Mate Pair and Paired-End Sequencing – Illumina Today I will post my recurring question about the

Mate Pair and Paired-End Sequencing – Illumina

Today I will post my recurring question about thedifferences on MatePair andPaired-End Sequencing technologies used in Next-Gensequences.

Unfortunately because of technology limitations you can notread long reads, just it their ends (from 35 to 100 bp depending of thechemical and sequencer), to workaround this serious limitation, it was designedthis two methodologies.

First of all my sources was the own Illumina Website, hereis the link for Mate Pair and the link for the Paired-End Seq.  Asappear in the website and many articles, these technologies are very usefulwhen you deal with De Novo Sequencing (Assembly a entire Genome) or repetitiveparts of genome, why once you have a well aligned sequence and you knowthe distance between the two sequences you can use the first one as a anchor todetermine the second sequence position.  I think the easiest one, is thePaired-End soI will start with it.

I recommend looking at this video for those who knownothing about Illumina technology.

Paired-End

 

Paired-End sequecing

• It is a modification of the shotgun sequencing(where yoursequences have no pairs)

• Once you have the DNA fragmented in 200-500 bp, you addadapter in both ends of the sequence of interest (A1 and A2),

• In the forth step you generate clusters (spotson flowcell of  same sequences  made by amplification).

• Finally after the cluster generation you go to sequencing step(fifth and sixth steps) where using modified dNTPs and primers for knowsequences (SP1 and SP2) you read the reads by light signals.

• Because you know in the preparation you made sequences ofknow distance you can/must input this information in your aligner or assembler(depend on your application), because its a very helpful information that willmake these softwares to make less mistakes.

• Remember that the orientation of a pair of reads (R1/R2)must appear in the aligner output like(→←) respectively.

Mate Pair

Mate Pair sequecing

• In the mate pair the sequence fragmentation is made inbigger fragments (2-5 kb).

• A addition of a Biotin in each 5′ ends is done (step 3).

• The sequence with correct addition of Biotin willcircularize and after a wash, the sequencing with non-circularized fragmentwill be thrown away (step 4)

• In step 5 and 6, the circularized fragments will be cuttedwith the biotin in the middle and size-selected (400-600 bp).

• And than the sequencing is done normally: adapter withprimer sequence addition (step 7), the fragments will be spoted and clutered(step 8), and sequencing (step 9 and 10).

• Because you know in the preparation you made sequences ofknow distance you can/must input this information in your aligner or assembler(depend on your application), because its a very helpful information that willmake these softwares to make less mistakes.

• Remember that the orientation of a pair of reads (R1/R2)must appear in the aligner output like(←→) respectively.