Detailed Information on Publication Record
2023
16S rDNA sequencing analysis of low-abundance samples spiked with bacterial mock community
VYKLICKÁ, Kateřina, Jan BÖHM, Petr ANDRLA, Eva BUDINSKÁ, Břetislav LIPOVÝ et. al.Basic information
Original name
16S rDNA sequencing analysis of low-abundance samples spiked with bacterial mock community
Authors
VYKLICKÁ, Kateřina (203 Czech Republic, belonging to the institution), Jan BÖHM (203 Czech Republic), Petr ANDRLA (203 Czech Republic), Eva BUDINSKÁ (703 Slovakia), Břetislav LIPOVÝ (203 Czech Republic) and Petra BOŘILOVÁ LINHARTOVÁ (203 Czech Republic, guarantor)
Edition
III. ČESKO-SLOVENSKÝ KONGRES MEZIOBOROVÉ PŘÍSTUPY V HOJENÍ RAN, 2023
Other information
Language
English
Type of outcome
Konferenční abstrakt
Field of Study
10608 Biochemistry and molecular biology
Country of publisher
Czech Republic
Confidentiality degree
není předmětem státního či obchodního tajemství
RIV identification code
RIV/00216224:14310/23:00133546
Organization unit
Faculty of Science
Keywords in English
low-abundance samples; microbiome; 16S rDNA sequencing; mock community
Změněno: 16/2/2024 09:52, Mgr. Terezie Slámová
Abstract
V originále
Background The use of negative and positive controls, such as bacterial mock community, is a crucial part of a well-established workflow for 16S rDNA sequencing. Several mock community standards (i.e., mixes with known composition of bacterial species) are available for purchase. To prevent interference with the sample, the selected mock community should ideally consist of species not occurring in the target sample. We aimed to set up a method for analysis of bacteriome of three human low-abundance matrices that takes advantage of spiking of these samples with non-human mock community in amounts not overlaying the sample bacteriome but, and at the same time, yielding sufficient sequence coverage. Methods Samples of urine, blood and bronchoalveolar lavage (BAL) from 3 patients, 3 bacterial cultures of Klebsiella pneumoniae (104 CFU/mL) serving as positive controls, and 3 negative controls (DNA free water) were isolated by QIAamp DNA Blood Mini Kit in triplicates. Parallelly, the mock community ZymoBIOMICS Spike-in Control I (High Microbial Load, MOCK), consisting of bacterial species Allobacillus and Imtechella (1:1), was isolated. All samples and controls were subsequently spiked with 1 µl of 500×, 1,000× or 2,000× diluted DNA from isolated mock (what more 3 negative controls without spiking were also used) and amplified. Two PCRs were performed – one with 30 and the other with 33 cycles. The final library was sequenced on the MiSeq instrument using MiSeq Reagent Kit v3 (600-cycle). Results The sequencing analysis was successful with both 30 and 33 cycles used for PCR. The results showed the contamination of reagents with Flavobacterium (~1,000 reads per sample) and Massilia (~250 reads per sample) species. These bacteria were, therefore, excluded from subsequent data analysis. In urine, blood, and BAL samples with 2,000× diluted mock, the average relative abundance of Allobacillus and Imtechella was 5 % (on average 5 % combined), while with the 1,000× and 500× diluted mock, the relative abundances of these mock community bacteria were 30 % and 60 % (on average), respectively. Conclusions 16S rDNA sequencing of urine, blood and BAL samples spiked with mock was successfully performed; however, we detected the presence of certain bacterial DNA in used reagents. For the studied matrices, we recommend: i) to monitor the possibility of sample contamination from reagents; ii) to use the mock dilution for these lowabundance matrices of 2,000×, which results in approx. 5 % relative abundance of the mock community in the sample. This mock dilution is sufficient to serve as an internal standard while not overlaying the bacteria, which are abundant more than 5 % in this type of samples.
Links
| EF17_043/0009632, research and development project |
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| LM2018121, research and development project |
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| 857560, interní kód MU (CEP code: EF17_043/0009632) |
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