Genome sequencing and sequence analysis I Flashcards
Give five examples of what biological sequencing can be used for.
- Sequencing PCR products
- De novo whole genome sequencing
- Isolating genetic differences (SNPs, other mutations)
- Profiling mRNA (RNAseq)
- Finding protein-nucleic acid interactions (ChIP seq)
What probing methods exist for assaying sequencing methods which apply “sequencing by synthesis”?
- Fluorescence
- Radio-labelling
- Phosphate detection (pyroseq)
- pH ([H+]) (nanopore)
Explain sanger sequencing (the chain termination method)
- Make four reactions containing the template, pol, master mix, all dNTPs, and one particular ddNTP.
- Fragments of different sizes are generated.
- Fragments are ran in gel/capillary.
Why do ddNTPs terminate the DNA
Without the second OH-group, the phosphodiester bond between PO4 and OH cannot occur.
Which NGS method uses emulsion PCR?
Roche’s, ion torrent
Describe how to prepare your samples for Illumina bridge amplification.
- DNA is fragmented into known lengths.
- Each fragment gets two adapters (a,b), including indicies (barcodes).
Describe Illumina seq cluster generation.
AS WELL AS I KNOW IT!!
1. There are oligonucleotides anchored to the illumina plate which are complementary to adapter a or b.
2. The fragments (w/ adapters) bind to their respective oligos on the flow cell.
3. PCR reaction synthesizes the complementary strand to the fragment which is attached through its adapter to the flow cell.
4. The dsDNA is denatured, and the unbound fragment (the newly synthesized fragment) is washed away.
5. The strand attached to the flow cell bends over due to heat cycling, and the second adapter binds to an anchored oligo.
6. PCR reaction occurs, either one-ended or pair-ended, leading to one-end or pair-end reads.
7. The dsDNA (in the bridge) is denatured, the flow cell is washed, then the process is repeated.
Describe Illumina seq sequencing
AS WELL AS I KNOW IT!!
Sequencing occurs in the clusters.
1. Sequencing primers are added (for the adapters).
2. Fluorescently labeled nucleotides are added (all have different fluorescences).
3. For every nucleotide, the reaction is exposed to exciting wavelengths for the different fluorphores.
Describe SMRT sequencing (PacBio)
- Target nucleic acid (RNA or DNA) is conjugated with circulating adapters.
- One circularized nucleic acid molecule is added to each well on the SMRT plate with polymerase dNTPs, Mg2+…
- dNTPs emit different fluorescences which are read from under the plate.
KOMPLETTERA
Describe the ion torrent method.
- Prepare a genomic library by fragmenting DNA and adding adapters to each fragment.
- Amplify the template via emulsion PCR, and attach the amplified fragments to beads
- Each bead is placed in a well on he ion torrent chip.
4.Chip is flooded with one type of nucleotides at the time. - Every time a nucleotide is incorporated into the DNA strand which is synthesized, there’s a proton expelled.
Explain nanopore sequencing.
(You have a lipid bilayer with a nanopore)
1. Unzip dsDNA fragment with a helicase which then feeds the ssDNA into the nanopore.
2. The pore can fit one strand of single stranded DNA.
3. Ions can pass through the pore, depending on how the channel is obstructed, different curents can pass the pore.
When resequencing, what proportion (%) of reads must differ from the consensus reference sequence in order to define the nucleotide(s) as SNPs?
90% of resequenced fragments must concur.
What is a contig sequence?
It’s a de novo assembly in which all the reads are combined.
Why are paired-end reads beneficial for aligning them?
- You know the length of the fragment as a whole.
- You know how long the reads are.
- You can by doing this, find the flanking regions of repetative regions easily.
