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

<strong>Description:</strong>This dataset contains sequencing reads of the wild type strain <em>S. suis</em> P1/7 and two knockout mutants 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 one 5bp deletion relative to the reference sequence (AM946016.1) published in GenBank. Moreover, we confirm that the two knockout strains lack the deleted <em>cpsEF </em>and s<em>ly</em> genes and do not have other mutations relative to the sequence of our laboratory stock P1/7. <strong>Explanation of variables:</strong>All files contain trimmed Illumina reads in the FASTQ (.fq) format in zipped (.gz) format.SsuisP17 refers to the wild type strain <em>Streptococcus suis </em>P1/7. The appendix _dCPS and _dSLY refers to a knockout strain with the <em>cpsE/cpsF</em> and <em>sly</em> genes deleted, respectivelyThe number of the sample name, '_1' and '_2' refers to each member of a paired-end read pair.<br><strong>Materials and methods:</strong>All strains were grown on Todd-Hewitt (Oxoid) agar plates supplemented with 0.2% Bacto™ yeast extract (BD Biosciences) (THY) at 37 °C with 5% CO2. All growth was collected from the agar plate using a sterile loop and resuspended in a bead tube containing cryoperservative (Microbank™, Pro-Lab Diagnostics UK, United Kingdom) following MicrobesNG strain submission procedures. The bead tubes containing the bacteria were sent to MicrobesNG for DNA extraction and sequencing.Five to forty microlitres of the bacterial suspension were lysed with 120 µL of TE buffer containing lysozyme (final concentration 0.1 mg/mL) and RNase A (ITW Reagents, Barcelona, Spain) (final concentration 0.1 mg/mL), incubated for 25 min at 37°C. Proteinase K (VWR Chemicals, Ohio, USA) (final concentration 0.1mg/mL) and SDS (Sigma-Aldrich, Missouri, USA) (final concentration 0.5% v/v) were added and incubated for 5 min at 65°C. Genomic DNA was purified using an equal volume of SPRI beads and resuspended in EB buffer (Qiagen, Germany). DNA concentration was quantified with the Quant-iT dsDNA HS kit (ThermoFisher Scientific) assay in an Eppendorf AF2200 plate reader (Eppendorf UK Ltd, United Kingdom).Genomic DNA libraries were prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, USA) following the manufacturer’s protocol with the following modifications: input DNA was increased 2-fold, and PCR elongation time was increased to 45 s. DNA quantification and library preparation were carried out on a Hamilton Microlab STAR automated liquid handling system (Hamilton Bonaduz AG, Switzerland). Pooled libraries were quantified using the Kapa Biosystems Library Quantification Kit for Illumina. Libraries were sequenced using Illumina sequencers (HiSeq/NovaSeq) using a 250bp paired end protocol. Reads were adapter trimmed using Trimmomatic 0.30 with a sliding window quality cutoff of Q15 [1]. De novo assembly was performed on samples using SPAdes version 3.7 [2], and contigs were annotated using Prokka 1.11 [3]. <br>References1. Bolger, A. M., Lohse, M., &amp; Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114–2120. http://doi.org/10.1093/bioinformatics/btu170 2. Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., ... Pevzner, P. A. (2012). SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. Journal of Computational Biology, 19(5), 455–477. http://doi.org/10.1089/cmb.2012.0021 3. Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics. 30(14):2068-9<br>

<strong>数据集描述：</strong>本数据集包含野生型<em>猪链球菌（Streptococcus suis）</em>P1/7菌株，以及两套针对猪链球菌的新型CRISPR-Cas9基因组编辑系统构建的敲除突变体的测序读段。本研究数据显示，本实验室保存的P1/7菌株与GenBank中发布的参考序列（AM946016.1）相比，存在2个单核苷酸多态性（Single Nucleotide Polymorphism, SNP）及1处5bp缺失。此外，本研究证实两株敲除菌株分别缺失了<em>cpsEF</em>与<em>sly</em>基因，且相对于本实验室保存的P1/7菌株序列，未携带其他突变。
<strong>变量说明：</strong>所有文件均为经修剪的Illumina测序读段，以FASTQ（.fq）格式压缩为GZ（.gz）格式存储。SsuisP17指代野生型<em>猪链球菌（Streptococcus suis）</em>P1/7菌株；后缀_dCPS与_dSLY分别指代缺失<em>cpsE/cpsF</em>基因与<em>sly</em>基因的敲除菌株；样本名称中的"_1"与"_2"分别指代双端测序读段对的两条读段。
<strong>材料与方法：</strong>所有菌株均接种于添加0.2% Bacto™酵母提取物（BD Biosciences）的Todd-Hewitt（Oxoid）琼脂平板（简称THY培养基），于37℃、5% CO₂条件下培养。使用无菌接种环从琼脂平板刮取菌落，重悬于含有冷冻保存液的Microbank™管（Pro-Lab Diagnostics UK，英国）中，遵循MicrobesNG菌株提交流程进行处理。将含菌的Microbank™管寄送至MicrobesNG进行DNA提取与测序。
取5~40 μL细菌悬液，使用120 μL含溶菌酶（终浓度0.1 mg/mL）与RNase A（ITW Reagents，西班牙巴塞罗那，终浓度0.1 mg/mL）的TE缓冲液进行裂解，于37℃孵育25分钟。随后加入蛋白酶K（VWR Chemicals，美国俄亥俄州，终浓度0.1 mg/mL）与十二烷基硫酸钠（SDS，Sigma-Aldrich，美国密苏里州，终浓度0.5% v/v），于65℃孵育5分钟。使用等体积SPRI磁珠纯化基因组DNA，并用EB缓冲液（Qiagen，德国）重悬。使用Quant-iT dsDNA HS定量试剂盒（ThermoFisher Scientific）在Eppendorf AF2200酶标仪（Eppendorf UK Ltd，英国）中对DNA浓度进行定量。
使用Nextera XT文库制备试剂盒（Illumina，美国圣地亚哥），按照厂商实验方案并进行如下修改：将投入DNA量提升2倍，PCR延伸时间延长至45秒，完成基因组DNA文库构建。DNA定量与文库制备均在Hamilton Microlab STAR自动化液体处理系统（Hamilton Bonaduz AG，瑞士）上完成。使用Kapa Biosystems Illumina文库定量试剂盒对混合文库进行定量。采用Illumina测序仪（HiSeq/NovaSeq）以250 bp双端测序方案完成文库测序。
使用Trimmomatic 0.30对测序读段进行接头修剪，滑动窗口质量阈值设为Q15 [1]。使用SPAdes 3.7版本对样本进行从头组装[2]，使用Prokka 1.11版本对重叠群进行注释[3]。
<strong>参考文献：</strong>
1. Bolger, A. M., Lohse, M., &amp; Usadel, B. (2014). Trimmomatic: 用于Illumina测序数据的灵活修剪工具. 生物信息学, 30(15), 2114–2120. http://doi.org/10.1093/bioinformatics/btu170
2. Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., ... Pevzner, P. A. (2012). SPAdes：新型基因组组装算法及其在单细胞测序中的应用. 计算生物学杂志, 19(5), 455–477. http://doi.org/10.1089/cmb.2012.0021
3. Seemann T (2014) Prokka：快速原核基因组注释. 生物信息学, 30(14):2068-9