Stool samples from mice were collected and genomic DNA was extracted using the QIAAmp Fast Stool Mini Kit (Qiagen, Redwood City, CA, USA) as per manufacturer instructions. DNA samples were sent to Research and Testing Laboratory (currently RTL Genomics, Lubbock, TX, USA) for processing and analysis based on a previously described protocol [31 (link),32 (link)]. 16S ribosomal RNA variable region was amplified and subjected to sequencing on an Illumina MiSeq as previously described [31 (link)]. Reads were processed and classified into operational taxonomic units (OTUs) as previously described [32 (link)]. Bacterial diversity between groups of mice were compared using the Shannon index (for alpha diversity) and the Bray–Curtis and Jaccard indices (for beta diversity).
Qiaamp fast stool mini kit
Manufactured by Qiagen
Sourced in United States
The QIAamp Fast Stool Mini Kit is a laboratory equipment product designed for the rapid and efficient extraction of DNA from stool samples. It provides a streamlined process for isolating high-quality DNA from small amounts of stool material.
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9 protocols using qiaamp fast stool mini kit
1
Microbiome Analysis of Murine Stool Samples
2
Fecal microbiome analysis by 16S rRNA sequencing
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Fecal samples were collected directly from the anus by massaging the distal rectum before CRD testing, and samples were frozen at −80°C until use. Microbial DNA was extracted using a QIAamp Fast Stool Mini Kit (Qiagen, Valencia, CA, USA). The V3–V4 region of the 16S rRNA gene was amplified with barcode‐indexed primers (338F and 806R), and paired‐end sequencing of the amplicons was performed using the Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA). The difference in the relative abundance of each taxon at different taxonomic levels was analyzed using the linear discriminant analysis effect size method, and predictions regarding the functions of the microbiome were based on 16S rRNA‐derived operational taxonomic units (OTUs) using a computational approach called phylogenetic investigation of communities using reconstruction of unobserved states.17
3
Fecal DNA Extraction and 16S Sequencing
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DNA was extracted from fecal pellets with the Qiagen QIAamp Fast Stool Mini Kit according to the manufacturer's instructions with several small modifications; briefly, 2–3 fecal pellets were homogenized in 500 μl InhibitEX Buffer using a disposable homogenizing pestle and vortex. The suspension was heated for 5 min at 70°C before stool particles were pelleted by 1‐min centrifugation at 20,000 g. The supernatant was thoroughly mixed with 20 μl proteinase K before 500 μl Buffer AL was added and the mix was incubated at 70°C for 10 min. After the addition of 500 μl of 100% ethanol, the lysate was applied to the QIAamp spin column and centrifuged for 1 min at 20,000 g. The filtrate was discarded before 500 μl Buffer AW1 was added to the spin column and centrifuged for 1 min at 20,000 g. This step was repeated with 500 μl Buffer AW2. The spin column was dried by centrifugation for 3 min at 20,000 g in a clean 2‐ml collection tube. The DNA was eluted in 200 μl of Buffer ATE, directly pipetted on the QIAamp membrane, and collected in a clean Eppendorf tube.
After DNA extraction from the fecal pellets, the Thermo Fisher Ion 16S™ Metagenomics Kit was used to amplify the hypervariable regions V2, V3, V4, V6, V7, V8, and V9 of bacterial 16S rRNA. The amplified fragments were then bar‐coded, sequenced on the Ion PGM sequencer system, and analyzed with the Ion Reporter Software.
After DNA extraction from the fecal pellets, the Thermo Fisher Ion 16S™ Metagenomics Kit was used to amplify the hypervariable regions V2, V3, V4, V6, V7, V8, and V9 of bacterial 16S rRNA. The amplified fragments were then bar‐coded, sequenced on the Ion PGM sequencer system, and analyzed with the Ion Reporter Software.
