Next-generation sequencing (NGS) enables the recovery of pathogen genomes from clinical samples without the need for culturing. Depletion of host/microbiota components (e.g., ribosomal RNA and poly-A RNA) and whole DNA/cDNA amplification are routine methods to improve recovery results. Using mixtures of human and influenza A virus (H1N1) RNA as a model, we found that background depletion and whole transcriptome amplification introduced biased distributions of read coverage over the H1N1 genome, thereby hampering genome assembly. Influenza serotyping was also affected by pretreatments. We propose that direct sequencing of noncultured samples without pretreatment is a favorable option for pathogen genome recovery applications.Reviewer: This article was reviewed by Sebastian Maurer-Stroh.Keywords: Pathogen genome recovery, Nonculture, Background Depletion, Whole Transcriptome Amplification, Next-generation sequencing
FindingsPathogen identification is a critical clinical application [1][2][3]. Identification methods based on culture have disadvantages, such as long turnaround time, increased biohazard risks, and culture bias. The high-throughput feature of NGS enables the recovery of pathogen genomes from noncultured samples, and offers the potential for highly accurate pathogen identification and rapid clinical diagnoses [4][5][6][7][8][9][10][11][12]. Many researchers have reported the NGS-based identification of pathogens from various noncultured samples [13][14][15][16][17][18][19][20][21] Two major challenges must be overcome when we seek to recover pathogen genomes from noncultured samples: noise from host and/or microbiota cells, and limited availability of DNA/RNA. Consequently, two pretreatments are usually employed before sequencing noncultured samples: background depletion (BD) to increase the signal-to-noise ratio [22,23], and alleged unbiased amplification to increase the amount of available nucleic acid in order to meet the requirement of NGS library preparation [24,25]. Despite of the benefits, how these pretreatments influence pathogen genome recovery during the sequencing of pathogenic DNA/ RNA from noncultured samples has not been fully investigated.