10.5061/DRYAD.ZW3R2284T
Nel Van Zyl, Kristien
0000-0001-5529-4526
Stellenbosch University
The effect of storage conditions on microbial communities in stool
Dryad
dataset
2019
NHLS Research Trust of South Africa*
GRANT004_ 94632
NHLS Research Trust of South Africa*
GRANT004_94679
Harry Crossley*
Harry Crossley Foundation
https://ror.org/03t1asa17
NHLS Research Trust of South Africa
GRANT004_ 94632
Harry Crossley
2020-08-28T00:00:00Z
2020-08-28T00:00:00Z
en
https://doi.org/10.1371/journal.pone.0227486
42474 bytes
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CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.
We performed qPCR to determine the abundance of various microbial
subpopulations, namely Bacteroidetes, Firmicutes, Enterobacteriaceae,
Bifidobacterium spp. and Lactobacillus 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 R2
values > 0.99. Group targeted Primer Sequence 5' to 3'
Amplicon size Reference 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 Bifidobacterium spp. F:
CGCGTCYGGTGTGAAAG 244 Delroisse et al., 2008 R: CCCCACATCCAGCATCCA
Lactobacillus spp. F: TGGAAACAGRTGCTAATACCG 231 - 233 Byun et al., 2004 R:
GTCCATTGTGGAAGATTCCC Fungi (ITS1) F: CTTGGTCATTTAGAGGAAGTAA 260 Bellemain
et al., 2010
Dataset details: Quality and Purity results: This dataset contains
A260/A280 and A230/A280 purity and quality metrics of extracted DNA, and
DNA yield as determined by Qubit. Cts: 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. Missing values: Cts: Sample 3,
Fungal amplification - all three replicates were lower than the detection
limit of the qPCR.