DNA Sequencing Exam Questions and Answers Graded A+
what is first generation sequencing and what did it involve? - Answers sanger sequencing
-> used radioactive dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators
-> gel-based and ran on gel
-> then moved to capillary polymers that would do that in sequencing farms
what is the Phred score? - Answers quality score for each nucleotide
Q = -10log10 P
P = probability that the base is incorrect
log transformed probability that the base is incorrect
we want a score of >20!
What is P (phred score) derived from? - Answers 1. peak spacing (ratio of largest to smallest in window
of 7)
2. uncalled/called ratio in a window of 7 peaks
3. uncalled/called ratio in a window of 3 peaks
4. peak resolution
what does a Q score of 10 vs 20 mean?> - Answers 10: 1 in 10 probability wrong
20: 1 in 100 probability wrong
how did the human genome project work? - Answers make BAC libraries from human genome and then
sequence that
how was sequencing used clinically? - Answers used to look for SNPs
-> homozygous would have double the signal of heterozygous
how is 1st gen and 2nd gen (and 3rd gen) sequencing different? - Answers 1st gen:
gel-based or capillary sequencing
- long
, - template is not single molecule (PCR)
- $1/read
- 37M/10X haploid genome
-non-reversible terminators
- not clonal
- analogue detection of fluorophores
2nd gen:
massively parallel sequencing
- short (2*100 bases)
- template clonally derived from single molecule
- $0.00125/read
- 10K / 30X haploid
-reversible terminators
-clonal sequencing
- automatic detection and base calling
3rd gen
- single molecule reads
- very long reads
- low quality and reliability
how to construct 2nd gen sequencer library? - Answers DNA/cDNA
-> shear into Pool of Random fragments
-> end repair and ligate w/ adapters (known sequences)
-> PCR amplify
what is first generation sequencing and what did it involve? - Answers sanger sequencing
-> used radioactive dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators
-> gel-based and ran on gel
-> then moved to capillary polymers that would do that in sequencing farms
what is the Phred score? - Answers quality score for each nucleotide
Q = -10log10 P
P = probability that the base is incorrect
log transformed probability that the base is incorrect
we want a score of >20!
What is P (phred score) derived from? - Answers 1. peak spacing (ratio of largest to smallest in window
of 7)
2. uncalled/called ratio in a window of 7 peaks
3. uncalled/called ratio in a window of 3 peaks
4. peak resolution
what does a Q score of 10 vs 20 mean?> - Answers 10: 1 in 10 probability wrong
20: 1 in 100 probability wrong
how did the human genome project work? - Answers make BAC libraries from human genome and then
sequence that
how was sequencing used clinically? - Answers used to look for SNPs
-> homozygous would have double the signal of heterozygous
how is 1st gen and 2nd gen (and 3rd gen) sequencing different? - Answers 1st gen:
gel-based or capillary sequencing
- long
, - template is not single molecule (PCR)
- $1/read
- 37M/10X haploid genome
-non-reversible terminators
- not clonal
- analogue detection of fluorophores
2nd gen:
massively parallel sequencing
- short (2*100 bases)
- template clonally derived from single molecule
- $0.00125/read
- 10K / 30X haploid
-reversible terminators
-clonal sequencing
- automatic detection and base calling
3rd gen
- single molecule reads
- very long reads
- low quality and reliability
how to construct 2nd gen sequencer library? - Answers DNA/cDNA
-> shear into Pool of Random fragments
-> end repair and ligate w/ adapters (known sequences)
-> PCR amplify
