Metagenomics sequencing shows microbiome differences in brain abscess samples
Description
The data reported here are the sequence information and microbiome characteristics of three culture-negative brain abscess samples. The processed sequences were used for Operational Taxonomic Units (OTU) analysis and sequences that possessed more than 97% similarity were grouped into the same OTUs.
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Genomic DNA was extracted from brain abscess samples using Nucleospin Tissue DNA extraction kit (Macherey Nagel, Germany) according to the manufacturer’s instruction. Samples were quantified using Qubit DNA HS Assay (Invitrogen) and diluted to 5ng and were amplified for full-length 16S rRNA (~1500bp) using universal primers . The PCR products were checked on agarose gel and used for further amplifying V3-V4 region of 16SrRNA (~465bp) using specific forward (5'-CCTACGGGNGGCWGCAG'-3) and reverse (5'-GACTACHVGGGTATCTAATCC'-3) primers. The V3-V4 PCR products were purified using AMPure XP beads (Beckman Coulter) and proceeded with DNA library preparation using NEBNext Ultra DNA Library Prep Kit (Illumina). The amplicons were end repaired and mono-adenylated at 3’ end in a single enzymatic reaction and NEB hairpin-loop adapters were ligated to the DNA fragments in a T4-DNA ligase-based reaction. Following ligation, the loop containing Uracil is linearized using USER Enzyme (a combination of UDG and Endo VIII), to make it available as a substrate for PCR based indexing in the next step. During PCR, barcodes were incorporated using unique primers for each sample. The DNA libraries were checked for fragment distribution on Fragment Analyzer using HS NGS Fragment Kit (1-6000bp) (Agilent) and loaded on to Illumina MiSeq V2 instrument to generate 0.5M, 250bp Paired end reads/sample. After quality checking using FASTQC toolkit, the sequenced reads were stitched to form long reads greater than 400 bp using the FLASH program and subjected to dereplication and chimera removal using the VSEARCH program. The reads were then mapped against the SILVA-132 database; sequences that possessed more than 97% similarity were grouped into the same OTUs using the RDP classifier. Taxonomy classification (phylum, order, family, genus, and species) and relative abundance were done using QIIME1.