NGS chemistry

Question 1

What is sequencing by synthesis (Illumina)?

Answer 1

Short read based sequencing assay. Involves measuring DNA polymerase incorporation of fluorescently labelled dNTPs into a DNA template strand during sequential cycles of DNA synthesis. During incorporation, the nucleotides are identified through fluorophore excitation

Question 2

What are the 4 steps in the sequencing by synthesis method (Illumina)?

Answer 2

1. Library preparation- Extract DNA, enrichment, fragmentation (sonification or enzymatic approaches), repair ends, ligate adapters and barcodes
2. Cluster generation- DNA Template motifs bind with oligos on flow cell> Bridge amplification>double stranded DNA>Repeat
3. Sequencing- Cleave reverse strands>fluorescently labelled nucleotides are incorporated and the excitation is recorded> Forward sequence cleaved and reverse read is sequenced
4. Data analysis- BCL file converted into FASTQ file

Question 3

What are the two methods of enrichment used in the sequencing by synthesis method?

Answer 3

1. Hybrid capture e.g. I’llumina’s streptavidin beads, which has probes complimentary to ROI
2. PCR amplification e.g. Long range PCRs to amplify across a ROI

Question 4

What are the pros and cons of the hybrid capture method? (4 and 3)

Answer 4

Pro-

1. Easier to target GC-rich regions
2. Works well for FFPE sampels
3. Cost effective for large target regions
4. Good uniformity of coverage

Con-

1. Time consuming to complete
2. Requires a lot of input DNA
3. More expensive for small target regions

Question 5

What are the pros and cons of PCR based amplification?

Answer 5

Pro-

1. Fast and simple to set up
2. Can work with smaller quantities of DNA
3. Cheaper for small target regions

Con-

1. Cannot distinguish PCR duplicates or other artifacts
2. Requires design of probe sequences
3. Susceptible to contaminants from FFPE
4. Subject to bias via PCR bias- Preferential amplification

Question 6

Name Pros and cons of sequencing by synthesis compared to Ion Torrent and PacBio ( 3 each )

Answer 6

Pro-

1. Cheaper per Gbase
2. Higher reads accuracy (mostly > Q30)
3. low error rate

Con-

1. Long run times (Miseq)
2. Large DNA input requirements (compared to PacBio)
3. Shorter read lengths (compared to PacBio)

Question 7

What is SMRT (PacBio)?

Answer 7

Long read sequencing technology 1500 bases. Single molecule real time sequencing (SMRT). Uses a Zero-mode waveguide (ZMW) to optically observe polymerase mediated synthesis in real time.

Question 8

What are the 6 steps in the PacBio workflow?

Answer 8

1. Fragment DNA
2. End repair
3. Ligate adapters using SMRTbell template prep
4. Purify DNA
5. Load on to SMRT
6. Detect fluorescence emitted

Question 9

What is nanopore sequencing (ONT)?

Answer 9

Single molecule sequencing using a protein nanopore 100s Kilobases. The nanopore protein is inserted into a synthetic membrane a potential is applied across the membrane. Single molecules that enter the nanopore cause characteristic disruptions in the current. Detecting these disruptions can be used to identify sequence.

Question 10

What steps are involved in nanopore sequencing? (chemistry)

Answer 10

1. Create hairpin structure with input DNA molecule
2. Strands to be sequenced are mixed with a processive (doesn’t release substrate) enzyme
3. Single stranded DNA pulled through the aperture
4. Enzyme passes DNA through 1 base at a time
5. Electrical current is measured to determine nucleotide- recorded in FAST5