Targeted resequencing using the MinION long read sequencing platform
Authors: McBride Martin W , Ellis Harriet , Fisher Simon , Asirvatham Amrita , Jones Marc 
Published: June 2021

MinION nanopore DNA sequencing is an emerging paradigm in portable, long read, economic sequencing technology. Using a targeted resequencing approach of the rat osteopontin (Spp1) region in the stroke‐prone spontaneously hypertensive (SHRSPGla) and Wistar Kyoto (WKYGla) rats. We aim to develop and validate a variant calling pipeline based on long reads produced by the MinION platform and to corroborate with published SHRSPGla and WKYGla promotor variants using the Rat Genome Database (RGD) (Rnor6.0).Using the Phusion high fidelity DNA polymerase, seven PCR amplifications of defined length were amplified across a 15kb region of the rat Spp1 promoter and gene from liver samples from two individual SHRSPGla and WKYGla animals. The library for MinION sequencing was prepared using Oxford Nanopore Ligation Sequencing Kit and Native Barcoding Expansion 1–12. Purified PCR amplicons were repaired and end‐prepped with dA‐tailing. Barcodes and adapters were ligated to the prepared DNA fragments before loading to the MinION flow cell for sequencing. Two Oxford Nanopore SpotON Flow cells were primed and duplicate prepared samples were each loaded onto flow cells on two separate MinION. The MinION raw data was analysed in real time during sequencing by cloud‐based EPI2ME software (Metrichor). This enabled base calling of raw data to produce DNA sequence from output measurements of current, converting raw fast5 files to base called fastq format. Reads were separated by barcode to identify reads by strain and animal. Quality control metrics for each sample were obtained from the BAM files produced by Samtools Mpileup performed on the merged MinION fastq files mapping to the Spp1 region (Chromosome 14, 6,675,281–6,690,258). A BWA/Bcftools pipeline was used to call variants (Phred>20). Variant calls from the SHRSPGla/WKYGla strains using our MinION pipeline were cross‐referenced with previously published Illumina BWA/GATK calls on RGD for this region (Rnor6.0).The MinION platform produced a mean read count of 1.49×104(± 6.21×103) per sample, with a mean mapping percentage of 49.9% (±10.1). The mean read length was 1130 bases (±132) with a mean quality of base calls of 12.83 (±0.16). Mean depth of coverage of each individual sample was 610x(±180). RGD describes 38 variants in this region of the osteopontin promotor. Across two sequencing runs, 27 of the 38 variants listed on the Rat Genome Database were called and identified several potential novel variants in the region.In conclusion, we confirm the MinION platform can be used as a targeted resequencing platform and variant calls can be made from the long reads produced, although read accuracy is dependent on read coverage.Support or Funding InformationBritish Heart Foundation and Stratified Medicine Scotland Innovation Centre is supported by the Scottish Funding Council, Scottish Enterprise and the Chief Scientist Office of ScotlandThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.