Shotgun metagenomic data was generated for 22 families and included 47 total samples (22 mothers and 25 daughters). The samples selected for shotgun metagenomics are indicated in Fig 1 . Families which either had similar taxonomic profiles or had species in common were selected for this analysis. Shotgun metagenomic sequence libraries were prepared from the extracted DNA using Illumina Nextera XT Flex kits according to manufacturer recommendations. The resulting libraries were sequenced on an Illumina HiSeq 4000 (10 per lane,150 bp paired-end mode) at the Genomic Resource Center at the University of Maryland School of Medicine. The average number of read pairs generated for each library was 37,000,000 (range: 24,500,000 to 81,800,000). Human reads were identified in the resulting sequence datasets using BMtagger and removed (ftp://ftp.ncbi.nlm.nih.gov/pub/agarwala/bmtagger/ ). Sequence datasets were further processed using sortmeRNA [51 (link)] to identify and remove ribosomal RNA reads and the remaining reads were trimmed for quality (4 bp sliding window, average quality score threshold Q15) using Trimmomatic v0.3653 [52 ]. Reads trimmed to less than 75bp were removed from the dataset.
Nextera xt flex kit
Manufactured by Illumina
The Nextera XT Flex kits are a set of laboratory equipment used for library preparation in next-generation sequencing. The kits enable the generation of sequencing-ready libraries from a variety of sample types, including genomic DNA, amplicons, and cDNA.
Automatically generated - may contain errors
Lab products found in correlation
Magattract powermicrobiome dna rna kit, by Qiagen (2 mentions)
Hiseq 4000 platform, by Illumina (2 mentions)
Novaseq 6000 s2 flow cell, by Illumina (2 mentions)
Hiseq 4000, by Illumina (1 mentions)
Quick dna fecal soil microbe kit, by Zymo Research (1 mentions)
Dna rna shield, by Zymo Research (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
6 protocols using nextera xt flex kit
1
Shotgun Metagenomic Analysis of Mother-Daughter Microbiomes
2
Vaginal Microbiome Metagenomic Sequencing
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Shotgun metagenomic data was generated for 194 samples (5 samples per subject). DNA was extracted from 200 μL of vaginal swab specimen resuspended in 1ml of Amies transport medium that had been preserved at − 80 °C. DNA extractions were performed using the MagAttract PowerMicrobiome DNA/RNA Kit (Qiagen) and bead-beating on a TissueLyser II according to the manufacturer’s instructions and automated onto a Hamilton STAR robotic platform. Shotgun metagenomic sequence libraries were constructed from the DNA extracts using Illumina Nextera XT Flex kits according to manufacturer recommendations and then sequenced on an Illumina HiSeq 4000 platform (150 bp paired-end mode) at the Genomic Resource Center at the University of Maryland School of Medicine. Eight uniquely barcoded samples were included in each HiSeq 4000 lane yielding an average of 35 million read pairs for each sample.
3
Shotgun Metagenomics Sequencing Protocol
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Shotgun metagenomic sequence libraries were constructed from the DNA extracts using Illumina Nextera XT Flex kits according to manufacturer recommendations and then sequenced on an Illumina HiSeq 4000 platform (150 bp paired-end mode) at the Genomic Resource Center at the University of Maryland School of Medicine. Each sample was uniquely barcoded in each HiSeq 4000 lane, yielding an average of 40 million read pairs for each sample. The sequencing data is publicly available (https://www.ncbi.nlm.nih.gov/sra/PRJNA997379).
4
Fecal Microbiome DNA Extraction and Sequencing
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The jejunum and colon tissues of the mice were dissected according to their gastrointestinal anatomical features. Intraluminal stool contents were collected from dissected tissues and stored immediately in DNA/RNA Shields (Zymo Research, Irvine, CA, USA) at −80°C to stabilize and protect the integrity of nucleic acids and minimize the need for immediate processing or freezing of specimens. DNA extraction was described previously [30 (link), 62 (link)]. In brief, 0.15–0.25 grams of fecal samples were extracted using the Quick-DNA Fecal/Soil Microbe kit (Zymo Research, Irvine, CA, USA). Negative extraction controls were included to ensure that no exogenous DNA contaminated the samples. Metagenomic sequencing libraries were constructed using the Nextera XT Flex Kit (Illumina), according to the manufacturer’s recommendations. Libraries were then pooled together in equimolar proportions and sequenced on a single Illumina NovaSeq 6000 S2 flow cell at Maryland Genomics at the University of Maryland School of Medicine.
5
Shotgun Metagenomic Analysis of Vaginal and Penile Microbiomes
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Shotgun metagenomic data were generated for 138 samples (74 vaginal and 64 penile) as described previously (84 (link)). Swabs were resuspended in 1 mL of Amies transport medium. Genomic DNA was extracted using 200 µL of resuspended material and the MagAttract PowerMicrobiome DNA/RNA Kit (Qiagen) with bead-beating using TissueLyser II. Sequence libraries were generated from extracted DNA using the Illumina Nextera XT Flex kit. Procedures were performed according to manufacturer instructions and automated on the Hamilton STAR robotic platform. Libraries were sequenced on an Illumina NovaSeq 6000 (150 bp, paired-end) at Maryland Genomics at UMSOM. Host reads were identified and removed using BMtagger and the GRCh38 human genome as reference (91 ). Non-host reads were processed using fastp (v.0.21) (92 (link)). Metagenome taxonomic compositions were determined via mapping to VIRGO (59 (link)), and CSTs were assigned using VALENCIA (2 (link)). VALENCIA is a supervised classifier for the vaginal microbiome. The penile CST assignments we observed are consistent with prior unsupervised clustering results (25 (link), 27 (link)– (link)29 (link)), so we feel it is reasonable to use VALENCIA with penile samples in STING.
6
Fecal Metagenomics DNA Extraction and Sequencing
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Genomic DNA was extracted from homogenized fecal samples stored in DNA/RNA shields using the fecal/soil microbe kits (Zymo, Irvine, CA) according to the manufacturer’s instructions. Briefly, a 500 µl aliquot of fecal material mixture was homogenized using bead lysis and centrifuged down to remove lysate debris. Metagenomic sequencing libraries were constructed from the same DNA using Illumina Nextera XT flex kit according to the manufacturer recommendations. Libraries were then pooled together in equimolar proportions and sequenced on a single Illumina NovaSeq 6000 S2 flow cell at the Genomic Resource Center at the University of Maryland School of Medicine.
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