cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing.
The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.
Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
Maria
M
Cartolano
Max Planck-Genome-centre Cologne, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
Bruno
B
Huettel
Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
Benjamin
B
Hartwig
Max Planck-Genome-centre Cologne, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
Richard
R
Reinhardt
Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
Korbinian
K
Schneeberger
D017360:Arabidopsis / Q000235:genetics; D003199:Computer Systems; D018076:DNA, Complementary / Q000235:genetics; D066289:Data Curation; D030541:Databases, Genetic; D015723:Gene Library; D018745:Genome, Plant; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*; D024363:Transcription Initiation Site; D014908:Triticum / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger