Next generation sequencing (15)

Illumina sequencing

PCR in a very small space

Gap between original strand and copy

Original is covalently bound and other is washed away

Fluorescent imaging

By adding fluorescently labeled dNTPs (one binds)

What does next gen sequencing allow?

-Multiple sample runs

-High sensitivity

Illumina sequencing

PCR for copies of the same molecule in a very small place (DNA clusters)

Step 1

Ligate linkers

Step 2

Denature and anneal to primers

Step 3

DNA synthesis

Step 4

Denature and wash

Step 5

Anneal

Step 6

DNA synthesis part 2

Sequencing of each strand (6)

1. dsDNA is cut

2. Fluorescently labeled dNTPs and new primer are added

3. Wash away excess dNTP

4. Fluorescent imaging = NTP determination

5. Chemically remove unbound fluorophore

6. Repeat

Sequence analysis info (4)

1. Common ancestry

2. Shared function

3. Timing events in evolution

4. Identify mutations

Purpose of sequence analysis

Homologous between 2 sequences

Sequence similarity

Degree of match between 2 sequences (%)

Homologous sequences

2 or more sequences derived from a common ancestral sequence

Analysis if length is not the same

Gaps are introduced to maximize the match

Programs for sequence analysis

Types of BLAST or basic local alignment search tool

BLASTN

Nucleotide sequence similarity

Steps of BLASTN (3)

1. Exact match

2. Extend match locally

3. Allow for short gaps in alignment

DNA shape for nanopore sequencing

Leader-Hairpin template

Does nanopore sequencing require PCR?

No

Change in current passing through a pore is…

proportional to the size of molecule (nucleotide)

Advantages of nanopore (4)

1. Sequencing single molecules

2. Very long reads = easier to map

3. Detects modifications (methylation)

4. Portable

Studying DNA methylation

Associated to generation and progression of cancer