Fusobacterium nucleatum and Bacteroides fragilis detection in colorectal tumours: optimal target site and correlation with total bacterial load

These data were generated to investigate detection of Fusobacterium nucleatum (F. nucleatum) and Bacteroides fragilis (B. fragilis) across different regions of human colorectal tumours. Relative abundance of each species in DNA extracted for clinical molecular mutation testing from formalin-fixed, paraffin-embedded (FFPE) tumour samples from 42 patients was assessed using targeted real-time PCR quantitative (qPCR) (the screening cohort).  DNA was then freshly extracted from specific regions of tumours testing positive for one or both species (n = 20) and from 31 additional patients, and relative abundance of each species assessed using qPCR (site investigation cohort). Total bacterial load at the tumour luminal surface (where F. nucleatum and B. fragilis were most frequently detected) was also assessed by qPCR using primers targeting amplification of 16S rRNA. 16S sequencing was performed on tumour luminal surface DNA samples from five patients as an orthogonal method to confirm the abi..., Bacterial detection was performed on the ViiA7 Real-time PCR System (Thermo Fisher Scientific) using PrimeTime® qPCR primers, probes and mastermix (IDT) according to the manufacturer’s instructions. Reactions were performed using 1X PrimeTime® Gene Expression Master Mix, 1X PrimeTime®qPCR Assay and up to 10ng of DNA. Cycling conditions were 95°C for 3 minutes, 60 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Amplification results were reviewed using QuantStudioTM Real-Time PCR Software version 1.1 (Thermo Fisher Scientific). Amplification of beta-actin and prostaglandin transporter (PGT) was used to determine relative abundance.

Diversity profiling was performed by AGRF (Australian Genome Research Facility, Melbourne Australia). Samples were amplified with universal primers to the V1-V3 region of the bacterial 16S gene (forward AGAGTTTGATCMTGGCTCAG; reverse GWATTACCGCGGCKGCTG). Amplicons were indexed using the Nextera XT Index Kit (Illumina, San Diego, CA, USA) followed by Pa..., Please refer to ReadMe file.

本数据集旨在探究人类结直肠肿瘤不同区域中具核梭杆菌（Fusobacterium nucleatum, F. nucleatum）与脆弱拟杆菌（Bacteroides fragilis, B. fragilis）的检测情况。研究首先从42例患者的福尔马林固定石蜡包埋（formalin-fixed, paraffin-embedded, FFPE）肿瘤样本中提取DNA，用于临床分子突变检测，并通过靶向实时荧光定量PCR（quantitative PCR, qPCR）分析两种菌的相对丰度，构建筛选队列（screening cohort）。随后，针对1种或两种菌均呈阳性的20例肿瘤特定区域样本，以及额外31例患者的肿瘤样本，重新提取新鲜DNA，再次通过qPCR分析两种菌的相对丰度，构建位点探究队列（site investigation cohort）。同时，针对两种菌检出频率最高的肿瘤管腔表面，利用靶向16S rRNA扩增的引物通过qPCR检测总细菌载量。选取5例患者的肿瘤管腔表面DNA样本进行16S测序，作为正交验证方法以确认实验结果（原文此处截断为"confirm the abi..."）。细菌检测在ViiA7实时PCR系统（Thermo Fisher Scientific）上开展，采用PrimeTime® qPCR引物、探针及预混液（IDT），严格遵循制造商说明书进行操作。反应体系包含1×PrimeTime®基因表达预混液、1×PrimeTime® qPCR检测试剂，以及最高10ng的DNA。循环程序为：95℃预变性3分钟，随后60个循环：95℃变性5秒，60℃退火延伸30秒。扩增结果通过QuantStudio™实时PCR软件v1.1（Thermo Fisher Scientific）进行分析。以β-肌动蛋白（beta-actin）与前列腺素转运蛋白（PGT）的扩增作为相对丰度计算的内参基因。

多样性谱分析由澳大利亚基因组研究设施（AGRF, Australian Genome Research Facility, 澳大利亚墨尔本）完成。使用针对细菌16S基因V1-V3区的通用引物进行扩增（上游引物：AGAGTTTGATCMTGGCTCAG；下游引物：GWATTACCGCGGCKGCTG）。扩增产物通过Nextera XT索引试剂盒（Illumina, 美国加利福尼亚州圣地亚哥）进行索引标记，随后进行[原文截断为"Pa..."]，具体详情请参阅ReadMe文件。