Ghost | Laboratory of Neurobiology

The brown ghost knifefish (Apteronotus leptorhynchus) is a weakly electric teleost fish of particular interest as a versatile model system for a variety of research areas in neuroscience and biology. The comprehensive information available on the neurophysiology and neuroanatomy of this organism has enabled significant advances in such areas as the study of the neural basis of behavior, the development of adult-born neurons in the central nervous system and their involvement in the regeneration of nervous tissue, as well as brain aging and senescence. Despite substantial scientific interest in this species, no genomic resources are currently available.

To overcome this limit, we performed, in collaboration with the laboratory of Professor Jeffrey N. Agar (Barnett Institute, Department of Chemistry and Chemical Biology, Northeastern University) a de novo assembly and annotation of the A. leptorhynchus transcriptome. After evaluating several trimming and transcript reconstruction strategies, de novo assembly using Trinity uncovered 42,459 unique contigs containing at least a partial protein-coding sequence based on alignment to a reference set of known Actinopterygii sequences. As many as 11,847 of these contigs contained full or near-full length protein sequences, providing broad coverage of the proteome. A variety of non-coding RNA sequences were also identified and annotated, including conserved long intergenic non-coding RNA and other long non-coding RNA observed previously to be expressed in adult zebrafish (Danio rerio) brain, as well as a variety of miRNA, snRNA, and snoRNA. Shotgun proteomics confirmed translation of open reading frames from over 2,000 transcripts, including alternative splice variants. Assignment of tandem mass spectra was greatly improved by use of the assembly compared to databases of sequences from closely related organisms.

The release of an annotated de novo transcriptome assembly from Apteronotus leptorhynchus provides a broad overview of RNA expressed in central nervous system tissue of this species. The assembly, which includes substantial coverage of a wide variety of both protein coding and non-coding transcripts, allows the scientific community the development of better tools to understand the mechanisms underlying unique characteristics of the knifefish model system, such as their tremendous regenerative capacity and negligible brain senescence.

Transcriptome assembly workflow and alignment to D. rerio reference proteome. A. RNA was extracted from brain and spinal cord tissue of adult A. leptorhynchus, fragmented and barcoded, and strand-specific cDNA libraries were created for Illumina sequencing. Reads were trimmed using several strategies and then normalized in silico prior to de novo assembly with Trinity. Transcript reconstruction was performed using several strategies and then benchmarked using BLAST to maximize transcripts aligning to a D. rerio reference proteome. The best assembly was then further annotated. B. Out of the transcripts from the entire A. leptorhynchus assembly with any alignment to a D. rerio reference protein, most transcripts aligned to <40% of the reference D. rerio proteins. C. When filtering the assembly for transcripts with FPKM ≥ 1, the relative proportion of transcripts with less than complete alignments was reduced. D. When selecting only the best alignment for a given D. rerio reference protein, this best alignment was more complete than many other fragmented alignments, which can be partially attributed to multiple transcripts from the same gene with less than complete ORFs. E. The distribution of the longest aligned sequences for each unique D. rerio reference sequence was preserved in the assembly containing only transcripts with FPKM ≥ 1. F. Out of the entire assembly, more than half of the transcripts had at least some alignment to a reference D. rerio sequence. Similarly, more than half of the sequences with FPKM ≥ 1 aligned to a D. rerio protein sequence. G. Using only transcripts with FPKM ≥ 1, nearly 60% of the reference D. rerio proteome had at least one assembly transcript with an alignment (24,112 sequences), which represented approximately 80% of the D. rerio sequences that were hit by the entire assembly (30,121 unique sequences). (From Salisbury et al., 2015.)

Publication:

Salisbury, J.P., Sîrbulescu, R.F., Moran, B.M., Auclair, J.R., Zupanc, G.K.H., Agar, J.N.: The central nervous system transcriptome of the weakly electric brown ghost knifefish (Apteronotus leptorhynchus): de novo assembly, annotation, and proteomics validation. BMC Genomics 16, 166 (2015), doi:10.1186/s12864-015-1354-2