Transcriptome landscape of human primary monocytes at different sequencing depth.

Area of Science:

• Genomics
• Molecular Biology
• Immunology

Background:

• Understanding gene and transcript expression in human primary monocytes is crucial for immunological research.
• Previous studies have explored RNA sequencing (RNA-Seq) but optimal sequencing depth for comprehensive profiling remains an area of investigation.

Purpose of the Study:

• To investigate the impact of varying sequencing depths (50M, 100M, 200M reads) on the profiling of differential gene and transcript expression in human primary monocytes.
• To identify novel transcripts and immune-related genes with increased sensitivity at higher sequencing depths.

Main Methods:

• RNA sequencing (RNA-Seq) was performed on human primary monocytes from healthy young subjects.
• Raw sequencing data (1.3 billion reads) were analyzed at three different depths: 50 million, 100 million, and 200 million reads.
• Differential gene and transcript expression analysis was conducted, with a focus on immune-related genes and splice junctions.

Main Results:

• A total of 17,657 genes and 75,392 transcripts were identified at 200 million reads.
• Increased sequencing depth led to the identification of more genes, known and novel transcripts, and a higher proportion of reads mapping across splice junctions.
• Comparative analysis revealed 217 differentially expressed (DE) protein-coding genes and 50 DE novel transcripts, with 40 DE protein-coding genes linked to the immune system.
• Sequence alignment quality was not affected by the increase in sequencing depth.

Conclusions:

• Higher sequencing depth significantly enhances the discovery of genes, transcripts, and immune-related markers in human monocytes.
• RNA-Seq at greater depths provides a more comprehensive view of the transcriptome, including novel transcript identification.
• The findings support the utility of increased sequencing depth for robust transcriptomic analysis in immunological studies.