Allprep 96 powerfecal dna rna kit

Stool samples, reagent-only negative controls, and mock community positive controls (Zymo Research) were extracted using either the AllPrep PowerFecal DNA/RNA 96 Kit (Qiagen) or the Maxwell HT 96 gDNA Blood Isolation System (Promega) [27 (link)]. SARS-CoV-2 viral load was quantified as per CDC guidelines [28 ] using the 2019-nCoV N1 primer and probe set [28 ], as well as human RNaseP as an internal control. Each RT-qPCR reaction contained TaqPath™ 1-Step RT-qPCR Master Mix (Thermo Fisher), RNA template, the CDC N1 or RNaseP forward and reverse primers (IDT), probe, and RNase-free water to a total reaction volume of 10 μl. Viral copy numbers were quantified using N1 quantitative PCR (qPCR) standards (IDT) in tenfold dilutions to generate a standard curve. The assay was run in triplicate for each sample with three no-template control wells per 384 well plate.

Stool samples, reagent-only negative controls, and mock community positive controls (Zymo Research) were extracted using either the AllPrep PowerFecal DNA/RNA 96 Kit (Qiagen) or the Maxwell HT 96 gDNA Blood Isolation System (Promega)^{53 (link)}. SARS-CoV-2 viral load was quantified as per CDC guidelines⁵⁴ using the 2019-nCoV N1 primer and probe set⁵⁴ , as well as human RNaseP as an internal control. Each RT-qPCR reaction contained TaqPath™ 1-Step RT-qPCR Master Mix (Thermo Fisher), RNA template, the CDC N1 or RNaseP forward and reverse primers (IDT), probe, and RNase-free water to a total reaction volume of 10 μl. Viral copy numbers were quantified using N1 quantitative PCR (qPCR) standards (IDT) in 10-fold dilutions to generate a standard curve. The assay was run in triplicate for each sample with three no-template control wells per 384 well plate.

Stool samples were collected at home the day before or on the day of blood collection, time was recorded, and samples were stored in the refrigerator at home. At the hospital visit before BCG vaccination, samples were immediately aliquoted and stored at −80 °C.
Nucleic acids were extracted the AllPrep 96 PowerFecal DNA/RNA kit from QIAGEN (custom product # 1114341). This method pairs bead-beating on a Tissuelyser II (QIAGEN) with a 96-well AllPrep protocol and is available through QIAGEN. Bead-beating is performed twice at 20 Hz for 5 minutes each round with a rotation of the plate in between rounds. Purified DNA was stored at –20 °C. Metagenomic sequencing libraries were prepared from 2 ng of input DNA using the Nextera XT DNA Library Preparation kit (Illumina) according to the manufacturer’s recommended protocol. Prior to sequencing, libraries were pooled by collecting equal volumes of each library. Insert sizes and concentrations for each pooled library were determined using an Agilent Bioanalyzer DNA 1000 kit (Agilent Technologies) prior to sequencing on an Illumina NovaSeq 6000 with 151 bp paired-end reads to yield ~ 10 million paired-end reads per sample. Data was analyzed using the Broad Picard Pipeline which includes de-multiplexing and data aggregation (https://broadinstitute.github.io/picard).