4
Comparative Evaluation of DNA Extraction Kits
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Three DNA extraction kits were evaluated: Zymo Research ZR Fecal DNA MiniPrep™ kit, Qiagen QIAamp® Fast Stool Mini Kit, and MO BIO Powersoil®. Tubes containing RNAlater were centrifuged to pellet stool, and RNAlater solution was removed before processing them in the same manner as the nonpreserved stool samples.
For ZR Fecal DNA MiniPrep™ kit extractions, samples were processed following the manufacturer's protocol using the provided 0.5mm glass bead tubes and performing a 6‐min bead beating on a “Disruptor Genie” vortex adapter. DNA was eluted after incubation with Elution buffer and passed through Zymo‐Spin™ IV‐HRC columns.
For the Qiagen QIAamp Fast Stool Mini KitTM extractions, samples were processed following the manufacturer's protocol, using Lysis buffer and a 70°C Lysis step. Samples were strongly vortexed before and after the 70°C incubation. DNA was eluted after incubation with the provided Elution buffer.
For MoBio protocol extractions using the MO BIO Powersoil® kit, C1 solution was added and the sample was bead beaten for 10 min on the MoBio Vortex adapter. The remaining procedures were done exactly per the MoBio protocol instructions. Samples were likewise eluted after 5 min of incubation with the provided Elution buffer.
For ZR Fecal DNA MiniPrep™ kit extractions, samples were processed following the manufacturer's protocol using the provided 0.5mm glass bead tubes and performing a 6‐min bead beating on a “Disruptor Genie” vortex adapter. DNA was eluted after incubation with Elution buffer and passed through Zymo‐Spin™ IV‐HRC columns.
For the Qiagen QIAamp Fast Stool Mini KitTM extractions, samples were processed following the manufacturer's protocol, using Lysis buffer and a 70°C Lysis step. Samples were strongly vortexed before and after the 70°C incubation. DNA was eluted after incubation with the provided Elution buffer.
For MoBio protocol extractions using the MO BIO Powersoil® kit, C1 solution was added and the sample was bead beaten for 10 min on the MoBio Vortex adapter. The remaining procedures were done exactly per the MoBio protocol instructions. Samples were likewise eluted after 5 min of incubation with the provided Elution buffer.
5
Bacterial and Fungal Community Analysis of Cecal Samples
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Total genomic DNA of cecal samples was extracted using the Qiagen QIAamp Fast Stool Mini Kit (Qiagen, Shanghai, China) according to the manufacturer’s protocol. V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified with the primer set 338F (5ʹ-ACTCCTACGGGAGGCAGCAG-3ʹ ) and barcoded 806R (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ ) [15 (link)] under the following PCR cycling conditions: initial denaturation at 94°C for 3 min followed by 5 cycles of denaturing at 94°C for 10 s, annealing at 55°C for 15 s, and extension at 72°C for 30 s before a final extension at 72°C for 5 min. Fungal ITS regions were amplified with the primers ITS3_KYO2 and ITS4_KYO3 [16 (link)]. The following PCR cycling conditions were used: an initial denaturation of 15 min at 95°C; followed by 30 cycles of 30 s at 95°C, 30 s at either 51°C or 55°C, and 30 s at 72°C; and final elongation for 5 min at 72°C. Purification, quantification, and sequencing of 16S rRNA and ITS amplicons were carried out following the procedure described by Song et al. [17 (link)]. The 16S rRNA and ITS amplicon sequences have been deposited in the NCBI Sequence Read Archive under the Submission ID: PRJNA523884.
6
Identifying Burkholderia pseudomallei from Environmental Samples
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The clinical isolate was confirmed as B. pseudomallei at the Virginia Division of Consolidated Laboratory Services and CDC by using the LRN algorithm, including biochemicals and PCR. All 29 environmental samples were sent to CDC for culture and identification. We directly inoculated all environmental samples into TBSS-C50 (Galimand) broth and incubated broths in a shaking incubator at 37ºC for 6 days. We then cultured enriched broths onto Ashdown agar, and extracted DNA by using a QIAamp Fast Stool Mini Kit (QIAGEN, https://www.qiagen.com ) or testing by real-time PCR. Suspected colonies from Ashdown agar were selected for further workup, and confirmation of isolates followed the LRN algorithm.
