Fecal specimens were collected from patients at onset and also from HCs and stored at -80°C in a sterile preservation tube containing an anti-DNA degradation solution.
Following the manufacturer’s instructions, DNA was extracted from fecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, CA, USA). Detection of the isolated DNAs was determined by spectrophotometry (Multiskan™ GO, Thermo Fisher Scientific, USA). The DNA extracts also were checked by 1.5% agarose gel electrophoresis in 1× Tris-Acetate-EDTA buffer. Reaction volumes for polymerase chain reaction (PCR)in a total of 20 μL consisted of 10 μL KAPA HiFi Hot Start Ready Mix (KAPA Biosystems, MA, USA), 2 μL DNA of approximately 30 ng/μL and 1 μL forward and reverse primers of 10 μM. The 16S rDNA primer sequences were forward 5’-GTGCCAGCMGCCGCGGTAA-3’; reverse 5′-GGACTACNVGGGTWTCTAAT-3′. PCR reaction conditions were configured as required. Products were analyzed using the Qubit 3.0 (Thermo Fisher Scientific, MA, USA) to determine concentrations and then mixed in equal amounts to create a sequencing library. Insert fragments and library molar concentrations were detected and quantified using a QSEP100 (Bioptic, Taiwan) and an ABI7300 fluorescence quantitative PCR instrument (Thermo Fisher Scientific, MA, USA) was used to generate a qualified library for sequencing on the MiniSeq Illumina platform (Illumina, CA, USA). Public access to the original datasets is accessible. This data can be found athttps://www.ncbi.nlm.nih.gov/sra/PRJNA906033 .
The raw reads were assembled with Flash (22 (link)). Primers were removed and clean tags were generated by deleting lower reads with the use of cutadapt (23 (link)). The sequences were clustered into operational taxonomic units (OTUs) at 97% similarity using UCHIME in reference mode (24 (link)). Representative OTU sequences were aligned in the Silva_132_97_16S database (25 (link)) for taxonomic classification using the RDP Classifier (26 (link)).
Following the manufacturer’s instructions, DNA was extracted from fecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, CA, USA). Detection of the isolated DNAs was determined by spectrophotometry (Multiskan™ GO, Thermo Fisher Scientific, USA). The DNA extracts also were checked by 1.5% agarose gel electrophoresis in 1× Tris-Acetate-EDTA buffer. Reaction volumes for polymerase chain reaction (PCR)in a total of 20 μL consisted of 10 μL KAPA HiFi Hot Start Ready Mix (KAPA Biosystems, MA, USA), 2 μL DNA of approximately 30 ng/μL and 1 μL forward and reverse primers of 10 μM. The 16S rDNA primer sequences were forward 5’-GTGCCAGCMGCCGCGGTAA-3’; reverse 5′-GGACTACNVGGGTWTCTAAT-3′. PCR reaction conditions were configured as required. Products were analyzed using the Qubit 3.0 (Thermo Fisher Scientific, MA, USA) to determine concentrations and then mixed in equal amounts to create a sequencing library. Insert fragments and library molar concentrations were detected and quantified using a QSEP100 (Bioptic, Taiwan) and an ABI7300 fluorescence quantitative PCR instrument (Thermo Fisher Scientific, MA, USA) was used to generate a qualified library for sequencing on the MiniSeq Illumina platform (Illumina, CA, USA). Public access to the original datasets is accessible. This data can be found at
The raw reads were assembled with Flash (22 (link)). Primers were removed and clean tags were generated by deleting lower reads with the use of cutadapt (23 (link)). The sequences were clustered into operational taxonomic units (OTUs) at 97% similarity using UCHIME in reference mode (24 (link)). Representative OTU sequences were aligned in the Silva_132_97_16S database (25 (link)) for taxonomic classification using the RDP Classifier (26 (link)).
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