Sanger Sequencing vs. Next-Generation Sequencing

Biotechnology Chapter 4 Day 3 Questions

1. Most of the PCR protocols we’ve learned about using two oligonucleotides and overlap PCR required three oligonucleotides. Why does a sequencing PCR require one single primer?
2. Would you expect a Sanger sequencing reaction to have equal numbers of deoxynucleotides and dideoxynucleotides? If not, which type of nucleotide should be more abundant?
3. Bill Clinton threw a party in the Rose Garden in the summer of 2000 to celebrate the publication of a draft of the human genome. Two groups were given credit for this accomplishment.  What was the difference in the approach taken by these groups to accomplish this feat?
4. Rooms full of sequencing machines containing 384 acrylamide gels each were used to sequence the human genome. Contrast this with “massively parallel sequencing” of next-generation sequencing technologies.
5. If more than one sample is included in a NGS “run”, how do you know which sample a sequencing signal came from?
6. Compare how PCR reactions end up spaced apart on a 2-dimensional (planar) surface in 454 and Illumina sequencing approaches.
7. What is the advantage of paired-end reads over single-end reads?
8. What is read depth?
9. What caveats come with the short read lengths in NGS compared to the longer read lengths in Sanger sequencing?