Draft De Novo Transcriptome of the Rat Kangaroo Potorous tridactylus as a Tool for Cell Biology.

Dylan B Udy,Mark Voorhies,Todd M Lowe,Patricia P Chan,Sophie Dumont,Aaron F Straight

doi:10.1371/journal.pone.0134738

Dylan B Udy, Mark Voorhies + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0134738

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Abstract

The rat kangaroo (long-nosed potoroo, Potorous tridactylus) is a marsupial native to Australia. Cultured rat kangaroo kidney epithelial cells (PtK) are commonly used to study cell biological processes. These mammalian cells are large, adherent, and flat, and contain large and few chromosomes—and are thus ideal for imaging intra-cellular dynamics such as those of mitosis. Despite this, neither the rat kangaroo genome nor transcriptome have been sequenced, creating a challenge for probing the molecular basis of these cellular dynamics. Here, we present the sequencing, assembly and annotation of the draft rat kangaroo de novo transcriptome. We sequenced 679 million reads that mapped to 347,323 Trinity transcripts and 20,079 Unigenes. We present statistics emerging from transcriptome-wide analyses, and analyses suggesting that the transcriptome covers full-length sequences of most genes, many with multiple isoforms. We also validate our findings with a proof-of-concept gene knockdown experiment. We expect that this high quality transcriptome will make rat kangaroo cells a more tractable system for linking molecular-scale function and cellular-scale dynamics.

Highlights

We performed an experimental test that helps validate the rat kangaroo transcriptome, and its usability for siRNA design and gene knockdown. We expect that this high quality transcriptome will make rat kangaroo cells a more tractable system for mechanistic experiments linking molecular-scale function and cellular-scale dynamics, and for transcriptome-wide gene expression analyses
We performed nextgeneration sequencing via a paired-end 150-cycle rapid run on the Illumina HiSeq2500, generating 679,303,792 raw reads (Table 1), corresponding to very high coverage depth
We sequenced over 99 billion nucleotides, and these had a Q20 of 98.4% and GC content of 49.9% (Table 1)

Summary

Introduction

Probing the molecular basis of these cellular processes in PtK cells has been challenging due to the absence of rat kangaroo gene sequence information. We performed high-throughput sequencing, assembly and annotation of this draft transcriptome based on PtK2 cell transcripts. 2 TPM values in the table correspond to the most abundant, full-length rat kangaroo Trinity transcript for each gene.

Results

Conclusion