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

Abstract

Normalization of cDNA is widely used to improve the coverage of rare transcripts in analysis of transcriptomes employing next-generation sequencing. Recently, long-read technology has been emerging as a powerful tool for sequencing and construction of transcriptomes, especially for complex genomes containing highly similar transcripts and transcript-spliced isoforms. Here, we analyzed the transcriptome of sugarcane, a highly polyploidy plant genome, by PacBio isoform sequencing (Iso-Seq) of two different cDNA library preparations, with and without a normalization step. The results demonstrated that, while the two libraries included many of the same transcripts, many longer transcripts were removed, and many new generally shorter transcripts were detected by normalization. For the same input cDNA and data yield, the normalized library recovered more total transcript isoforms and number of predicted gene families and orthologous groups, resulting in a higher representation for the sugarcane transcriptome, compared to the non-normalized library. The non-normalized library, on the other hand, included a wider transcript length range with more longer transcripts above ∼1.25 kb and more transcript isoforms per gene family and gene ontology terms per transcript. A large proportion of the unique transcripts comprising ∼52% of the normalized library were expressed at a lower level than the unique transcripts from the non-normalized library, across three tissue types tested including leaf, stalk, and root. About 83% of the total 5,348 predicted long noncoding transcripts was derived from the normalized library, of which ∼80% was derived from the lowly expressed fraction. Functional annotation of the unique transcripts suggested that each library enriched different functional transcript fractions. This demonstrated the complementation of the two approaches in obtaining a complete transcriptome of a complex genome at the sequencing depth used in this study.

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“…2 c). Genes identified only by Iso-seq of normalized cDNAs were those with low expression levels in our previous study [ 49 ], similar to the detection of low-expressed transcripts by normalizing cDNAs described recently [ 52 ]. The overall size distribution of expressed genes identified by Iso-seq or RNA-seq was largely similar between the two methods (Fig.…”

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“…For PCR 2, the conditions were 95 • C for 5 min; 10 cycles of 95 • C for 45 s, 66 • C for 30 s, and 72 • C for 45 s; and a final extension at 72 • C for 2 min. For comparison, the Illumina 16S rRNA protocol was used as described by Hoang et al [36]. The final PCR was cleaned using AMPureXP beads (Beckman Coulter, Brea, CA, USA), and samples were pooled into sequencing libraries for quantification.…”

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“…Thus, cDNA normalisation reduces the prevalence of highly represented transcripts and equalises the number of unique transcripts in a cDNA sample, thereby dramatically increasing the probability of detecting rare transcripts. This approach is widely used for sequencing transcriptomes, but can also be useful for analysing actively translated mRNAs [24].…”