mNGS: Quality control of EVs-derived from human umbilical cord mesenchymal stem cells

Metagenomic next-generation sequencing of MSC-EVsQuality control of EVs derived from MSCs (donor samples 1-5) was performed using metagenomic next-generation sequencing (mNGS) by Hugobiotech Co., Ltd. (Beijing, China), as previously documented. Briefly, DNA was extracted and purified from 200 uL (approximately 150 ug) of MSC-EVs according to the manufacturer's instructions for the QIAamp DNA Micro Kit (50) (56304, Qiagen, Germany). The DNA concentration and quality were assessed using Qubit Fluorometric Quantitation and agarose gel electrophoresis. DNA libraries were prepared using the QIAseq Ultralow Input Library Kit (180492, Qiagen, Germany). The library concentration and quality were confirmed through Qubit and agarose gel electrophoresis. Libraries, each tagged with a unique barcode, were pooled and sequenced on an Illumina NextSeq platform (Illumina HiSeq X10, Illumina, USA). Sequencing data underwent quality control to eliminate adapter sequences, low-quality reads, low-complexity reads, and short reads. Human reads were filtered out by mapping against the human reference genome using the SNAP software tool. The filtered reads were then aligned to a microbial genome database using the Burrows-Wheeler Alignment (BWA) tool. This database included over 20,000 microbial genomes sourced from NCBI, comprising 11,910 bacterial, 7,103 viral, 1,046 fungal, and 305 parasitic genomes. Finally, the microbial compositions of the samples were determined and analyzed using mNGS.