Qiasymphony system

DNA was isolated from cell lines using the GenElute Mammalian Genomic DNA Mini-Prep Kit (Sigma-Aldrich). DNA from the TNBC tumours used in this study were previously described^{72 (link)} as was the procedure for extracting patient and control plasma DNA^{44 (link)}. DNA was extracted from plasma or serum using the QiaSymphony system (Qiagen), with the QIAmp circulating nucleic acid kit (Qiagen). All samples were bisulfite converted using the EpiTect Fast DNA Bisulfite Kit (Qiagen).

Genomic DNA was extracted from cultures of STEC O157:H7 using the QIAsymphony system (Qiagen). The sequencing library was prepared using the Nextera XP kit (Illumina) for sequencing on the HiSeq 2500 instrument (Illumina), run with the fast protocol. fastq reads were processed using Trimmomatic v0.27 [17 (link)] to remove bases with a PHRED score of <30 from the leading and trailing ends, with reads <50 bp after quality trimming discarded.

Genomic DNA was extracted from cultures of STEC O157:H7 using the QIAsymphony system (Qiagen). The sequencing library was prepared using the Nextera XP kit (Illumina) for sequencing on the HiSeq 2500 instrument (Illumina), run with the fast protocol. FASTQ reads were processed using Trimmomatic v0.27 (Bolger et al., 2014 (link)) to remove bases with a PHRED score of <30 from the leading and trailing ends, with reads <50 bp after quality trimming discarded.

DNA was purified from EDTA blood using the QiaSymphony system (Qiagen, Hilden, Germany). Genome-wide single nucleotide polymorphism (SNP) genotyping was performed with the Genome Wide Human SNP array 50K (Affymetrix, Santa Clara, USA) and analysed using PLINK v1.07 [14 (link)]. For homozygozity mapping, we searched for any region >3 Mb, with minimum of 30 SNPs and less than four heterozygous calls. Recessive SIL1 mutations were excluded by haplotype analysis and direct sequencing of the SIL1 gene.

An observational, epidemiological case-to-case study was carried out in patients hospitalized due to severe acute influenza infection. A severe case of laboratory-confirmed influenza virus infection was defined as a case requiring hospitalization due to pneumonia, septic shock, multi-organ failure, acute respiratory distress, death or any other severe condition including ICU admission, or who developed these clinical signs during hospitalization for other reasons⁸ . The diagnosis was confirmed by RT-PCR and/or culture on nasopharyngeal swab samples.
Respiratory tract samples were processed at each hospital laboratory within 24 h of receipt. A 300 μl aliquot was taken for total nucleic acid extraction and eluted in 25 μl of RNase-free elution buffer using the automatic QIAsymphony system (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Subsequently, two specific one-step multiplex real-time PCR using Stratagene Mx3000P QPCR Systems (Agilent Technologies, Santa Clara, CA, USA) were carried out for typing A/B influenza virus (sensitivity was 10 and 103 copies/μl, respectively) and subtyping influenza A virus (sensitivity was 102, 103 and 10 copies/μl for H1, H3 and H5 RNA, respectively)^{48 (link)}. For H1 positive detections to identify H1N1pdm09, specific in-house primers were used.

In Sweden, all invasive cases of meningococcal disease, according to the European Union case definition, are mandatorily reported by clinicians to the Public Health Agency of Sweden (24 ) and the corresponding isolates are sent to the National Reference Laboratory for Neisseria meningitidis at Örebro University Hospital. The bacteria are subsequently cultured overnight on chocolate agar plates at 37°C in 5% CO₂, serogrouped by coagglutination (25 (link)), and stored at −70°C. In this study, all invasive MenW isolates collected in Sweden between 1 January 1995 and 30 June 2017 (n = 86) were subjected to whole-genome sequencing (WGS). DNA from cultured isolates was automatically extracted with the QIAsymphony system (Qiagen, Hilden, Germany) using the QIAsymphony DSP virus/pathogen kit, according to the manufacturer's instructions, with added RNase treatment and elution with Tris-HCl (pH 8).

Illumina sequencing was performed at UKHSA and followed the same protocol as described by Chattaway et al. (2017) (link). The QIAsymphony system (Qiagen) was used to extract genomic DNA from selected DEC samples. The Nextera XP kit (Illumina) was used to prepare the sequence library for sequencing on the HiSeq 2,500 instrument (Illumina), run with the fast protocol. Trimmomatic v0.27 was utilised to remove bases with a PHRED score of <30 from the leading and trailing ends on the FASTQ reads, with reads <50 bp after quality trimming discarded (Bolger et al., 2014 (link)).
Sequence type (ST) was determined from reads using MOST (v1.0) as previously described by Tewolde et al. (2016) (link) and eBurst Group (eBG) as described in Achtman et al. (2012 (link); Figure 1).