Genomic DNA was extracted from cultures of STEC O157:H7 using the Qiagen Qiasymphony (Qiagen, Hilden, Germany). The sequencing library was prepared using the Nextera XP kit (Illumina, San Diego, USA) for sequencing on the Illumina HiSeq 2500 (Illumina, San Diego, USA) instrument run with the fast protocol. High-quality trimmed (leading and trailing trimming at link)]). Illumina reads (read length 80–100 bp) were mapped to the STEC O157:H7 reference genome Sakai (GenBank accession BA000007) using BWA-MEM v0.7.13 [9 (link)]. The Sakai STEC O157:H7 reference genome (BA000007) contains 18 prophages of which two are Stx-encoding (stx1a and stx2a) and six prophage like-regions including the locus of enterocyte effacement [10 (link)]. SNPs were identified using GATKv2.6 in unified genotyper mode [11 (link)]. Core-genome positions that had a high-quality SNP (>90 % consensus, minimum depth 10×, MQ >=30) in at least one isolate were extracted for further analysis. Genomes were compared to the sequences held in the PHE STEC O157:H7 WGS database, (SnapperDB v0.2.5. STEC O157:H7) and isolates with five SNP differences or less within their core genome were considered closely related and likely to have an epidemiological link [7, 12 (link)].
Nextera xp kit
Manufactured by Illumina
Sourced in United States
The Nextera XP kit is a library preparation kit designed for next-generation sequencing. It enables the efficient and rapid generation of sequencing-ready libraries from DNA samples.
Automatically generated - may contain errors
6 protocols using nextera xp kit
1
Genomic Profiling of STEC O157:H7 Strains
2
Genomic DNA Extraction and Sequencing of STEC O157:H7
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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.
3
Genomic DNA Extraction and Sequencing of STEC O157:H7
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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.
4
Illumina Sequencing Workflow for Genomic DNA
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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 ).
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);
5
Genomic DNA Extraction and Sequencing of STEC O157:H7
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Genomic DNA was extracted from cultures of STEC O157:H7 using the QIAsymphony system (Qiagen, Hilden, Germany). The sequencing library was prepared using the Nextera XP kit (Illumina, San Diego, USA) for sequencing on the HiSeq 2500 instrument (Illumina, San Diego, USA), run with the fast protocol. FASTQ reads were processed using Trimmomatic v0.2730 (link) as previously described31 (link).
6
Sequencing of Shigella species
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Four strains of Shigella Shigella sonnei Shigella flexneri Shigella Table 1 ). If the isolate formed part of a cluster, the earliest isolate was selected with respect to faecal sample collection date (or date of isolation if sample data were unavailable). For Illumina sequencing, Genomic DNA was extracted from cultures 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 [23 (link)] to remove bases with a PHRED score of <30 from the leading and trailing ends, with reads <50 bp after quality trimming discarded.
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