Question

1. If you were creating a library using the restriction endonucleases SbfI and EcoRI, for which restriction site would you design a fork-tailed adapter? Can you imagine any characteristic of a genome that would cause you to design a fork-tailed adapter for the other restriction site? If you were to use this set of enzymes/adapters to create a library in corn, how many reads would you need for adequate coverage of each individual?

2. How many reads would you need using SbfI/EcoRI in zebrafish to provide adequate cover for each individual? How many would you need for a stickleback fish? Does this make sense, given the size of the respective genomes? How could you explain this observation? Does looking at the results for different enzyme combinations for these two species help to explain the observation?

3. How many individuals could you analyze on a single MiSeq run, if you were using the combination of NlaIII and MluCI on mice?

4. If you were interested in doing a study of population structure in corn, which enzyme combination would you choose?  How many individuals could you analyze with a single MiSeq run? How about with a single lane of an Illumina HiSeq 2000?

5. Suppose you were using paired-end sequencing with 250 cycles. How much of the genome of a stickleback fish would you expect to sequence at >7X coverage if you used EcoRI/MspI?

6. You amplify an EcoRI/MspI fragment using MspI 2.1.0 and EcoRI 1.1.0 adapters and the the PCR primers NGS_i5_S505 and NGS_i7_N719. What would the final amplicon look like? (Write the sequence.)  

7. What happens to MspI/MspI fragments in the ddRadSeq protocol we are following? How about EcoRI/EcoRI fragments?

8. We are using three different MspI and EcoRI adapters in our protocol. There are a couple of advantages to using more than one adapter for each end; what are they?

9. Taking into consideration the different indices available, and the use of three different adapters for each end of genomic fragments, how many different individuals could be analyzed with a single sequencing run?

10. Suppose you were analyzing mice just as depicted in Table 1. of Petersen et al., using the enzyme pair EcoRI-MspI  You want to increase the number of fragments you are analyzing by 50%; how would you adjust your protocol?

11. For SNP discovery, there are concerns that ddRadSeq may limit analysis to regions of the genome with low point mutation rates. Please explain why this would be so.

12. Explain the similarities between ddRadSeq and two-step PCR. Why isn’t a PCR step (instead of ligation) used on the genomic fragments in ddRadSeq?

13. If you were using single step PCR, how many different primers would you need to take full advantage of the indices available from Illumina (S502, N701, etc.) for one locus? How many would be needed if you were using two-step PCR for one locus? How would the answers change for 10 loci?

14. What are the advantages of two-step PCR over one-step PCR? Are there any conditions under which one-step PCR is more efficient?

15. A typical number of cycles for the second PCR in two-step PCR is 10. How many copies of each input molecule would be generated in ten cycles?

16. What would happen if you used two-step PCR, and the primers used in the first step were both compatible with i7 primers? Would amplification happen in the second step of the two-step PCR process? Could the amplicon be sequenced?

Answer 1

12: ddRadSeq is used for SNP discovery and genotyping. It is a variation on the RAD sequencing protocol and replacing fragment shearing with a second restriction digestion to improve the tunability and accuracy of size-selection step. Except use of two restriction enzymes and ligation step, the two techniques are similar in some ways like digestion with restriction enzyme, amplification. Ligation precise size selection and reduce chances of duplicate readings.

14: Advantages of two-step PCR:

1. It is highly sensitive and more efficient because random primers and oligo d(T) can be used.
2. It uses seperate conditions for cDNA synthesis and RT PCR.
3. It provides a stock of cDNA for future use.
4. Quantification of multiple genes from a limited number of RNA samples is possible.

One-step PCR is convenient and can reduce chances of contamination because in a single tube, RNA is converted to cDNA and all of the cDNA is immediately used for qPCR quantification. It is perfect fot the amplification of few genes of interest.

15: The number of each input molecule is double in each cycle so, after 10 cycles there is 1024 copies of each input molecule.