DNA Sequencing of Parathermosynechococcus lividus PCC 6715 cultivated in a Floating Light Ball Reactor (FLBR)

Next Generation Sequencing was conduct by STAR*SEQ GmbH laboratory (Johann-Joachim-Becher-Weg 30a, 55128 Mainz). Harvesting cells from the cyanobacteria strain PCC6715, preparation of genomic DNA using the Maxwell® RSC Fecal Microbiome DNA Kit, Qiagen QIAamp PowerFecal DNA Kit or Quick-DNA Fecal/Soil Microbe Kit. Grinding of samples will be done using Bead Tubes (glass beads). Preparation of a DNA-seq library and paired-end sequencing, 10 M reads, read length 150 nt, NextSeq 2000, XLeap SBS chemistry, the estimated output is up to 10 M reads in total (2x5 M reads) and up to 1.5 Gb per sample. Sequencing library preparation with NEBNext® UltraExpress™ FS DNA Library Prep Kit. Kraken2 Metagenomic Analysis: The Kraken Metagenomics workflow assigns taxonomic labels to short DNA sequences with high sensitivity and speed using exact alignments of k-mers and a novel classification algorithm. The DNA of strain PCC6715, as well as contaminants of both eukaryotic and prokaryotic origin, were sequenced and analyzed. Sequencing individual DNA fragments allows for both qualitative and quantitative analysis of the contaminants.

Sample FL-013-020 obtained from isolated wet biomass of non-sterile cultivaton of Parathermosynechococcus lividus PCC6715 at 55 °C in 1-Liter glass bottles in BG-11 medium.
Sample FL-010-221 obtained from isolated wet biomass of sterile cultivaton of Parathermosynechococcus lividus PCC6715 at 40 °C in 1-Liter glass bottles in BG-11 medium.
Sample FL-009-063 and Sample FL-009-064 are duplicates obtained from isolated wet biomass of non-sterile cultivaton of Parathermosynechococcus lividus PCC6715 at 45 °C in BG-11 medium in a Floating Light Ball Reactor (FLBR) at 22-Liter scale.

本研究的下一代测序（Next Generation Sequencing）由STAR*SEQ GmbH实验室（地址：约翰-约阿希姆-贝歇尔路30a号，55128美因茨）完成。实验流程如下：从蓝细菌菌株PCC6715中收获细胞，采用Maxwell® RSC粪便微生物组DNA试剂盒（Maxwell® RSC Fecal Microbiome DNA Kit）、Qiagen QIAamp PowerFecal DNA试剂盒或Quick-DNA粪便/土壤微生物试剂盒（Quick-DNA Fecal/Soil Microbe Kit）提取基因组DNA；使用填充玻璃微珠的玻璃珠管（Bead Tubes）完成样本研磨；通过NEBNext® UltraExpress™ FS DNA文库制备试剂盒（NEBNext® UltraExpress™ FS DNA Library Prep Kit）构建DNA测序文库，随后采用NextSeq 2000测序仪搭配XLeap SBS化学试剂进行双端测序，单样本预计总产出可达10M读段（2×5M读段），数据量最高1.5Gb，读长为150 nt。

Kraken2宏基因组分析：Kraken宏基因组流程通过k-mer精确比对与新型分类算法，可对短DNA序列实现高灵敏度、高速的分类标签分配。本研究对PCC6715菌株的DNA以及真核、原核来源的污染物进行了测序与分析，对单个DNA片段的测序可实现污染物的定性与定量分析。

样本FL-013-020：取自55℃下于BG-11培养基、1L玻璃瓶中进行非无菌培养的Parathermosynechococcus lividus PCC6715的分离湿生物质。
样本FL-010-221：取自40℃下于BG-11培养基、1L玻璃瓶中进行无菌培养的Parathermosynechococcus lividus PCC6715的分离湿生物质。
样本FL-009-063与FL-009-064为生物学重复样本，取自45℃下于BG-11培养基、22L规模浮动光球反应器（Floating Light Ball Reactor, FLBR）中进行非无菌培养的Parathermosynechococcus lividus PCC6715的分离湿生物质。