An opinionated guide to the proper care and feeding of your transcriptome

1 Characterizing transcriptomes in both model and non-model organisms has resulted in a 2 massive increase in our understanding of biological phenomena. This boon, largely made 3 possible via high-throughput sequencing, means that studies of functional, evolutionary 4 and population genomics are now being done by hundreds or even thousands of labs 5 around the world. For many, these studies begin with a de novo transcriptome assembly, 6 which is a technically complicated process involving several discrete steps. Each step 7 may be accomplished in one of several different ways, using different software packages, 8 each producing different results. This analytical complexity begs the question – Which 9 method(s) are optimal? Using reference and non-reference based evaluative methods, I 10 propose a set of guidelines that aim to standardize and facilitate the process of transcrip11 tome assembly. These recommendations include the generation of between 20 million 12 and 40 million sequencing reads from single individual where possible, error correction of 13 reads, gentle quality trimming, assembly filtering using Transrate and/or gene expres14 sion, annotation using dammit, and appropriate reporting. These recommendations have 15 been extensively benchmarked and applied to publicly available transcriptomes, result16 ing in improvements in both content and contiguity. To facilitate the implementation 17 of the proposed standardized methods, I have released a set of version controlled open18 sourced code, The Oyster River Protocol for Transcriptome Assembly, available at 19 http://oyster-river-protocol.rtfd.org/. 20