Next Generation Sequencing and Sequence Analysis

L15

What is particular about next generation sequencing?

• they do PCR in solid support to obtain lots of copies if the same molecule in a very small space

  • these DNA clusters are then used for sequencing

• allows us to obtain many more seqs in comparison to Sanger Sequencing

what is an example of a next gen sequencing dvlped 20yrs ago in response to the need for a higher throughput sequencing technique

Illumina sequencing

Mechanism of next gen sequencing

(1)

• Double-stranded DNA is cut

• Strand is denatured and washed

• Leaving only one strand to be sequenced

(2)

• Add new primer

• then fluorescently labelled dNTPs

• one dNTP binds

  • then wash away excess

(3)

• Use fluorescent imaging to see which dNTP is bound

  • easier to see fluorescence when there is a lot of DNA (that’s why we do this amplificaiton)

(4)

• chemically remove fluophore and wash

(5)

• repeat until the DNA strand is replicated

Reversible terminators

what we call the nucleotides wiht fluophores conected to them

How many samples are there per experiment in next gen sequencing?

lots!

how did Dr. Pääblo’s grouop exploit Next Gen Sequencing (NGS) ?

• sequenced the whole genome of Neanderthals

• NGS provided them

  • high sensitivity

  • requiring small amts of sample

  • huge number of reads per experiment

What 4 things does sequence analysis provide us info about considering the basis that DNA and proteins are common to all organisms

1. common ancestry of gene and organisms

2. shared function or structure of proteins (assign to unknown)

3. timing events in evolution (aka when smthg occured)

4. identify mutations linked to disease (useful in clinic)

what are two basic concepts of seq analysis

• sequence similarity

• homologous sequences

  • very often the purpose of analysis is to test for homology between two seqs

Sequence similarity

• degree of match between two sequences.

• This is generally measured as a %

homologous sequences

• two or more seqs derived from a common ancestral seq

  • generally have high similarity

• two seqs can either be homologous or not

how do we search for max similarity between sequences?

• we look at the options for alligning sequences that have the most matches

what increases the difficulty of finding similarity between sequences?

length

• difficulty increases as seq is longer

• and when sequences aren’t the same length it’s hard to compare them

  • often have lost or gained some nucleotides

how can we maximize match when looking at seq similarity

• introduce gaps

Dot plot what is it and what is its use

• a type of visualization that represents the allignment of two sequences

• can be helpful to visualize changes in seqs that have occured over evolution

  • inversions

  • duplications

example of a program for seq analysis to search sequence database and its two variants

BLAST

• basic

• local

• allignment

• search

• tool

—> BLASTN: nucleotide seq similarity

—> BLASTP: protein seq similarity

How does BLAST work? example with BLASTN (3 steps)

1. query sequences: look for exact match by “BLASTed”ing it in the database of DNA seqs (look for HIT)

2. Extend the match locally

3. Allow for short gaps in alignment

What are two newer approaches to sequencing that use single molecule sequencing and what is particular about them?

• Pacific Biosciences

• Oxford Nanopore Technologies

• they skip” the PCR step

Nanopore Sequencing

• membrane w pore

  • electrical current through pore that can be measured

    • each base has difft voltage: when molecules go through pore there is chaneg in electric potential of current

• can pass DNA molecule through pore

  • helicase separates it but strands are linked as the end —> reads both strands like in a line

• we can read the sequences w/ changes in the electrical curent

• no need for PCR amplificaiton before sequencing

• not based on DNA replication

  • no use of DNA polymerase, dNTPs, or primers

what are the advantages of nanopore sequencing?

• sequencing single molecules opens possibility to study new bio Qs

• very long reads minimize need for genome assembly and allows mapping of repetitive sequences

• detects modifications like methylation in DNA 

  • you can train programs to detect changes in the DNA

• portable 

  • you can take it wherever you can take a laptop

  • sequencing is j a small casette

What is the advantage of having a long read for sequencing

• Previous sequencing techniques result in short sequences at every read

  • from tons of bp to abt 1kpb

  • when broken apart hard to tell where in genome seq belongs

• significantly longer seqs done by Nanopore (tens of kbp) allow the sequencing of repeated seqs along w/ flanking seqs

  • making it easier to map them in the genome

    • e.g. now we can sequence telomeres

Studying DNA methylation with Nanopore

• Methylation patterns on DNA are associated to the generation and progression of cancer (and other diseases linked to aging)

• Use of DNA sequencing techniques, like Nanopore, is now being used to help make decisions at the clinic