The Bellerophon pipeline, improving de novo transcriptomes and removing chimeras

Abstract

Transcriptome quality control is an important step in RNA-seq experiments. However, the quality of de novo assembled transcriptomes is difficult to assess, due to the lack of reference genome to compare the assembly to. We developed a method to assess and improve the quality of de novo assembled transcriptomes by focusing on the removal of chimeric sequences. These chimeric sequences can be the result of faulty assembled contigs, merging two transcripts into one. The developed method is incorporated into a pipeline, that we named Bellerophon, which is broadly applicable and easy to use. Bellerophon first uses the quality-assessment tool TransRate to indicate the quality, after which it uses a Transcripts Per Million (TPM) filter to remove lowly expressed contigs and CD-HIT-EST to remove highly identical contigs. To validate the quality of this method, we performed three benchmark experiments: 1) a computational creation of chimeras, 2) identification of chimeric contigs in a transcriptome assembly, 3) a simulated RNAseq experiment using a known reference transcriptome. Overall, the Bellerophon pipeline was able to remove between 40 to 91.9% of the chimeras in transcriptome assemblies and removed more chimeric than non-chimeric contigs. Thus, the Bellerophon sequence of filtration steps is a broadly applicable solution to improve transcriptome assemblies.