next generation sequencing

HIRA BATOOL

MAIRA KHAN

MARYAM ZAHRAH

NADIA ASHRAF

NEXT GENERATION SEQUENCING

Determining the sequence of the nucleotides i.e. A,T,C,G along a DNA strand

Why DNA to be sequenced…???

To know the order of nucleotides which will enable us to:› To know about genes

› Find information about the architecture of the genome

› Comparative genome analysis

Plus-minus strand sequencing

Maxam-Gilbert chemical sequencing

Sanger`s chain termination sequencing (Dye termination sequencing)

The most used method for last 30 years!

By the passage of time new techniques were developed which replaced old techniques because they were:

› Old

› Time consuming

› Laborious

› Expensive

› Hazardous(used hazardous reagents)

› Volume of reagents and space

Roche’s (454) GS FLX Genome Analyzer

Illuminas Solexa 1G sequencer

Applied Biosystem’s SOLiD system

Helicos

A DNA sequence that initially binds the RNA polymerase.

Upstream to the transcription start site.

Core promoter refers to the minimal set of sequence elements required for accurate transcription Initiation.

Usually -35 to +35

- 37 TO -32

Consensus sequence: G/C G/C G/A CGCCC

Recognized by TFIIB.

The TFIIB–BRE interaction facilitates the assembly of a TFIIB–TBP–TATA complex

-31 to – 26

Consensus sequence : T A T A A/T A A/T

Recognized by TBP( a subunit of TFIID)

In Humans, 32% of 1031 potential promoter regions have one.

Primary role is formation of pre-initiation complex(promoters + General TF).

-2 TO +4

C/T C/T A+1 N T/A C/T C/T

Recognized by TFIID

Nucleates PIC formation in TATA less promoter

facilitates the binding of Transcription Factor II D (TBP)..

+ 28 to +32

A/G G A/T CGTG

Recognized by TFIID

DPE plays a major role at TATA-less promoters.

+18 TO +29

Consensus sequence: CSARSSAACGC

cooperate with the initiator to stimulate transcription.

NO TATA in these promoters.

DNA sequences in which Four guanine bases can associate through Hoogsteen hydrogen bonding to form a square planar structure called a G-tetrad, and two or more G- tetrads can stack on top of each other to form a G-quadruplex.

Repeats of at least 3 guanine residues are separated by loops of 1-7 other base pairs

Present in DNA, RNA, LNA (locked), PNA (peptide)

Across a wide range of species, G4 DNA motifs were found in telomeres, G-rich micro- and mini-satellites, near promoters, and within the ribosomal DNA (rDNA)

Important components of human telomeres, and play a role in regulation of transcription and translation.

They are also interesting as nanotechnologicaldevices..

Generally, a simple pattern match is used for searching for possible quadruplex forming sequences:

G3+N1-7G3+N1-7G3+N1-7G3+

where N is any base (including G)

.

Text = Promoter or Telomere sequence

Pattern= GGG

N=text.length( )

M=pattern.length( )

Count=0 [ count the no of G’s]

Array[i][j] [ stores start and end position]

i=0 [stores start]

For(t=0;t<=N;) [scans Text]

{

for(p=0;p<=M;) [scans Pattern]

{

if(text[t]==Pattern[p])

{

p++;

t++;

count++;

}

else

t++; count=0

if(count>=M)

{

j=0;

array[i][j]=t -2; [stores start]

array[i][j+1]=t; [stores end]

i++;

}

t= prefix function( )

if(arr[i][j] – arr[i-1][j+1] >0 && arr[i][j]- arr[i-1][j+1]!<=7)

{

t= prefix function( )

}

}

}

http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1000861

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1636468/

Quadruplex.org

R Purine (A or G) Y Pyrimidine (C or T) N Any nucleotide W Weak (A or T) S Strong (G or C) M Amino (A or C) K Keto (G or T) B Not A (G or C or T) H Not G (A or C or T) D Not C (A or G or T) V Not T (A or G or C)

PNA's backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The bases are linked to the backbone by methylene carbonyl bonds.

LNA The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon