nanopore sequencing of MEFV gene: F24
Genomic DNA from 200 L blood was isolated using QIAamp DNA Blood Mini Kit (QIAGEN, MD, USA), according to the manufacturers instructions. Five pairs of multiplex primers each targeting approximately 4 kb region of the MEFV gene were selected using the primal scheme tool for multiplex primer selection  (Quick et al., 2017). Amplicons were generated using Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, USA), LongAmp Hot Start Taq 2X Master Mix (New England Biolabs, USA), and BioMaster LR HS-PCR (2x) Mix (Biolabmix, Russia). Sequencing libraries were prepared according to the manufacturers (Oxford Nanopore Technologies, Oxford, UK) protocol 1) using native barcoding kits (EXP-NBD104, EXP-NBD114) in combination with a ligation sequencing kit (SQK-LSK109) or 2) Native Barcoding Kit 24 V14 (SQK-NBD114.24). The final sequencing library amount was 20-50 fmol. Raw sequencing data was basecalled and filtered with guppy basecaller (version 6.0.1) with a high-accuracy model. Basecalled data was then demultiplexed for downstream analysis. The alignment was done with a minimap2 aligner (version 2.24-r1122) the ONT genomic reads option on the reference sequence of the MEFV gene (accession: NG_007871) downloaded from the RefSeq (LRG_190).
SUB14864225
F24_MEFV_R09