The Impact of cDNA Normalization on Long-Read Sequencing of a Complex Transcriptome

Area of Science:

• Transcriptomics
• Genomics
• Molecular Biology

Background:

• Next-generation sequencing (NGS) and long-read technologies like PacBio Iso-Seq are crucial for transcriptome analysis, especially in complex plant genomes.
• cDNA normalization is a common technique to enhance the detection of rare transcripts in sequencing data.
• Sugarcane, a highly polyploid plant, presents a complex genome that benefits from advanced sequencing strategies.

Purpose of the Study:

• To evaluate the impact of cDNA normalization on PacBio Iso-Seq transcriptome analysis in sugarcane.
• To compare the transcript recovery, length distribution, and functional representation between normalized and non-normalized cDNA libraries.
• To assess the complementary roles of both approaches in achieving a comprehensive sugarcane transcriptome.

Main Methods:

• PacBio Iso-Seq was performed on two sugarcane cDNA libraries: one normalized and one non-normalized.
• Transcriptome data from both libraries were analyzed for transcript isoforms, gene families, orthologous groups, and transcript length.
• Differential expression and functional annotation were conducted on unique transcripts from each library across leaf, stalk, and root tissues.

Main Results:

• Normalization identified more total transcript isoforms and gene families but removed many longer transcripts.
• The non-normalized library captured a wider range of transcript lengths, including more transcripts >1.25 kb.
• Normalized libraries enriched lowly expressed and long noncoding transcripts, while non-normalized libraries showed higher isoform diversity per gene.
• Each library approach captured distinct functional transcript fractions, highlighting their complementary nature.

Conclusions:

• cDNA normalization is effective for detecting rare and long noncoding transcripts but may reduce the representation of longer isoforms in complex plant transcriptomes.
• Combining normalized and non-normalized PacBio Iso-Seq data provides a more comprehensive view of the sugarcane transcriptome than either method alone.
• This integrated approach is valuable for deep transcriptome sequencing of complex genomes.