GENEX workflow

GENEX workflow identifies and maps microbial-like and human-like sequences embedded within eukaryotic and prokaryotic reference genomes to improve taxonomic profiling of ancient environmental DNA.

Key Features:

• Detection and Mapping: Detects coordinates of microbial-like and human-like sequences in reference genomes, accepting FASTA input and producing BED output with genomic positions.
• Indexing and Alignment: Constructs a Bowtie2 index from each reference genome and aligns pre-computed pseudo-reads to the indexed genome.
• Pseudo-read Sources: Uses pre-computed pseudo-reads derived from microbial databases GTDB v.214 and NCBI RefSeq release 213, and from the human reference hg38.
• Custom Analysis Scripts: Employs custom scripts to extract covered regions' positions and quantify the most abundant microbial species in eukaryotic genomes.
• Annotated Outputs: Produces BED files with genomic coordinates and associated taxonomic annotations for detected microbial-like regions.

Scientific Applications:

• Ancient environmental DNA reconstruction: Enables more accurate reconstruction of past ecosystems by detecting and allowing masking of microbial-like contamination in reference genomes used for metagenomic profiling.
• Mitigation of reference contamination bias: Reduces bias in taxonomic assignments caused by microbial contamination of eukaryotic reference genomes, particularly when plant and animal DNA are low-abundance.

Methodology:

GENEX constructs a Bowtie2 index for each reference genome, aligns pre-computed pseudo-reads (from GTDB v.214, NCBI RefSeq release 213, and hg38), uses custom scripts to identify covered regions and quantify abundant microbial species, and outputs BED files with coordinates and taxonomic annotations; it was applied to nearly 3,000 eukaryotic reference genomes from NCBI RefSeq and GenBank and 1,323 PhyloNorway plant genome assemblies.