The DNA was extracted according to the protocol set by Ausubel et al. (1992) in sterile conditions. All the chemicals used were previously sterilized using an autoclave and filtered through 0.2 μm sterile filters. For the shotgun library preparation, the manufacture’s (Roche Applied Science) standard protocol was replaced with the optimized protocol for limited DNA samples (Džunková et al., 2014 (link)). The exact number of molecules present in the 454 shotgun library concentration was determined by qPCR by probes specific for custom “Y” 454 library adaptors, as described by Zheng et al. (2011) (link). After the quantification step, the emPCR and sequencing with a GS FLX Titanium Sequencing XLR70 Kit (Roche Applied Science, Ref. 5233526001) were carried out following the standard protocols on 1/8 of the 454 picotiterplate. The workflow of the sequencing library preparation is shown in Figure 2 .
Gs flx titanium sequencing kit xlr70
Manufactured by Roche
Sourced in Germany
The GS FLX Titanium Sequencing Kit XLR70 is a laboratory equipment product designed for DNA sequencing. It provides the necessary reagents and consumables to perform high-throughput, massively parallel sequencing of DNA samples.
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5 protocols using gs flx titanium sequencing kit xlr70
1
DNA Extraction and Shotgun Library Preparation
2
Preparation and Sequencing of MID Samples
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MDAsample and DSsample were prepared using different MIDs by combining the two approaches on the same 1/8 PTP plate. We calculated the sample volume needed to obtain 340,000 beads (170,000 molecules per one MID) with 5–15% enrichment as recommended by Roche. The emPCR was prepared with the Small Volume emPCR kit (Roche Applied-Science. Penzberg, Germany, #05618444001) and later sequenced using a GS FLX Titanium Sequencing XLR70 Kit (Roche Applied Science, #5233526001).
3
Amplicon Sequencing and Variant Analysis
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The amplicon library was sent to emPCR amplification and 454 sequencing as described previously [25 , 26 (link)]. Briefly, Lib-L emPCR Kit (Roche) was used for emPCR according to the emPCR Amplification Method Manual. GS FLX Titanium Sequencing Kit XLR70 (Roche) was used for 454 sequencing following the protocol. Reads for each sample were sorted according to the MID. Sequence reads were mapped against the GFPm gene with 454 Life Sciences GS Reference Mapper (Version 2.6). Coverage was calculated and variants were called. Variants were filtered based on the coverage, variant frequency, and homopolymer. Only high confidence single nucleotide variants that have the following features were selected: (1) at least 3 non-duplicate reads have the nucleotide substitution; (2) the substitution frequency is greater than 5%; (3) the substitution is not located at homopolymer sites.
4
Genomic Sequencing of M. iowae Serovar K
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Genomic DNA from M. iowae serovar K was sequenced at the Ohio State University Plant-Microbe Genomics Facility using the GS FLX system (454 Life Sciences). It was prepared with the GS FLX Titanium Rapid Library Preparation Kit (Roche) and sequenced using GS FLX Titanium Sequencing Kit XLR70. Shotgun sequencing data were assembled with the GS De Novo Assembler version 2.5.3 (Roche). Annotation was performed as previously described [23] . The draft genome project has been deposited at DDBJ/EMBL/GenBank under accession number AWQU00000000. The draft genomes of serovars K and I were compared using wgVISTA [24] .
5
Sequencing and Assembly of S. Heidelberg
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We performed shotgun sequencing of the 44 S. Heidelberg using the Genome Sequencer FLX 454 (Roche, Branford, CT) and the GS FLX Titanium Sequencing Kit XLR70 according to the manufacturer’s protocol to generate an average genome coverage of 23×. De novo assemblies were performed using Roche’s Newbler software (v.2.6) with the resulting contigs being annotated using the NCBIs Prokaryotic Genomes Automatic Annotation Pipeline (Klimke et al. 2009 (link)).
Using DNA from the 2011 clinical outbreak isolate 41578, we also sought to close the genome using the Pacific Biosciences (PacBio) sequencing platform. Specifically, we prepared a single 10-kb library that was sequenced using the C2 chemistry on eight single-molecule real-time cells with a 90-min collection protocol on the PacBio RS. The 10-kb continuous-long-read data were de novo assembled using the PacBio hierarchical genome assembly process/Quiver software package, followed by Minimus 2, and they were polished with Quiver.
Using DNA from the 2011 clinical outbreak isolate 41578, we also sought to close the genome using the Pacific Biosciences (PacBio) sequencing platform. Specifically, we prepared a single 10-kb library that was sequenced using the C2 chemistry on eight single-molecule real-time cells with a 90-min collection protocol on the PacBio RS. The 10-kb continuous-long-read data were de novo assembled using the PacBio hierarchical genome assembly process/Quiver software package, followed by Minimus 2, and they were polished with Quiver.
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