Question

1. Compare and contrast first generation sequencing techniques (Sanger sequencing and pyrosequencing) and the second generation sequencing technique ForenSeq using the MiSeq. Include a discussion of reagents, time, cost, optimal sequence length, detection technologies, data output and post-testing analysis in your discussion. Tabulate your answers and be sure to complete the table.

2. Describe the processes that are occurring in PCR1 and PCR2. Why are each of these steps important. Describe the roles of i5 and i7, forward and reverse indexes and forward and reverse tags in the sequencing process.

3. How is the Analytical Threshold (AT) and the Interpretation Threshold (IT) determined in ForenSeq?  How is it different than how these thresholds are determined in capillary electrophoresis (CE) based STR techniques?

4. Why is the STR analysis using CE limited to approximately 30 loci?  How does ForenSeq overcome this problem?  Why is the STR data obtained with ForenSeq more discriminating than CE based data?

5. What questions can ForenSeq help to answer?  

6. What are the components that are necessary for a complete research project design that will result in usable data for analysis?

7. Describe the mechanism of sequencing by synthesis?

Answer 1

First generation Reagents - DNA pol enzyme,ssDNA, 4 dna nucleotides second generation time-4 hrs time 4-55 hrs cost-less costly costly seq length- 25-30 bases 2*25 bases 15 GB output

2. PCR steps involves

Denauration-unwinding double helix

anneling-priming DNA

Extension-adding complementary nuclotides

3.Analytical thresholdis the lowest RFU value at which DNA can be distingushed from noise while stochastic threshold applies to low DNA input data

4.Accurate detection STR expansion is limited by the sequence read length .