Metabarcoding of Hungarian sourdough samples

Total DNA from the sourdough samples wasextracted by the Macherey-Nagel (Duren, Germany) Genomic DNA From Food kitfollowing the manufacturers' protocols. 16Smetabarcoding was carried out using the 16S long-read metabarcoding kit(SQK-16S024) of Oxford Nanopore Technologies (Oxford, UK) according to themanufacturer's instructions. In the first step of the library preparation aPCR was performed for the amplification of the 16S rRNA gene target region(~1500 bp) and to add a unique barcode to each sample. DNA concentrations werequantified by Qubit fluorometer (Invitrogen, Waltham, MA). The initial DNAconcentration was 10 ng per sample, the final PCR mix contained 25 ul LongAmpHot Start Taq 2x Master Mix, 10 ul input DNA, 10 ul 16S barcode primers (each)and 5 ul nuclease free water. The reaction was performed using a thermal cycler(Biometra TAdvanced, Analytik Jena, Jena, Germany) with the following PCRconditions: initial denaturation at 95C for 1 min, 25 cycles of 95C for 20 s,55C for 30 s, 65C for 2 min, followed by a final extension step at 65C for 5min. The amplicons were purified with AMPure XP beads (Beckman Coulter, Brea,CA, USA) and eluted in 10 mM Tris-HCl pH 8.0 with 50 mM NaCl. DNA concentrationof the samples was quantified with a Nabi spectrophotometer (MicroDigital,Seongnam-si, South Korea). Approximately 100 fmol of the combined library wasloaded into an ONT SpotON flow cell. Super accurate basecalling andde-barcoding in the Guppy v6.4 software were performed after the sequencing.本研究采用德国杜伦市Macherey-Nagel公司的食品基因组DNA提取试剂盒（Genomic DNA From Food kit），严格遵循制造商操作手册，从酸面团样本中提取总DNA。随后采用英国牛津市牛津纳米孔科技公司（Oxford Nanopore Technologies）的16S长读长宏条形码测序试剂盒（SQK-16S024），依照制造商操作指南完成16S宏条形码测序。在文库制备的第一步中，通过PCR扩增16S rRNA基因的目标区域（约1500 bp），并为每个样本添加独特的条形码序列。使用Qubit荧光定量仪（Invitrogen，美国马萨诸塞州沃尔瑟姆市）对DNA浓度进行定量。每个样本的初始DNA投入量为10 ng；最终的PCR反应混合液包含25 μL 2×LongAmp Hot Start Taq 预混酶（LongAmp Hot Start Taq 2x Master Mix）、10 μL 模板DNA、10 μL 16S条形码引物（正向与反向引物各取等量体积）以及5 μL 无核酸酶水。PCR反应使用德国耶拿市Analytik Jena公司的Biometra TAdvanced型热循环仪进行，反应程序设置如下：95℃预变性1分钟；随后进行25个循环，每个循环包含95℃变性20秒、55℃退火30秒、65℃延伸2分钟；最后于65℃终延伸5分钟。扩增产物使用美国加利福尼亚州布雷亚市贝克曼库尔特公司（Beckman Coulter）的AMPure XP磁珠（AMPure XP beads）进行纯化，并用含50 mM NaCl的10 mM Tris-HCl（pH 8.0）缓冲液洗脱。使用韩国城南市MicroDigital公司的Nabi分光光度计对纯化后样本的DNA浓度进行定量。将约100 fmol的混合文库加载至ONT SpotON测序流通池。测序完成后，使用Guppy v6.4软件进行高精度碱基识别与解条形码操作。