7
Fecal Microbiome Profiling Protocol
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Fecal DNA was individually extracted from subsamples at more than 3 different regions of the original sample, using the QIAGEN QIAamp Fast Stool Mini Kit according to the manufacturer's instructions. DNA was amplified by targeting the V3‐V4 region of the bacterial 16S rRNA gene using primers, 341F (5′‐CCTAGGGGNGGCWGCAG‐3′) and 805R (5′‐GACTACHVGGGTATCTAATCC‐3′) (Klindworth et al., 2013 (link)), and amplification was performed using the following protocol: one denaturation step at 94°C for 3 min, five cycles of denaturation at 94°C for 15 s and extension at 65°C for 60 s, 20 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 20 s and extension at 72°C for 30 s, and a final extension at 72°C for 5 min. Sequencing library construction and amplicon sequencing were performed at Macrogen (Seoul, South Korea) using a 2 × 300 bp Illumina MiSeq sequencing system (Illumina, USA).
8
Gut Microbiome Analysis in Parkinson's Disease
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All patients’ fecal samples were collected at the Clinic of Neurology II, EVK Hattingen Germany and were snap frozen immediately at −80 °C after defecation. In 16 patients with PD, a sample was collected before, during and after combined treatment including dietary intervention and bowel cleansing.
For the extraction of DNA, a commercial system (QIAamp Fast Stool Mini Kit, Qiagen, Hilden, Germany) was used. In brief, the samples were thawed at 4 °C and approximately 500 mg was suspended in Inhibitex buffer (3 mL). Suspensions were heated (95 °C, 5 min), spun (20,000× g) and the supernatant was used for DNA preparation according to the protocol of the manufacturer. Purity was checked by determining the OD260/OD280 ratio, damage and degradation by agarose gel electrophoresis and absence of inhibitors was verified by amplification with the primers 27F and 534R, which amplify the V1-V3 regions. The 16SrDNA region was amplified with the same primers and sequenced by Illumina technology by a commercial vendor (GATC Biotech, Konstanz, Germany).
For the extraction of DNA, a commercial system (QIAamp Fast Stool Mini Kit, Qiagen, Hilden, Germany) was used. In brief, the samples were thawed at 4 °C and approximately 500 mg was suspended in Inhibitex buffer (3 mL). Suspensions were heated (95 °C, 5 min), spun (20,000× g) and the supernatant was used for DNA preparation according to the protocol of the manufacturer. Purity was checked by determining the OD260/OD280 ratio, damage and degradation by agarose gel electrophoresis and absence of inhibitors was verified by amplification with the primers 27F and 534R, which amplify the V1-V3 regions. The 16SrDNA region was amplified with the same primers and sequenced by Illumina technology by a commercial vendor (GATC Biotech, Konstanz, Germany).
9
Modified QIAamp Stool DNA Extraction
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We used the QIAamp Fast Stool Mini kit protocol (QIAGEN) to extract DNA from samples preserved using the two-step method, following the manufacturer’s instructions. We modified this protocol to extract DNA from the swabbed samples, as follow: (i) the initial sample (swab tip in buffer solution) was vortexed and centrifuged for 2 minutes (14,100 g) before discarding the swab, (ii) 250 μl of Inhibitex were added to the supernatant, (iii) samples were incubated with proteinase K for 1 hour at 56°C, (iv) 500 μl of CT capture buffer (Isohelix extraction kit, Cell Project) were added to the sample (replacing ethanol), and (v) DNA was eluted in 75 μl of buffer ATE (S1 Table ). For every batch of samples, we used DNA extraction blanks to monitor contamination. All DNA extracts were purified using OneStep PCR inhibitors Removal Kits (Zymo research).
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