Cost effective, experimentally robust differential-expression analysis for human/mammalian, pathogen and dual-species...

Area of Science:

• Genomics
• Bioinformatics
• Molecular Biology

Background:

• Advancements in sequencing technology have led to increased read lengths.
• Researchers are increasingly adopting longer sequencing reads for gene expression studies.
• The optimal read length for differential gene expression analysis remains an area of investigation.

Purpose of the Study:

• To evaluate the impact of sequencing read length on differential gene expression analysis.
• To determine if longer reads are consistently warranted across various genomic contexts.
• To provide recommendations for optimal read length selection based on experimental factors.

Main Methods:

• Analysis of 14 pathogen or host-pathogen differential gene expression datasets.
• Assessment of genomic attributes (gene density, operons, gene length, introns/exons, intron length).
• Comparison of paired and unpaired reads trimmed to 36, 54, 72, and 101 bp using PCA, hierarchical clustering, and regression analyses.

Main Results:

• No significant influence of genomic attributes on differential gene expression analysis outcomes was observed.
• Read pairing had the most substantial effect in datasets with low sample variation.
• 54 bp and 72 bp reads generally yielded the most similar results to longer reads.

Conclusions:

• Recommend 54 bp reads for organisms with few or no introns, and 72 bp reads for others, considering cost and mapping efficiency.
• Single-end reads are robust for differential gene expression analysis in many cases.
• Paired-end reads are recommended for eukaryotes to analyze splice variants and for community datasets intended for secondary analysis.