March 25

Question 1

How does illumina sequencing work?

What are 2 issues?

Answer 1

1. Fragement DNA, and add adaptor to each end
2. Bind DNA to complementary adaptors on slide, then it bridges and does PCR to form a polony (colony of PCR products)
3. Then add only fluoresently labeled ddNTPs, so only 1 based added to primer (for almost all of the PCR products), then take image.
4. Then add “unblock” by adding OH group and remove fluoresent label, so now the next ddNTP can be added, take picture.

This method only gives short reads as we have phasing. Not all nucleotides are added to each PCR product, so if you have ATCG sequenced so far, some labels in the image might show just the A, T, or C still, so they are behind. This inaccuracy gets worse the longer the read is, so reads are kept short.

Also regions with repeats. We only have reads of about 200 bp, so 500 long A repeat will take multiple reads, but then hard to assemble when doing final genome assembly with short reads

Question 2

How does PacBio work? Issues? Long or short reads?

Answer 2

1. Do PCR
2. Fragment DNA
3. Add 3’ Poly A tail
4. Get Poly A tails on each end of each strand to circularize
   5.Have wells with 1 DNA polymerase at bottom of each. These wells are narrower than wavelength of light, so light only penetrates a bit from the bottom, just covers the DNA polymerase.
5. Add fluorescent dNTPs, so when it is added is is close enough to bottom of well to be detected, then after dNTP leaves DNA polymerase and onto the released DNA strand, it can’t be excited anymore

Has irregular tempo, it is just “chance” of if nuclotide reaches DNA poymerase. Get really long reads

Question 3

How does Nanopore sequencing work? Long or short reads, accuracy is how good compared to others?

Answer 3

1. Have porin from E. coli.
2. 1 strand from ds DNA is threaded thorugh the pore, and ion flow through pore as well. Then the base added will change conductivity. It has the lowest accuracy, but is cheap and gives long reads.

Question 4

What are 4 similarities of massive parallel sequencing (MPS) tech?

Answer 4

1. Random fragmentation
2. Ligate adaptors to DNA
3. Paired end read capability, can read from either end
4. Digital info (can assemble genomes in silico)