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Fig. 2 | BMC Neurology

Fig. 2

From: The application of shotgun metagenomics to the diagnosis of granulomatous amoebic encephalitis due to Balamuthia mandrillaris: a case report

Fig. 2

Result of metagenomic NGS, PCR and phylogenetic analysis. A, Analysis of metagenomic next-generation sequencing (NGS) results from a patient. The total reads distribution is shown on the left and the distribution of reads without the human genome is shown on the right. Genomic DNA was extracted from the biopsied brain samples using an extraction kit (QIAGEN, Tokyo, Japan). Shotgun metagenomic sequencing was performed using the MiSeq® NGS system (NextraXT prep kit®, Illumina) and a paired-end sequencing approach. For the identification of non-human DNA, the CLC Genomics Workbench was first used to query a human genome database (UCSC: hg38) and a human mRNA database (RefSeq release 54) and eliminate most human sequences. The remaining unmapped reads were subsequently analyzed by Nucleotide BLAST against the nt database from NCBI. A total of 129 reads showed a striking homology to B. mandrillaris under conditions of E-value <1e− 20, hit length > 80, and identity > 80%. B, Polymerase chain reaction analysis using species-specific primers revealed a positive reaction for B. mandrillaris in the brain and skin samples from the patient and a negative reaction for B. mandrillaris in the CSF from the patient and the water control. C, Phylogenetic analysis of B. mandrillaris mitochondrial small subunit rRNA gene was performed based on the neighbor-joining method using MEGA X software. The percentage trees in which the associated taxa cluster together in the bootstrap test (1000 replicates) are shown next to the branches. Scale bars indicate the genetic distance

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