---
title: "Hands-On Session 1: From raw reads to isoforms"
canonical: "https://help.biobam.com/space/BTP/3494215689/Hands-On%20Session%201%3A%20From%20raw%20reads%20to%20isoforms"
format: markdown
---
# Introduction
## Dataset
[https://drive.google.com/drive/folders/1TImxh7w5ZR0-gcnIwAgzl7hMza_rqwfj?usp=sharing](https://drive.google.com/drive/folders/1TImxh7w5ZR0-gcnIwAgzl7hMza_rqwfj?usp=sharing)
## Goals and Algorithms/Tools
- Alignment of Long Reads to a reference genome
- minimap2
- Isoform definition from Long Reads
- FLAIR
- Characterization of a Long-Read Transcriptome
- SQANTI3
# Task 1: Aligning reads to reference using minimap2
In this task, you will be aligning the reads of our dataset to the provided reference genome. We will achieve this using minimap2, as we are of course dealing with long reads.
The interface of minimap2 in OmicsBox is very intuitive and should be self-explanatory, and if any questions arise, you can consult (1) the :question_mark:-Icons next to the inputs, (2) the [OmicsBox User Manual](https://manual.omicsbox.biobam.com/user-manual/omicsbox-modules/module-transcriptomics/long-read-data-analysis-tools/long-read-alignment-with-minimap2/), or (3) Fabian. For the purposes of our workshop, the default options of minimap2 (using “Long-Read Splice Alignment for PacBio CCS Reads” as the Preset) as configured in the interface should be sufficient, but feel free to explore and experiment.
## Questions
- How can the resulting .bam files be inspected within OmicsBox?
Hint
General Tools > Genome Browser > Load BAM
# Task 2: Defining Isoforms using FLAIR
Next, you will use FLAIR to define isoforms based on our aligned reads. We would have also been able to do this without running minimap2 ourselves first, but now we can supply our own aligned sequences, since we already have them.
After introducing the reads and reference genome as well as annotation, and selecting to use our own alignment files, the algorithm options can remain at their default value, although you are once again encouraged to explore and experiment.
On the last page of the wizard, simply select the target locations of the outputs.
## Questions
- How can the resulting transcript models (.gtf) be inspected and compared to our reads using OmicsBox?
Hint
In the Genome Browser, load the .gtf as a new track to compare to the previously loaded BAM file.
- How can we investigate the quality of our obtained transcript models?
Hint
See Task 3: Transcriptome Characterization using SQANTI3
# Task 3: Transcriptome Characterization using SQANTI3
As we have just learned a lot about SQANTI3 today, we will keep this section short and simply showcase how to use this powerful tool within OmicsBox.
Having just received our transcript models from FLAIR, we can investigate these results in more detail by providing the transcriptome .gtf file to SQANTI3, along with our reference genome and annotations. SQANTI3 thrives when also provided with supporting orthogonal data, but for our use case, we will leave it at the bare minimum.
## Questions
- What insights can you gain from exploring the outputs of SQANTI3?