.

Fast and Easy Nanopore Sequencing Workflow for Rapid Genetic Testing of Familial Hypercholesterolemia

“…Consistent with other studies, we assume that tandem repeats lead to indistinguishable conductance states due to their inherent nature of repeating identical bases ( Rang et al, 2018 ; Noakes et al, 2019 ). Additionally, these errors may be caused by sequence-dependent kinetics of the motor enzyme ( Craig et al, 2017 ) complicating the resolution of tandem repeat length ( Noakes et al, 2019 ; Whitford et al, 2021 ). Consequently, nanopore sequencing is reported to cause an increased frequency of homopolymer deletions ( Cretu Stancu et al, 2017 ; Whitford et al, 2021 ).…”

“…Additionally, these errors may be caused by sequence-dependent kinetics of the motor enzyme ( Craig et al, 2017 ) complicating the resolution of tandem repeat length ( Noakes et al, 2019 ; Whitford et al, 2021 ). Consequently, nanopore sequencing is reported to cause an increased frequency of homopolymer deletions ( Cretu Stancu et al, 2017 ; Whitford et al, 2021 ).…”

“…Through the use of Oxford Nanopore sequencing as a cost-effective method without the need for major system requirements and specialized laboratory equipment, our technique is an attractive easy-to-use tool for all diagnostic laboratories ( Jain et al, 2016 ; Petersen et al, 2019 ). Identification of LDLR variants by traditional Sanger sequencing can be still challenging, costly and slow, especially when multiple patient samples need to be analyzed ( Whitford et al, 2021 ). Also, other NextGen sequencing technologies for genetic diagnostics still are expensive ( Petersen et al, 2019 ).…”

Nanopore sequencing enables multigenic family reconstruction despite highly frequent PCR-induced recombination

“…Lately, PacBio long reads have successfully enabled to identify new isoforms from RNAseq data, thanks to new tools [5]; improvement of the Nanopore basecallers with either Nanopolish [6] or Nanocaller [7] made possible the use of Nanopore sequencing for identification of inter-individual single nucleotide polymorphisms (SNPs) and variations in gene copy numbers [8], and error correction enabled using Nanopore technology for reference-free transcriptome analysis [9]. Nanopore sequencing has also recently been used for multiplex amplicon sequencing, decreasing the financial cost by 200x as compared to Sanger sequencing [10]. A persistent problem for identification of new genetic variants is the non-random distribution of long read sequencing errors, these errors being more frequent in homopolymer regions (representing approximately half of sequencing errors) and in GC-rich regions [4], making it hard to discriminate true mutations from sequencing errors even when increasing the depth of sequencing coverage.…”