This presentation will give an overview of how RNA is prepared for sequencing and describe three modifications or options in the sequencing process that can help solve alignment problems. Activity 1 will introduce these modifications.
Activities 2 and 3 will focus on showing how the modifications can help with the problem of multimappers (reads that are aligned to more than one location on the genome). How do you figure out which alignment is the correct alignment?
1. What do you have to do to prepare a large RNA molecule before it can be sequenced?
2. What are three things researchers can do during RNA-seq so that read alignment or mapping will be more successful?
3. Challenge question
Scenario: You have sequenced only one end of your RNA fragments (i.e., you have single-end data instead of paired-end data). You are trying to align your fragments to the genome. Your technician tells you that the fragment lengths in your sample are about 240 base pairs long.
Does this information about the lengths of your fragments help you align your single-end reads to the genome? Explain your answer.