The conventional culture methods include bacterial culture, fungal culture, acid-fast bacterial culture, and blood culture. Blood cultures contain aerobic and anaerobic cultures. We performed conventional culture and mNGS methods according to the patient’s medical conditions, and finally selected cases with the same specimens for examination to be included in this study. However, it was not required that all types of cultures be performed on each patient. Microbial culture and automated microbial identification systems were utilized.
Following standard operating procedures, nucleic acid extraction, nucleic acid fragmentation, end repair, end adenylation, primer ligation, and purification were performed from each sample to form a sequencing library using kits from an automated workstation (Amar et al., 2021 (link)). Libraries were assessed for quality using kits quantified by real-time PCR and loaded onto an illumina Nextseq CN500 sequencer for 75 cycles of single-end sequencing, producing approximately 20 million reads per library (Miller et al., 2019 (link)). Furthermore, peripheral hematopoietic cell specimens from healthy donors were used as negative controls simultaneously, and sterile deionized water was represented as non-template controls concurrently with each batch (Miller et al., 2019 (link)).
Following standard operating procedures, nucleic acid extraction, nucleic acid fragmentation, end repair, end adenylation, primer ligation, and purification were performed from each sample to form a sequencing library using kits from an automated workstation (Amar et al., 2021 (link)). Libraries were assessed for quality using kits quantified by real-time PCR and loaded onto an illumina Nextseq CN500 sequencer for 75 cycles of single-end sequencing, producing approximately 20 million reads per library (Miller et al., 2019 (link)). Furthermore, peripheral hematopoietic cell specimens from healthy donors were used as negative controls simultaneously, and sterile deionized water was represented as non-template controls concurrently with each batch (Miller et al., 2019 (link)).
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