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Background   De novo  assembly of non-model organism’s transcriptomes has recently been on the rise in concert with the number of  de novo  transcriptome assembly software programs. There is a knowledge gap as to what assembler software or  k-mer  strategy is best for construction of an optimal  de novo  assembly. Additionally, there is a lack of consensus on which evaluation metrics should be used to assess the quality of  de novo  transcriptome assemblies.    Result  Six different assembly strategies were evaluated from four different assemblers. The Trinity assembly was used in its default 25 single  k-mer  value while Bridger, Oases, and SOAPdenovo-Trans were performed with multiple  k-mer  strategies. Bridger, Oases, and SOAPdenovo-Trans used a small multiple k-mer (SMK) strategy consisting of the k-mer lengths of 21, 25, 27, 29, 31, and 33. Additionally, Oases and SOAPdenovo-Trans were performed using a large multiple k-mer (LMK) strategy consisting of k-mer lengths of 25, 35, 45, 55, 65, 75, and 85. Eleven metrics were used to evaluate each assembly strategy including three genome related evaluation metrics (contig number, N50 length, Contigs >1 kb, reads) and eight transcriptome evaluation metrics (mapped back to transcripts (RMBT), number of full length transcripts, number of open reading frames, Detonate RSEM-EVAL score, and percent alignment to the southern platyfish, Amazon molly, BUSCO and CEGMA databases). The assembly strategy that performed the best, that is it was within the top three of each evaluation metric, was the Bridger assembly (10 of 11) followed by the Oases SMK assembly (8 of 11), the Oases LMK assembly (6 of 11), the Trinity assembly (4 of 11), the SOAP LMK assembly (4 of 11), and the SOAP SMK assembly (3 of 11).    Conclusion  This study provides an in-depth multi  k-mer  strategy investigation concluding that the assembler itself had a greater impact than  k-mer  size regardless of the strategy employed. Additionally, the comprehensive performance transcriptome evaluation metrics utilized in this study identified the need for choosing metrics centered on user defined research goals. Based on the evaluation metrics performed, the Bridger assembly was able to construct the best assembly of the testis transcriptome in  Fundulus heteroclitus .

Comparison of De Novo Transcriptome Assemblers and k-mer Strategies Using the Killifish, Fundulus heteroclitus