Biome representational in silico karyotyping

Abstract

Metagenomic characterization of complex biomes remains challenging. Here we describe a modification of digital karyotyping-biome representational in silico karyotyping (BRISK)-as a general technique for analyzing a defined representation of all DNA present in a sample. BRISK utilizes a Type IIB DNA restriction enzyme to create a defined representation of 27-mer DNAs in a sample. Massively parallel sequencing of this representation allows for construction of high-resolution karyotypes and identification of multiple species within a biome. Application to normal human tissue demonstrated linear recovery of tags by chromosome. We apply this technique to the biome of the oral mucosa and find that greater than 25% of recovered DNA is nonhuman. DNA from 41 microbial species could be identified from oral mucosa of two subjects. Of recovered nonhuman sequences, fewer than 30% are currently annotated. We characterized seven prevalent unknown sequences by chromosome walking and find these represent novel microbial sequences including two likely derived from novel phage genomes. Application of BRISK to archival tissue from a nasopharyngeal carcinoma resulted in identification of Epstein-Barr virus infection. These results suggest that BRISK is a powerful technique for the analysis of complex microbiomes and potentially for pathogen discovery.

Figure 1.

(A) Schematic of BRISK technique. (B) Representative ethidium bromide-stained agarose gel of BRISK products. (Lane 1) 1 kb DNA ladder; (lane 2) 100 bp DNA ladder; (lane 3) PCR amplification of BRISK fragments following ligation of asymmetric adaptors; (lane 4) amplification of unbound material from biotin column; (lane 5) amplification of beads following melt and elution of single-stranded DNA; (lane 6) ampification of material eluted from beads (desired product containing one long and one short adaptor); (lane 7) negative PCR control.

Figure 2.

Schematic of bioinformatic analysis applied to BRISK sequencing results. (ED) Levenstein edit distance.

Figure 3.

Observed vs. expected recovery of sequence tags by chromosome using BRISK from human whole blood sample. (Closed circles) unamplified DNA; (open circles) phi29 amplified DNA.

Figure 4.

Distribution of genomically unknown sequences (GUS) in the oral mucosa of three normal human volunteers. PCR primers were designed for each of eight GUS tags and performed on salivary DNA samples from three individuals (S1–S3). S1 was the individual from whom each GUS was originally identified. (B) is PCR performed on blood-derived DNA from subject 1; (C) is PCR performed on DNA derived from HEK293 cells. (NC) is a no-template DNA negative control. Universal bacterial 16S primers were used as positive control for the presence of bacterial DNA. Melanopsin (OPN4)-specific primers were used as positive control for the presence of human DNA. After sequence extension by vectorette-assisted genome walking, GUS 8 was identified as a human DNA sequence from clone RP11-318L16 on chromosome 1.