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

Citation

Rye, Marie et al. (2022), Data from: Fusobacterium nucleatum and Bacteroides fragilis detection in colorectal tumours: optimal target site and correlation with total bacterial load, Dryad, Dataset, https://doi.org/10.5061/dryad.qrfj6q5gn

Abstract

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 ability to detect the targeted species by qPCR.

Methods

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 Paired End sequencing on a MiSeq next generation sequencer (Illumina). Paired-end reads were assembled by aligning the forward and reverse reads using PEAR1 (version 0.9.5). Primers were identified and trimmed. Trimmed sequences were processed using Quantitative Insights into Microbial Ecology (QIIME 1.8) USEARCH (version 8.0.1623) and UPARSE software. Sequences were quality filtered and sorted by abundance after removal of full-length duplicate sequences. Singletons or unique reads were discarded. Sequences were clustered and then chimera filtered using “rdp_gold” database as reference. Reads were mapped back to Operational Taxonomic Units with a minimum identity of 97% and taxonomy was assigned using the QIIME 1 default classifier, pre-trained against Greengenes database5 (Version 13_8, Aug 2013).

Usage Notes

Please refer to ReadMe file.

Funding

St John of God Subiaco Hospital, Award: N/A