Considerations for reference vs reference-free transcriptome analysis

Whether you work with microbes that have a sequence reference genome or you have one without a reference, please know that there are several reasonably complete workflows that you can use for both types of analyses. No need to re-invent the wheel.

We will attempt to cover both approaches using available workflows and then point you to additional resources so that you can choose the one that best fits your needs.

Reference-based transcriptome analysis

The general approach for reference-based (if you already have an available sequenced genome) is as follows (can use different tools though):

Ref based workflow outline

Several workflows can be utilized for this type of analysis, including:

Reference-free transcriptome analysis

The approach for reference-free (no good reference genome and not enough mRNA-seq data for assembled transcriptomes) is slightly different and an entirely different animal than reference-based approaches. The process is related and generally can follow this type of workflow:

Ref free workflow

This is usually an iterative process that may require additional tools and computational time to work through the data. But it can be done! A useful presentation from Meg Staton on de novo assembly.

There are fewer workflows readily available for de novo transcriptome assembly:

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