DOI: 10.5061/dryad.zw3r2284t
title: The effect of storage conditions on microbial communities in stool

description: Microbiome research has experienced a surge of interest in recent years
 due to the advances and reduced cost of next-generation sequencing
 technology. The production of high quality and comparable data is
 dependent on proper sample collection and storage and should be
 standardized as far as possible. However, this becomes challenging when
 samples are collected in the field, especially in resource-limited
 settings. We investigated the impact of different stool storage methods
 common to the TB-CHAMP clinical trial on the microbial communities in
 stool. Ten stool samples were subjected to DNA extraction after 48-hour
 storage at -80°C, room temperature and in a cooler-box, as well as
 immediate DNA extraction. Three stool DNA extraction kits were evaluated
 based on DNA yield and quality. Quantitative PCR was performed to
 determine the relative abundance of the two major gut phyla Bacteroidetes
 and Firmicutes, and other representative microbial groups. The bacterial
 populations in the frozen group closely resembled the immediate extraction
 group, supporting previous findings that storage at -80°C is equivalent to
 the gold standard of immediate DNA extraction. More variation was seen in
 the room temperature and cooler-box groups, which may be due to the growth
 temperature preferences of certain bacterial populations. However, for
 most bacterial populations, no significant differences were found between
 the storage groups. As seen in other microbiome studies, the variation
 between participant samples was greater than that related to differences
 in storage. We determined that the risk of introducing bias to microbial
 community profiling through differences in storage will likely be minimal
 in our setting.
description: We
 performed qPCR to determine the abundance of various microbial
 subpopulations, namely Bacteroidetes, Firmicutes, Enterobacteriaceae,
 <i>Bifidobacterium</i> spp. and
 <i>Lactobacillus</i> spp., relative to Eubacterial 16S rRNA
 gene amplification. We also amplified fungal species by targeting the ITS1
 region. Amplification was performed on the Rotor-Gene Q thermocycler
 (Qiagen) as singleplex reactions using 1X KAPA 2G SYBR Fast Uni Kit (KAPA
 Biosystems), 0.2 µM of each primer (see below) and nuclease free water
 (Qiagen) in 20 µL reactions. DNA input was standardized to 30 ng per
 reaction and all reactions were performed in triplicate. The adapted
 cycling conditions for all bacterial populations were as follows:
 denaturation at 95°C for 3 minutes, followed by 40 cycles of denaturation
 (95°C for 5 seconds) and annealing/extension (60°C for 30 seconds). The
 cycling conditions for fungal amplification were denaturation at 95°C for
 3 minutes, followed by denaturation (95°C for 5 seconds) and
 annealing/extension at 64, 62 and 60°C for 30 seconds for 10, 10 and 20
 cycles respectively. Fluorescence was acquired to the green channel during
 the annealing step. The Rotor-Gene software was used to calculate the
 efficiency and detection threshold for each primer set using individual
 standard curves. The efficiencies ranged between 0.91 and 1.04 with
 R<sup>2</sup> values &gt; 0.99.
 <b>Group
 targeted</b> <b>Primer Sequence 5'
 to 3'</b> <b>Amplicon
 size</b> <b>Reference</b> Bacteria (16S rRNA) F: ACTCCTACGGGAGGCAGCAGT 174 - 199 Walter et al., 2000 R: GTATTACCGCGGCTGCTGGCAC Bacteroidetes F: CGATGGATAGGGGTTCTGAGAGGA 238 Guo et al., 2008 R: GCTGGCACGGAGTTAGCCGA Firmicutes F: GGAGYATGTGGTTTAATTCGAAGCA 126 Guo et al., 2008 R: AGCTGACGACAACCATGCAC Enterobacteriaceae F: CATTGACGTTACCCGCAGAAGAAGC 195 Bartosch et al., 2004 R: CTCTACGAGACTCAAGCTTGC <i>Bifidobacterium </i>spp<i>.</i> F: CGCGTCYGGTGTGAAAG 244 Delroisse et al., 2008 R: CCCCACATCCAGCATCCA <i>Lactobacillus</i> spp. F: TGGAAACAGRTGCTAATACCG 231 - 233 Byun et al., 2004 R: GTCCATTGTGGAAGATTCCC Fungi (ITS1) F: CTTGGTCATTTAGAGGAAGTAA 260 Bellemain et al., 2010  
description: <b>Dataset details:</b> <strong>Quality and Purity
 results:</strong> This dataset contains A260/A280 and
 A230/A280 purity and quality metrics of extracted DNA, and DNA yield as
 determined by Qubit. <strong>Cts:</strong> These data are comma separated values representing the Cycle threshold (Ct) values of 7 microbial qPCRs tested for 4 different storage conditions. 10 samples were tested in triplicate for each storage condition. <strong>Missing values:</strong> Cts: Sample 3, Fungal amplification - all three replicates were lower than the detection limit of the qPCR.