Multimappers can be more complicated than what's shown in the example in Activity 2. In Activity 3, we look at the case where a read aligns to three different locations on the genome. How do you tell which alignment is correct now?
1. In the Activity 3 presentation, what happened when you increased the read length? Did knowing the identity of more of the base pairs in the read help you decide whether Alignment 1, 2, or 3 was the correct alignment?
2. Did adding paired-end sequencing information allow the researcher to tell whether Alignment 1 or Alignment 2 was correct? Describe the situation in the example where the paired-end sequencing information can match both Alignments 1 and 2.
3. In the example, could you tell if Alignment 1 or 2 was correct when you have the following information? Explain why.
a) Additional fragment length information: The RNA fragments that you sequenced were 100—200 base pairs long (bp).
b) Fragment length based on possible alignments of paired-end reads: Alignment 1 = 150 bp, Alignment 2 = 500 bp
4. Challenge question
You are analyzing RNA from an organism with a small genome that has very few repeated sequences. There is almost no chance of seeing multimappers. Which modifications (increasing read length, using paired-end data, using fragment length information) would you choose, if any?