16S rRNA gene V4 region amplicons from fecal DNA isolates as described were sequenced on the Illumina MiSeq 2 × 250 bp platform. Quality filtering and analysis were performed using the QIIME software pipeline following established protocols (Caporaso et al., 2010 (link)).
Miseq 2 250 bp platform
Manufactured by Illumina
Sourced in United States
The MiSeq 2 × 250 bp platform is a benchtop DNA sequencing system designed for targeted gene sequencing, small genome sequencing, and amplicon-based studies. The platform can generate paired-end reads up to 2 × 250 base pairs in length.
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Lab products found in correlation
4 protocols using miseq 2 250 bp platform
1
16S rRNA Gut Microbiome Sequencing
2
16S rRNA Amplicon Sequencing of Stool DNA
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Total DNA from stool specimens in the Carle cohort was extracted using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Valencia, CA, USA) with the bead-beating method previously described [33 (link)]. The V3-V4 region of the 16S rRNA gene was amplified with dual-indexed primers (Supplementary Materials Table S1 ) and sequenced at the Roy J. Carver Biotechnology Center at the University of Illinois on the Illumina MiSeq 2 × 250 bp platform (San Diego, CA, USA). DNA extraction and library preparation protocols for the TGH stool samples were as previously published [22 (link)]. Briefly, total DNA was extracted using the QIAamp® PowerFecal DNA Kit (QIAGEN, Valencia, CA, USA; previously known as MoBio PowerFecal DNA kit). The V4 region of the 16S rRNA gene was amplified using modified 515F and 806R primers [22 (link)] (Supplementary Materials Table S1 ) and sequenced on Illumina’s MiSeq 2 × 300 bp platform.
3
Genome Sequencing and Annotation of S. griseorubens JSD-1
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The genome sequencing of JSD-1 was performed by Illumina MiSeq 2×250 bp platform with insert sizes of 300 bp, 360 bp, and 700 bp paired-end as well as 3 kb and 8 kb mate-paired libraries. Assembly of all sequence reads applying Newbler 2.8 assembler resulted in a draft genome. Glimmer 3.0 were used to predict open reading frames (ORFs) with BLASTP annotation [30 (link),31 (link)]. The functional annotation was determined with the KEGG, COG, and Swiss-Prot databases [32 (link)-34 (link)]. Finally, the draft genome map of the S. griseorubens JSD-1 was submitted to the GenBank for further investigation.
4
16S rRNA Gene Amplicon Sequencing
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Sequencing libraries were prepared from the V3-V4 hypervariable region of 16S rRNA gene, following the protocol entitled "16S Metagenomic Sequencing Library Preparation" from Illumina [17] (link). The V3-V4 hypervariable region of 16S rRNA gene was amplified using the following primers: forward: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3'; reverse: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3'). The 25 l PCR reaction was performed using a KAPA HiFi HotStart ReadyMix (Roche) and contained 1 l of extracted fecal microbial DNA and 1 M of each primer. The reaction cycles consisted of initial denaturation at 98°C for 2 min; followed by 25 cycles of denaturation at 98°C for 15 sec, annealing at 56°C for 30 sec, and elongation at 72°C for 30 sec; and a final elongation at 72°C for 5 min.
Next, a second PCR was performed using Illumina index primers and the following reaction cycle: initial denaturation at 95°C for 3 min; followed by 8 cycles of denaturation at 95 °C for 30 sec, annealing at 55°C for 30 sec and elongation at 72°C for 30 sec; and a final elongation at 72°C for 5 min. The amplicons were quantified using MultiNA (Shimadzu, Japan), a microchip electrophoresis system for DNA/RNA analysis. The amplicons were sequenced using the Illumina MiSeq 2 × 250 bp platform with a MiSeq Reagent Nano Kit V2 (Illumina).
Next, a second PCR was performed using Illumina index primers and the following reaction cycle: initial denaturation at 95°C for 3 min; followed by 8 cycles of denaturation at 95 °C for 30 sec, annealing at 55°C for 30 sec and elongation at 72°C for 30 sec; and a final elongation at 72°C for 5 min. The amplicons were quantified using MultiNA (Shimadzu, Japan), a microchip electrophoresis system for DNA/RNA analysis. The amplicons were sequenced using the Illumina MiSeq 2 × 250 bp platform with a MiSeq Reagent Nano Kit V2 (Illumina).
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