Dna purification kit

DNA was extracted from all bla_NDM-positive A. baumannii isolates using QIAGEN DNA purification kit (Qiagen, Valencia, CA). This was further manipulated by Nextera DNA Sample Preparation kit (Nextera, United States) for preparation of the DNA library according to the manufacturer’s recommended protocol. Sequencing was performed using the paired end 2×150bp reads sequencing technology on an Illumina MiSeq platform (Illumina Inc., San Diego, CA, United States). Reads quality was assessed using FastQC v0.11.9 (Brown et al., 2017 (link)) before trimming with Trimmomatic v0.35 to cut away remaining adaptors and low-quality reads (Bolger et al., 2014 (link)). Trimmed reads were de novo assembled using SPAdes 3.14.1 (Bankevich et al., 2012 (link)) with default parameters. The quality of genomes assembly was evaluated using QUAST v5.0.2 (Gurevich et al., 2013 (link)). Functional annotations of the draft genomes were generated during submission to the National Center for Biotechnology Information (NCBI) genome database using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP; Tatusova et al., 2016 (link)). Plasmid sequences were identified using plasmidSPAdes (Antipov et al., 2016 (link)) and Unicycler (Wick et al., 2017 (link)) for raw reads assembly and Bandage (Wick et al., 2015 (link)) for visualization of circular contigs.

All recombinant DNA manipulations were conducted according to standard procedures [24 ] and the recommendations of the enzyme manufacturer (Thermo Scientific). Plasmid and chromosomal DNA were isolated using the Qiagen Miniprep kit (Qiagen) and the Qiagen DNA purification kit (Qiagen), respectively, according to the manufacturer’s instructions.
Transformation of B. subtilis competent cells, PCR amplifications and DNA sequence analyses were performed as previously described [17 (link)]. Primers were purchased from Evrogen (Moscow, Russia). All constructions involving a PCR step were verified by DNA sequencing. Chromosomal deletion of yutF was confirmed by PCR (S1 Fig) and DNA sequencing.

Plasmids were constructed by Gibson Assembly for which primers were designed using NEBuilder (https://nebuilder.neb.com/#!/). DNA was amplified from WT genomic DNA by PCR using Advantage 2 Polymerase (#639202, Takara Bio, USA). Plasmid backbones were double digested with the necessary restriction enzymes and purified using the Qiagen DNA purification kit (#28704 × 4, QIAGEN GmbH, Germany). DNA inserts were cloned into the digested vectors using the NEBuilder Hifi DNA Assembly Mastermix (#E2621L, New England Biolabs).

DNA samples were extracted from peripheral leukocyte by phenol-chloroform method or using QIAGEN DNA purification kit (QIAGEN, CA, USA). Fluorescently-labelled PCR of the expanded repeat alleles of the ATXN1 (ataxin 1), ATXN2, ATXN3, CACNA1A (alpha 1A subunit, P/Q type voltage-dependent calcium channel: SCA6), ATXN7 and ATN1 (atrophin 1: DRPLA) genes were performed by using primer sets as previously described methods [9 (link),11 (link),25 (link)-28 (link)]. The allele sizes were then determined by running the PCR products on Beckman CEQ8800 DNA Analysis System (Beckman Coulter, CA, USA). Sizes of CAG repeat of both normal and pathological alleles of each chromosome were calculated by comparing results with the size of a normal control sample, of which the sizes were prior defined by direct sequencing. In order to avoid a false negative result caused by a very large expanded allele failing to be amplified by using regular PCR [29 (link)]. all samples whose results showed patterns of homozygous wild-type alleles of ATXN1, ATXN2 and ATXN3, were subsequently re-analyzed by performing long-range PCR using LA Taq polymerase (TaKaRa, Chiba, Japan).

Blood samples of all participants were collected and stored at −80°C until use. Genomic DNA was extracted from the whole blood using a commercial kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Patients’ DNA was purified by Qiagen DNA purification kit (Qiagen, Hilden, Germany). Genotyping was identified by MassArray method (Sequenom, USA).

After DNA extraction using the QIAGEN DNA purification kit (Qiagen, Valencia, CA) and library preparation using the Nextera DNA Sample Preparation kit (Nextera, USA), WGS was performed on an Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). Pre-assembly processing of the generated reads was carried out by FastQC (Andrews, 2010 ) for quality assessment and Trimmomatic v0.32 (Bolger et al., 2014 (link)) for the trimming of low-quality reads. De novo assembly of trimmed reads was carried out using SPAdes 3.14.1 (Bankevich et al., 2012 (link)). Post-assembly metrics were generated by QUAST v5.0.2 (Gurevich et al., 2013 (link)). Draft genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016 (link)). Plasmids were assembled from Illumina reads using PlasmidSPAdes (Antipov et al., 2016 (link)), a software for reading coverage-assisted plasmid identification. Assembly graphs (Fastg files) generated by PlasmidSPAdes were visualized on a bandage (Wick et al., 2015 (link)). Plasmid sequences were extracted from circular contigs or groups of contigs forming circular paths containing plasmid replication and/or mobilization genes. Contigs forming circular but overlapping paths were BLASTed for closest plasmids that were subsequently used for reference mapping using the short reads mapping tool BWA-MEM (Li and Durbin, 2010 (link)).

Bacterial genomic DNA was extracted using a Qiagen DNA purification kit (QIAGEN, Hilden, Germany), and the quality of the extracted DNA was evaluated using a Nanodrop™ spectrophotometer (Thermo Fisher Scientific, Massachusetts, United States) (Zheng et al., 2014 (link)). Then the genomic DNA was sent to Novogene (Beijing Novogene Bioinformatics Co., Ltd., Beijing, China) for WGS using Illumina HiSeq 4,000 (Illumina, San Diego, CA, United States) combined with the Oxford Nanopore MinION platform (Nanopore Technologies, Oxford, United Kingdom) (Xiaoliang et al., 2019 (link)). The sequencing process was up to 48 h long, and raw fast 5 files were generated using the MinKNOW software (Vijayakumar et al., 2020 (link)). De novo assembly was performed using Unicycler (Wick et al., 2017 (link)) and SPAdes genome assembler (Prjibelski et al., 2020 (link)). Finally, a complete and accurate genome assembly was generated by Pilon (Walker et al., 2014 (link)), and the quality, completeness and contiguity of it were verified using QUAST (Gurevich et al., 2013 (link)). The whole-genome sequence of DY1928 was deposited in GenBank¹ under the following accession numbers: CP090429 for the chromosome and CP090430 for the plasmid.