Oxford Nanopore long-read genome sequencing of mutants in Streptococcus suis strain P1/7 constructed using pSStarget, a novel plasmid-based CRISPR/Cas9 genome editing system

Description: This dataset contains ONT sequencing reads of the wild type strain S. suis P1/7 and four derived mutant strains constructed using a novel CRISPR-Cas9 genome editing system for S. suis. Our data shows that our laboratory stock of strain P1/7 has 2 SNPs and a 5 bp deletion relative to the reference sequence (AM946016.1) published in GenBank. We confirm that the precise deletion of the cpsEF and sly genes in the respective mutants as well as in the double knockout mutant. Moreover, we confirm the precise introduction of targeted mutations resulting in a single amino acid change in the Enolase gene product. No other mutations or structural variations have been detected. This dataset contains the following 5 files: WT_raw_reads.fastq.gz dCps_raw_reads.fastq.gz dSly_raw_reads.fastq.gz dCps_dSly_raw_reads.fastq.gz enoK261A_raw_reads.fastq.gz Explanation of variables: All files contain raw ONT reads in the FASTQ (.fq) format in zipped (.gz) format. WT refers to the wild type strain Streptococcus suis P1/7. The prefix dCps and dSly refers to a knockout strain with the cpsE/cpsF and sly genes deleted, respectively. The double knockout mutant, in which both loci were deleted, is denoted with the prefix dCps_dSly. Finally, prefix enoK261A denotes the strain with a single amino acid mutation in the essential eno gene. Materials and methods: All strains were grown in Todd-Hewitt broth (Oxoid) supplemented with 0.2% Bacto™ yeast extract (BD Biosciences) (THY) without agitation. Overnight cultures (10ml) were harvested by centrifugation at 4500x for 10min and the pellets were transferred to bead beating tubes tube containing 0.1mm silica beads (Lysing matrix B, MP Biomedicals) and lysed by bead beating for 40 seconds at 4.0 m/s using a FastPrep-24 5G (MP Biomedicals). The lysates were cleared by centrifugation at 16.000x for 10 minutes and the genomic DNA (gDNA) has been isolated using the PowerSoil DNA Isolation Kit (Qiagen) according to manufacturer’s instructions. The concentration, purity and integrity of the extracted gDNA were determined using the Qubit BR DNA assay, DeNovix spectrophotometer and gel electrophoresis, respectively. The concentrations were normalized to 50ng/μl and sent to Plasmidsaurus (https://www.plasmidsaurus.com/) for further processing. Briefly, genomic DNA was minimally fragmented, amplification-free libraries were constructed from the fragmented DNA using the v14 library prep chemistry and the libraries were sequenced using R10.4.1 flow cells. The raw reads have been delivered in FASTQ format. License This dataset is published under the CC BY-SA (Attribution ShareAlike) license.

Bibliographic Details

Main Authors:	Gussak, Alex, Ferrando, Maria Laura, Schrama, Mels, van Baarlen, Peter, Wells, Jerry
Format:	Dataset biblioteca
Published:	Wageningen University & Research
Subjects:	CRISPR, Cas9, Nanopore, ONT, Streptococcus, Streptococcus suis, genetic engineering, genomic manipulation, infectious disease, transformation, whole genome sequencing, zoonotic agent,
Online Access:	https://research.wur.nl/en/datasets/oxford-nanopore-long-read-genome-sequencing-of-mutants-in-strepto
Tags:	Add Tag No Tags, Be the first to tag this record!