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Recent advances in next-generation sequencing technologies have paved the path for a considerable amount of sequencing data at a relatively low cost. This has revolutionized the genomics and transcriptomics studies. However, different challenges are now created in handling such data with available bioinformatics platforms both in assembly and downstream analysis performed in order to infer correct biological meaning. Though there are a handful of commercial software and tools for some of the procedures, cost of such tools has made them prohibitive for most research laboratories. While individual open-source or free software tools are available for most of the bioinformatics applications, those components usually operate standalone and are not combined for a user-friendly workflow. Therefore, beginners in bioinformatics might find analysis procedures starting from raw sequence data too complicated and time-consuming with the associated learning-curve. Here, we outline a procedure for de novo transcriptome assembly and Simple Sequence Repeats (SSR) primer design solely based on tools that are available online for free use. For validation of the developed workflow, we used Illumina HiSeq reads of different tissue samples of Santalum album (sandalwood), generated from a previous transcriptomics project. A portion of the designed primers were tested in the lab with relevant samples and all of them successfully amplified the targeted regions. The presented bioinformatics workflow can accurately assemble quality transcriptomes and develop gene specific SSRs. Beginner biologists and researchers in bioinformatics can easily utilize this workflow for research purposes.
Here we present a complete workflow of free software that can be executed in a Linux environment. All the tools that we have used were developed by various other research groups and scientists, and are freely available online to download and execute (Supplementary File 01). Some of the software are even open-source, which allows users to suggest improvements and fix bugs, inherently improving the performance of the tools in return. Our workflow spans from acquiring sequenced reads, through quality control and assembly of data, up to assembly quality assessment and SSR primer design. For the current study, transcriptomic data was downloaded from a published study on Santalum album (sandalwood)70. We generated SSRs targeting few important oil biosynthetic genes with the objective of identifying makers for future breeding efforts. A batch of designed primers were validated with laboratory experiments.
We presented a complete workflow of free software from acquiring sequenced reads, through quality control and assembly of data, up to assembly quality assessment and SSR primer design. All the tools used are freely available online to download and execute while some of the software are open-source. 2b1af7f3a8