title: Illumina MiSeq sequencing; qiita_ptid_8990:13187.218
design: Fecal samples from patients suffering from bipolar disorder, healthy relatives (considered at high risk for developing bipolar disorder), and healthy controls were collected for microbiome analysis using 16S rDNA sequencing.
library_construction_protocol: DNA was extracted from fecal samples using NucleoSpin® 96 Soil (Macherey-Nagel). Bead beating was done for 1 min at 1000 g in a 1600 MiniG (SPEX SamplePrep). A minimum of one negative control was included per batch of samples from the DNA extraction and throughout the laboratory process (including sequencing). A ZymoBIOMICS™ Microbial Community Standard (Zymo Research) was also included in the analysis. PCR was done with the forward primer S-D-Bact-0341-b-S-17 and reverse primer S-D-Bact-0785-a-A-21 with Illumina adapters attached. These are universal bacterial 16S rDNA primers, which target the V3-V4 region. The following PCR program was used: 98 °C for 30 sec, 25x (98° C for 10 s, 55 °C for 20 s, 72 °C for 20 s), 72 °C for 5 min. Amplification was verified by running the products on an agarose gel. Indices was added in a subsequent PCR using the Nextera Index Kit V2 (Illumina) with the following PCR program: 98 °C for 30 sec, 8x (98° C for 10 s, 55 °C for 20 s, 72 °C for 20 s), 72 °C for 5 min. Attachment of indices was verified by running the products on an agarose gel. Products from the nested PCR were pooled based on band intensity and the resulting library cleaned with magnetic beads. The DNA concentration of pooled libraries was measured fluorometrically. Sequencing was done on an Illumina MiSeq desktop sequencer using the MiSeq Reagent Kit V3 (Illumina) for 2x 300 bp pairedend sequencing.
attributes.tag: barcode
attributes.value: AAAAA
attributes.tag: center_name
attributes.value: Region Hovedstadens Psykiatri
attributes.tag: center_project_name
attributes.value: rhpbip
attributes.tag: experiment_design_protocol
attributes.value: Fecal samples from patients suffering from bipolar disorder, healthy relatives (considered at high risk for developing bipolar disorder), and healthy controls were collected for microbiome analysis using 16S rDNA sequencing.
attributes.tag: primer
attributes.value: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC
attributes.tag: run_prefix
attributes.value: rhpbip-218_S32_L001
attributes.tag: sequencing_meth
attributes.value: Sequencing by synthesis
attributes.tag: target_gene
attributes.value: 16S rRNA
attributes.tag: target_subfragment
attributes.value: V4