Minion technology

Mucispirillum schaedleri ASF 457 variant MCS was provided to Bärbel Stecher by Charles River Laboratories, Inc. (Wilmington, MA, USA). Nucleic acids were extracted using a phenol-chloroform-based extraction method (80 (link)). DNAs were sequenced using the Illumina HiSeq2000 with 3-kb mate pair libraries and MinION technology (Oxford Nanopore Technologies, Oxford, United Kingdom). Illumina data were quality filtered with prinseq-lite (81 (link)), and MinION data were filtered with poretools (82 (link)) and prinseq-lite. De novo genome assembly was performed using SPAdes (83 (link)).

Genomic DNA was isolated using the MasterpureTM Yeast DNA purification kit (Lucigen) and sequenced using MinION technology from Oxford Nanopore at VANTAGE (Vanderbilt Technologies for Advanced Genomics). Libraries from twelve samples were generated for MinION sequencing using the Rapid Barcoding Kit (RBK-004) per manufacturer’s instructions (Oxford Nanopore). Sequencing was performed on a total of 3 flow cells (type R9.4.1). Sequences were demultiplexed using porechop v0.2.4 (https://github.com/rrwick/Porechop) with default settings.
Sequence reads were analyzed with tools available at UseGalaxy.org. Briefly, each dataset was converted to a BLAST database using NCBI BLAST+ makeblastdb [58 (link),59 (link)], which allowed a list of reads matching chromosome 9 sequences centromere-proximal to the SiRTA to be generated. Iterative BLAST searches with the tool in the Saccharomyces Genome Database (SGD; https://www.yeastgenome.org) were used to pinpoint the site of the rearrangement and/or to identify sequence reads containing intact chromosomes. Breakpoints were verified by PCR. Primer sequences and additional information about each of the 12 clones can be found in supplementary data (S1 Data). All sequence reads obtained have been submitted to the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA557764.