16S rDNA V4 Amplicon Sequencing
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Corresponding Organization :
Other organizations : Boston Children's Hospital, Harvard University, Brigham and Women's Hospital, Cedars-Sinai Medical Center
Protocol cited in 1 other protocol
Variable analysis
- Additional bead beater step using the Faster Prep FP120 at 6 meters/second for 1 min
- Incubation with buffers C2 and C3 for 10 min at 4°C
- DNA yields
- Bacterial genomic DNA extraction using the Mo Bio Power Fecal DNA Isolation kit according to the manufacturer's instructions
- Determination of starting nucleic acid concentrations by a Qubit Fluromoter
- Amplicon library preparation using dual-index barcodes
- Size selection of the aggregated library pool from 300-500 base pairs on a pippin prep 1.5% agarose cassette
- Concentration measurement of the pool by qPCR and loading onto the MiSeq Illumina instrument (300 bp kit) at 6-9pM with 40% phiX spike-in
- 40% phiX spike-in to compensate for low base diversity
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