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What happens when RNA reads map to more than one location in the genome? How do you know which alignment is correct? Activity 2 shows you how the sequencing modifications described in Activity 1 can help you when your read maps to two different locations in the genome.


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Discussion Questions

1. When the read length was increased from 30 base pairs to 60 bp, you could tell that the Alignment 1 was wrong. How did sequencing more nucleotides (bp) allow you to tell which alignment was correct?

2. Explain how having paired-end sequence data helped tell which alignment was correct.

3. From your paired-end data, you know that your read can only align with Alignment 2. The student helping you in your lab calculates that your paired reads at Alignment 2 must have come from a fragment that was 250 bp long. You know that all the fragments in the sample used for your experiment were 200—300 bp long. What does this knowledge about the distance between your paired ends tell you?