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According to the standard procedure for collecting samples from patients with pleural effusion, DNA was extracted using the TIANamp Micro DNA kit (DP316, TIANGEN BIOTECH). Ultrasonic fragmentation was employed to extract DNA fragments of 150-200 bp in length, which were then repaired and ligated for PCR amplification to construct a DNA library. Quality control of the DNA libraries (average length 200-300bp, >2ng/uL) was performed using the Agilent 2100 and Qubit 2.0 systems. The qualified libraries were sequenced on the MGISEQ-2000 platform.
Low-quality reads (<35 bp in length) were removed using the Burrows-Wheeler Aligner (version 0.7.10-r789), and human host sequences were eliminated. Subsequently, low-complexity reads were filtered out using the prinseq tool (version 0.20.3). The remaining data underwent classification through simultaneous alignment with a locally constructed reference database called the Microbial Genome Database.
The microbial classification downloaded from NCBI reference database included 4061 viruses associated with human disease classification, 2473 bacterial genomes or groups of whole genome sequence scaffolds, as well as 199 species of fungi and 152 species of parasites.
An algorithm for data analysis was applied to exclude non-significant microbes related to contact infection. Genus-/species-specific reads uniquely corresponding to microorganisms associated with infection are reported. |
WGS |
METAGENOMIC |
RANDOM PCR |
SINGLE
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