Computational protocol: Cryopreservation of artificial gut microbiota produced with in vitro fermentation technology

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Protocol publication

[…] The microbial community was analysed in faecal samples and fermentation effluents. The V4 hypervariable 16S region was amplified using specific primers 515F (5′‐GTGCCAGCMGCCGCGGTAA‐3′) and 806R (5′‐GGACTACHVGGGTWTCTAAT‐3′). Sequencing was carried out on an Illumina MiSeq (StarSEQ, Mainz, Germany) using V4 chemistry for 2 × 250 bp read length. Raw sequencing reads were processed by merging the paired reads using USEARCH (v8.1.1756) with a minimum length of the merged read of 100 bp, an expected error threshold of 1 and minimum overlapping of 15 bp. Raw sequencing reads were filtered using PRINSEQ‐lite (v0.20.4) based on selected quality criteria such as: (i) no ambiguous bases; (ii) read lengths between 247 and 257 base pairs (bp); and (iii) the average quality score at 10 bp and a complexity threshold of 10. Sequences that passed quality filtering were clustered into OTUs at 97% identity level using UPARSE (usearch v8.0.1623). Representative sequences (the most abundant) for each OTU were aligned using PyNAST (QIIME‐1.8.0) and taxonomically assigned using UTAX (usearch v8.1.1756). [...] Statistical analysis of the HPLC and qPCR data (log10‐transformed) was performed using IBM SPSS Statistics 23.0 (IBM SPSS, Armonk, New York, USA). Concerning the HPLC data, the initial acetate and propionate concentration present in the medium were subtracted from the concentrations measured after 3, 5, 7 and 24 h batch fermentation. Data are expressed as means ± SD of three different batch fermentations inoculated with three aliquots from the same treatment or control. Data were tested for normal distribution using the Shapiro–Wilk test, and equality of variance was assessed with the Levene test. ANOVA tests were performed on every treatment to compare qPCR and HPLC data. Treatments were compared to control with a Dunnett (two‐sided) test. A nonparametric Kruskal–Wallis test was performed when data were not normally distributed or the assumption of equality of variance was violated. A paired sample t‐test was performed to compare data prior to freezing with postfreezing data within one treatment. Differences were considered significant for the risk α ≤ 0.05. […]

Pipeline specifications

Software tools USEARCH, PRINSEQ, UPARSE, PyNAST, QIIME, SPSS
Applications Miscellaneous, 16S rRNA-seq analysis
Organisms Bacteria, [Eubacterium] rectale, Faecalibacterium prausnitzii
Chemicals Butyrates, Glycerol