According to the WHO, the risk of developing complications after SARS-CoV-2 infection is 10–20 %. Long COVID leads to a decrease in patients’ quality of life, and changes in immune system reactivity play a significant role in this. This pilot study of the PBMC transcriptome in patients with long COVID using nanopore sequencing aims to provide preliminary data on the functional status of PBMC and identify potential targets for the development of new effective personalized therapies for this disease.

The transcriptome analysis protocol included RNA extraction (GeneJET RNA Purification Kit), DNA removal (DNase I), double-stranded cDNA synthesis (Mint kit-2), sequencing library preparation (SQK-NBD114.24), nanopore sequencing (PromethION 2 Solo, FLO-PRO114M (R10.4.1) cells), basecalling (Dorado), read quality assessment (nanoQC/FastQC), adapter trimming and length/quality filtering (Cutadapt (5.2)/seqkit(2.12.0)), and data preparation for differential gene expression assessment (minimap2 in map-ont mode, samtools (1.10.2), Picard tools (CollectRnaSeqMetrics), featureCounts (2.1.1), biomaRt (2.62.1)), differential expression analysis (RStudio 2025.05.0 Build 418, DESeq2 (1.46.0), pheatmap (1.0.13), dplyr (1.1.4), RColorBrewer (1.1.-3), ggplot2 (3.5.2), ggrepel (0.9.6), apeglm (1.28.0) packages), GSEA-analysis (clusterProfiler (4.14.6), org.Hs.eg.db (3.20.0), enrichplot (1.26.6), DOSE (4.0.1), igraph (2.2.1)), semantic similarity of GO terms (biological process (BP) analysis (REViGO)), BP functional network creation (Cytoscape 3.10.4).

A pilot study of the peripheral blood leukocyte transcriptome in patients with long COVID using nanopore sequencing revealed significant differences in gene expression (n = 2,102) involved in 163 biological processes. Analysis of these changes in activity indicated a significant decrease in the overall functional activity of leukocytes, accompanied by a decrease in proliferative activity, weakening of innate and adaptive immunity, and a restructuring of metabolic processes toward cellular stabilization against the background of increased ribosome biogenesis, activation of oxidative phosphorylation and suppression of proteolysis.

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