Detection of fungal sequences in human brain: rDNA locus amplification and deep sequencing

Abstract

The aetiology of Alzheimer's disease (AD) and Parkinson's disease (PD) are unknown and tend to manifest at a late stage in life; even though these neurodegenerative diseases are caused by different affected proteins, they are both characterized by neuroinflammation. Links between bacterial and viral infection and AD/PD has been suggested in several studies, however, few have attempted to establish a link between fungal infection and AD/PD. In this study we adopted a nanopore-based sequencing approach to characterise the presence or absence of fungal genera in both human brain tissue and cerebrospinal fluid (CSF). We observed the presence of small fungal burden DNA in two AD brains and a control case (extensive amyloid angiopathy). This approach would be well-placed to investigate potential links between microbial infection and neurodegenerative disease.

Fig. 1

Schematic representation of the fungal ribosomal operon region to be sequenced. Forward (blue arrow) and reverse (orange arrow) primers targeting the fungal ribosomal operon and their sequences are shown. Oxford Nanopore universal tag sequences preceding the 18S and 28S rDNA-specific primer sequences are underlined in blue. The resulting ~ 3.5 kb amplicon covers hypervariable internal transcribed spacer regions 1 and 2 (ITS1 and ITS2), as well as variable domains V3–V9 of the 18S rDNA gene and D1–D3 of the 28S rDNA gene, which can refine taxonomic assignment. Figure modified from D’Andreano and Francino. Presence of PCR products was assessed using 0.5% agarose (Sigma-Aldrich, USA, A9539) gel electrophoresis with SYBR Safe DNA Gel Stain (Invitrogen, USA, S33102), and 1 kb DNA ladders (New England Biolabs, N3232S). Amplicons were subsequently purified using the Genomic DNA Clean and Concentrator®-10 (D4011). Amplicon quantification and size assessment were performed using TapeStation 2200 (Agilent Technologies, G2965A) and the D5000 ScreenTape System (Agilent Technologies, 5067-5588, 5067-5589).

Fig. 2

Metabarcoding analysis of samples. (A) Genus-level relative abundance of fungi and human (~ 3.5 kb amplicon region) of sequences taken from patients with/without either Alzheimer’s or Parkinson’s (disease and control), alongside analysis of DNA-free standards (negative) and known mock community standards (positive). X-axis indicates each sample, the first y-axis (left-hand side) indicates relative abundance as a percentage and the second y-axis (right-hand side) indicates total read count. Samples marked with a black triangle are from CSF samples, the remaining are from patient brains. Black circles depicts the number of reads per barcode. (B) Box-plot depicting total read numbers for each condition.

Fig. 3

Fungal relative abundance at genus level at three samples (Barcodes 5—AD brain, 10,171 reads; 6—control brain, 7543 reads and 8—AD brain, 12,307 reads) that had the highest burden of fungal DNA. Colours indicated classification of reads across the top 20 most abundant genera.